Structured Review

Thermo Fisher hiv 1 rt
Yonetani–Theorell plot of the combination of 33 and 3 on <t>HIV-1</t> RT RNase H activity. HIV-1 RT was incubated in the presence of 33 (3.3 μM, 1.1 μM, 0.55 μM, 0.27 μM, and 0.13 μM) combined with increasing concentrations of 3 : 0.27 μM (●), 0.55 μM (○), 1.1 μM (▼), and 3.3 μM (Δ). Data were obtained by a single experiment, made in duplicate.
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Images

1) Product Images from "Studies on Cycloheptathiophene-3-Carboxamide Derivatives as Allosteric HIV-1 Ribonuclease H inhibitors"

Article Title: Studies on Cycloheptathiophene-3-Carboxamide Derivatives as Allosteric HIV-1 Ribonuclease H inhibitors

Journal: ChemMedChem

doi: 10.1002/cmdc.201600015

Yonetani–Theorell plot of the combination of 33 and 3 on HIV-1 RT RNase H activity. HIV-1 RT was incubated in the presence of 33 (3.3 μM, 1.1 μM, 0.55 μM, 0.27 μM, and 0.13 μM) combined with increasing concentrations of 3 : 0.27 μM (●), 0.55 μM (○), 1.1 μM (▼), and 3.3 μM (Δ). Data were obtained by a single experiment, made in duplicate.
Figure Legend Snippet: Yonetani–Theorell plot of the combination of 33 and 3 on HIV-1 RT RNase H activity. HIV-1 RT was incubated in the presence of 33 (3.3 μM, 1.1 μM, 0.55 μM, 0.27 μM, and 0.13 μM) combined with increasing concentrations of 3 : 0.27 μM (●), 0.55 μM (○), 1.1 μM (▼), and 3.3 μM (Δ). Data were obtained by a single experiment, made in duplicate.

Techniques Used: Activity Assay, Incubation

2) Product Images from "A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1"

Article Title: A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0020208

Schematic of dislocation mutagenesis and the development of the K65R mutation in subtype C HIV-1. Step 1 : DNA synthesis approaches the end of a homopolymeric nt stretch that ends precisely at the location of K65R development. Step 2 : At the end of the sequence, the RT enzyme exhibits characteristic pausing of DNA synthesis. The template-strand folds onto itself and exposes a C in the folded-over template strand. Step 3 : A dGTP nt correctly binds opposite the C base of the misaligned template strand as DNA synthesis continues. Step 4 : The primer and template strands realign and the same C base becomes re-exposed on the now correctly aligned template strand. Step 5 : A second dGTP becomes incorporated opposite the re-exposed C on the correctly aligned primer/template strands and DNA synthesis continues normally. This series of events yields the AAG-to-AGG change that is responsible for the more facilitated appearance of the K65R mutation in subtype C HIV-1.
Figure Legend Snippet: Schematic of dislocation mutagenesis and the development of the K65R mutation in subtype C HIV-1. Step 1 : DNA synthesis approaches the end of a homopolymeric nt stretch that ends precisely at the location of K65R development. Step 2 : At the end of the sequence, the RT enzyme exhibits characteristic pausing of DNA synthesis. The template-strand folds onto itself and exposes a C in the folded-over template strand. Step 3 : A dGTP nt correctly binds opposite the C base of the misaligned template strand as DNA synthesis continues. Step 4 : The primer and template strands realign and the same C base becomes re-exposed on the now correctly aligned template strand. Step 5 : A second dGTP becomes incorporated opposite the re-exposed C on the correctly aligned primer/template strands and DNA synthesis continues normally. This series of events yields the AAG-to-AGG change that is responsible for the more facilitated appearance of the K65R mutation in subtype C HIV-1.

Techniques Used: Mutagenesis, DNA Synthesis, Sequencing

Sequence comparison of the RT region of the pol gene derived from subtype B and C HIV-1 spanning codons 62 to 68. (A) Outline of the reverse-transcription process in subtype B HIV-1 leading to either wild-type or K65R-containing transcripts. The K65R mutation results from an AAA-to-AGA mutation in subtype B HIV-1. (B) Outline of the reverse-transcription process in subtype C HIV-1 leading to either wild-type or K65R-containing transcripts. The K65R mutation results from an AAG-to-AGG mutation in subtype C HIV-1. The genomic sequences of the subtype B and C RT segments of pol spanning codons 62 to 68 are indicated. (−)ssDNA synthesis from the viral (+)ssRNA genome and (+)dsDNA synthesis from the (−)ssDNA intermediate are also depicted. Underlined are the homopolymeric nt stretches of both templates at their specific locations. In bold are the nt polymorphisms that exist between both subtypes in this region. Highlighted in a square is the base that, once mutated, gives rise to the K65R drug resistance mutation.
Figure Legend Snippet: Sequence comparison of the RT region of the pol gene derived from subtype B and C HIV-1 spanning codons 62 to 68. (A) Outline of the reverse-transcription process in subtype B HIV-1 leading to either wild-type or K65R-containing transcripts. The K65R mutation results from an AAA-to-AGA mutation in subtype B HIV-1. (B) Outline of the reverse-transcription process in subtype C HIV-1 leading to either wild-type or K65R-containing transcripts. The K65R mutation results from an AAG-to-AGG mutation in subtype C HIV-1. The genomic sequences of the subtype B and C RT segments of pol spanning codons 62 to 68 are indicated. (−)ssDNA synthesis from the viral (+)ssRNA genome and (+)dsDNA synthesis from the (−)ssDNA intermediate are also depicted. Underlined are the homopolymeric nt stretches of both templates at their specific locations. In bold are the nt polymorphisms that exist between both subtypes in this region. Highlighted in a square is the base that, once mutated, gives rise to the K65R drug resistance mutation.

Techniques Used: Sequencing, Derivative Assay, Mutagenesis, Genomic Sequencing

3) Product Images from "Primer ID Informs Next-Generation Sequencing Platforms and Reveals Preexisting Drug Resistance Mutations in the HIV-1 Reverse Transcriptase Coding Domain"

Article Title: Primer ID Informs Next-Generation Sequencing Platforms and Reveals Preexisting Drug Resistance Mutations in the HIV-1 Reverse Transcriptase Coding Domain

Journal: AIDS Research and Human Retroviruses

doi: 10.1089/aid.2014.0031

Primer ID method to estimate the HIV-1 population. (A) A unique Primer ID sequence is incorporated into each viral genome along with a sample-specific barcode ( blue ) during cDNA synthesis. For each clinical subject sample, two independent cDNA synthesis
Figure Legend Snippet: Primer ID method to estimate the HIV-1 population. (A) A unique Primer ID sequence is incorporated into each viral genome along with a sample-specific barcode ( blue ) during cDNA synthesis. For each clinical subject sample, two independent cDNA synthesis

Techniques Used: Sequencing

4) Product Images from "Diversity-oriented solid-phase synthesis and biological evaluation of oligonucleotide hairpins as HIV-1 RT RNase H inhibitors"

Article Title: Diversity-oriented solid-phase synthesis and biological evaluation of oligonucleotide hairpins as HIV-1 RT RNase H inhibitors

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkh948

Schematic representation of the mode of inhibition of HIV-1 RT RNase H-mediated degradation of viral RNA by small-molecule hairpin aptamers. The hairpins bind to the RNase H domain, found in the C-terminus of the p66 subunit of RT.
Figure Legend Snippet: Schematic representation of the mode of inhibition of HIV-1 RT RNase H-mediated degradation of viral RNA by small-molecule hairpin aptamers. The hairpins bind to the RNase H domain, found in the C-terminus of the p66 subunit of RT.

Techniques Used: Inhibition

( A ) Three-dimensional graph showing % inhibition (at 40 μM [aptamer]) versus helical conformation for hairpins with various loops and having the same stem compositions. More pronounced inhibitory activity (‘hits’) against HIV-1 RT RNase H is observed for hairpins containing 2′,5′-RNA loops, although the most potent member contains a 3′,5′-RNA loop. Hairpins containing DNA loops, regardless of their stem composition, do not show any degree of inhibition. ( B ) Effects of loop base sequence, point insertions and stem length.
Figure Legend Snippet: ( A ) Three-dimensional graph showing % inhibition (at 40 μM [aptamer]) versus helical conformation for hairpins with various loops and having the same stem compositions. More pronounced inhibitory activity (‘hits’) against HIV-1 RT RNase H is observed for hairpins containing 2′,5′-RNA loops, although the most potent member contains a 3′,5′-RNA loop. Hairpins containing DNA loops, regardless of their stem composition, do not show any degree of inhibition. ( B ) Effects of loop base sequence, point insertions and stem length.

Techniques Used: Inhibition, Activity Assay, Sequencing

( A ) Gel autoradiogram illustrating representative RNase H inhibitory activity resulting from screening DONAS hairpin library members. A [ 32 P]-RNA:DNA hybrid was incubated with HIV-1 RT in the presence of varying amounts of hairpins (0–50 μM). ( B ) Inhibition assay of DNA synthesis catalyzed by (a) DNA-dependent DNA polymerase and (b) RNA-dependent DNA polymerase activities of HIV-1 RT. Unlabeled hairpin (80 μM) was pre-incubated with RT for 20 min at room temperature prior to initiating the polymerization reaction by adding the proper DNA or RNA template hybridized to a 5′-[ 32 P]-labeled primer in the presence of dNTPs (see Materials and Methods). The various lanes show full-length DNA products synthesized after 15 min in the absence (−) and presence (+) of unlabeled hairpins.
Figure Legend Snippet: ( A ) Gel autoradiogram illustrating representative RNase H inhibitory activity resulting from screening DONAS hairpin library members. A [ 32 P]-RNA:DNA hybrid was incubated with HIV-1 RT in the presence of varying amounts of hairpins (0–50 μM). ( B ) Inhibition assay of DNA synthesis catalyzed by (a) DNA-dependent DNA polymerase and (b) RNA-dependent DNA polymerase activities of HIV-1 RT. Unlabeled hairpin (80 μM) was pre-incubated with RT for 20 min at room temperature prior to initiating the polymerization reaction by adding the proper DNA or RNA template hybridized to a 5′-[ 32 P]-labeled primer in the presence of dNTPs (see Materials and Methods). The various lanes show full-length DNA products synthesized after 15 min in the absence (−) and presence (+) of unlabeled hairpins.

Techniques Used: Activity Assay, Incubation, Inhibition, DNA Synthesis, Labeling, Synthesized

5) Product Images from "Inhibition of 5?-UTR RNA Conformational Switching in HIV-1 Using Antisense PNAs"

Article Title: Inhibition of 5?-UTR RNA Conformational Switching in HIV-1 Using Antisense PNAs

Journal: PLoS ONE

doi: 10.1371/journal.pone.0049310

Effects of PNA oligomers on the efficiency of dimerization of HIV-1 RNA transcripts. The samples from dimerization assays were analyzed on the 4% non-denaturing gels and the intensity of monomer and dimer bands on each gel was quantified. The dimerization efficiency was calculated as d/D × 100%, where d is the percentage intensity of dimer with different concentration of PNA and D is the percentage intensity of dimer in the absence of PNAs. Error bars indicate standard deviation from triplicate runs.
Figure Legend Snippet: Effects of PNA oligomers on the efficiency of dimerization of HIV-1 RNA transcripts. The samples from dimerization assays were analyzed on the 4% non-denaturing gels and the intensity of monomer and dimer bands on each gel was quantified. The dimerization efficiency was calculated as d/D × 100%, where d is the percentage intensity of dimer with different concentration of PNA and D is the percentage intensity of dimer in the absence of PNAs. Error bars indicate standard deviation from triplicate runs.

Techniques Used: Concentration Assay, Standard Deviation

PNA effect on the equilibrium between the LDI and BMH conformations of HIV-1 leader RNA. The RNA samples from conformer assays were analyzed on the non-denaturing gels and the intensity of bands corresponding to the LDI and BMH conformers on each gel was quantified. The relative shift from LDI to BMH conformation was calculated as e/E × 100%, where ‘e’ is the percentage intensity of LDI conformer in TEN buffer with different concentration of PNA oligomer and ‘E’ is the percentage intensity of LDI conformer in TEN buffer without PNA oligomers. Error bars indicate standard deviation of triplicate runs.
Figure Legend Snippet: PNA effect on the equilibrium between the LDI and BMH conformations of HIV-1 leader RNA. The RNA samples from conformer assays were analyzed on the non-denaturing gels and the intensity of bands corresponding to the LDI and BMH conformers on each gel was quantified. The relative shift from LDI to BMH conformation was calculated as e/E × 100%, where ‘e’ is the percentage intensity of LDI conformer in TEN buffer with different concentration of PNA oligomer and ‘E’ is the percentage intensity of LDI conformer in TEN buffer without PNA oligomers. Error bars indicate standard deviation of triplicate runs.

Techniques Used: Concentration Assay, Standard Deviation

Effects of PNA oligomers on gel mobility of HIV-1 leader RNA (447nts). The RNA transcript was incubated in dimerization buffer (5 mM MgCl 2 ) at 65°C in the absence or presence of increasing concentration of PNAs. The samples were analyzed on TBM gels. Each gel has the following loading pattern - Lane 1: Dimerization buffer without PNA oligomer, Lane 2–6: Dimerization buffer with the increasing template ratios, as indicated.
Figure Legend Snippet: Effects of PNA oligomers on gel mobility of HIV-1 leader RNA (447nts). The RNA transcript was incubated in dimerization buffer (5 mM MgCl 2 ) at 65°C in the absence or presence of increasing concentration of PNAs. The samples were analyzed on TBM gels. Each gel has the following loading pattern - Lane 1: Dimerization buffer without PNA oligomer, Lane 2–6: Dimerization buffer with the increasing template ratios, as indicated.

Techniques Used: Incubation, Concentration Assay

Effect of PNA oligomers on HIV-1 template-switching. Samples were analyzed on the 6% denaturing TBE gel. Each gel has the following loading pattern- Lane 1: HIV-1 leader transcripts including the AUG codon of gag gene, Lane 2: Donor transcript, Lane 3–7: Donor and acceptor transcripts with increasing concentration of anti-sense PNAs in molar ratio (with respect to the concentration of donor transcript) as indicated on top of each gel (Reverse transcribed with 0.2 unit of HIV-1RT/µl), Lane 8- Donor and acceptor transcripts without PNA oligo {Reverse transcribed with 0.2 unit of HIV-1RT/µl (from Lane 3 to 8 in each panel )},. FT- Full-length transcript, DT- Donor transcript, T- Template-switched product (Lane 3 to 8), F- Full-length donor product (Lane 3 to 8).
Figure Legend Snippet: Effect of PNA oligomers on HIV-1 template-switching. Samples were analyzed on the 6% denaturing TBE gel. Each gel has the following loading pattern- Lane 1: HIV-1 leader transcripts including the AUG codon of gag gene, Lane 2: Donor transcript, Lane 3–7: Donor and acceptor transcripts with increasing concentration of anti-sense PNAs in molar ratio (with respect to the concentration of donor transcript) as indicated on top of each gel (Reverse transcribed with 0.2 unit of HIV-1RT/µl), Lane 8- Donor and acceptor transcripts without PNA oligo {Reverse transcribed with 0.2 unit of HIV-1RT/µl (from Lane 3 to 8 in each panel )},. FT- Full-length transcript, DT- Donor transcript, T- Template-switched product (Lane 3 to 8), F- Full-length donor product (Lane 3 to 8).

Techniques Used: Concentration Assay

Effects of PNA on the template-switching efficiency between the acceptor and donor HIV-1 RNA templates. The RNA samples from the coupled dimerization-template switching experiments were analyzed on the 6% TBE denaturing gels and the intensity of bands corresponding to the template-switched products and the full-length product of the donor on each gel was quantified The template-switching efficiency for each sample was calculated as t/T × 100%, where ‘t’ is the % intensity of template-switching product in the primer extension reaction with different concentration of PNA oligomer and ‘T’ is the % intensity of the template-switching product in the primer extension reaction without PNA oligomers. Error bars indicate standard deviation of triplicate runs.
Figure Legend Snippet: Effects of PNA on the template-switching efficiency between the acceptor and donor HIV-1 RNA templates. The RNA samples from the coupled dimerization-template switching experiments were analyzed on the 6% TBE denaturing gels and the intensity of bands corresponding to the template-switched products and the full-length product of the donor on each gel was quantified The template-switching efficiency for each sample was calculated as t/T × 100%, where ‘t’ is the % intensity of template-switching product in the primer extension reaction with different concentration of PNA oligomer and ‘T’ is the % intensity of the template-switching product in the primer extension reaction without PNA oligomers. Error bars indicate standard deviation of triplicate runs.

Techniques Used: Concentration Assay, Standard Deviation

Schematic representation of RNA transcripts showing motifs within the 5′ UTR of HIV-1 RNA genome. (A)5′ leader HIV-1 RNA transcripts including the 5' end of the gag gene (position +1 to 447 relative to the transcriptional start site +1). (B) Acceptor and donor RNA templates used in the template-switching assays.
Figure Legend Snippet: Schematic representation of RNA transcripts showing motifs within the 5′ UTR of HIV-1 RNA genome. (A)5′ leader HIV-1 RNA transcripts including the 5' end of the gag gene (position +1 to 447 relative to the transcriptional start site +1). (B) Acceptor and donor RNA templates used in the template-switching assays.

Techniques Used:

RNA (447nts) in presence of increasing concentration of PNA oligomers. The radiolabeled HIV-1 leader transcript was incubated in TEN buffer with increasing concentration of respective PNA as well as in formamide and dimerization buffer. The samples were analyzed on non-denaturing TBE gels. Each gel has the same loading pattern. Lane 1: Formamide buffer, Lane 2: TEN buffer without PNA oligomer, Lane 3–7: TEN buffer with the increasing concentration of PNA (in molar ratio) as indicated on top of each gel, Lane 8: Dimerization buffer. F – Formamide buffer, D – Dimerization buffer.
Figure Legend Snippet: RNA (447nts) in presence of increasing concentration of PNA oligomers. The radiolabeled HIV-1 leader transcript was incubated in TEN buffer with increasing concentration of respective PNA as well as in formamide and dimerization buffer. The samples were analyzed on non-denaturing TBE gels. Each gel has the same loading pattern. Lane 1: Formamide buffer, Lane 2: TEN buffer without PNA oligomer, Lane 3–7: TEN buffer with the increasing concentration of PNA (in molar ratio) as indicated on top of each gel, Lane 8: Dimerization buffer. F – Formamide buffer, D – Dimerization buffer.

Techniques Used: Concentration Assay, Incubation

Dimerization efficiency of the HIV-1 RNA (1–447) transcript. The RNA transcripts were incubated in monomer (no MgCl 2 ) and dimerization buffer with varying concentration of MgCl 2 . All samples were analyzed on TBM gels. Lane1: monomer buffer, Lane 2: Dimerization buffer with 1 mM MgCl 2 Lane 3: Dimerization buffer with 2 mM MgCl 2 , Lane 4: Dimerization buffer with 5 mM MgCl 2 , Lane 5: Dimerization buffer with 10 mM MgCl 2.
Figure Legend Snippet: Dimerization efficiency of the HIV-1 RNA (1–447) transcript. The RNA transcripts were incubated in monomer (no MgCl 2 ) and dimerization buffer with varying concentration of MgCl 2 . All samples were analyzed on TBM gels. Lane1: monomer buffer, Lane 2: Dimerization buffer with 1 mM MgCl 2 Lane 3: Dimerization buffer with 2 mM MgCl 2 , Lane 4: Dimerization buffer with 5 mM MgCl 2 , Lane 5: Dimerization buffer with 10 mM MgCl 2.

Techniques Used: Incubation, Concentration Assay

Proposed stem-loop structure of the 5′untranslated region of HIV-1. Target loop regions are highlighted. PNAs targeted against different sites of 5′UTR of HIV-1 are shown in the diagram (Abbink et. al. 2003).
Figure Legend Snippet: Proposed stem-loop structure of the 5′untranslated region of HIV-1. Target loop regions are highlighted. PNAs targeted against different sites of 5′UTR of HIV-1 are shown in the diagram (Abbink et. al. 2003).

Techniques Used:

Mobility of HIV-1 leader RNA (447nts) in different buffers. The RNA transcripts were incubated in different buffer conditions and were analyzed on 4% non-denaturing TBE gel. Lane 1: Formamide buffer, Lane 2: Tris-Cl buffer, Lane 3: TN buffer, Lane 4: TN buffer with 0.1 mM MgCl 2 , Lane 5: TN buffer with 1.0 mM MgCl 2 , Lane 6: TN buffer with 5 mM MgCl 2 , Lane 7: TEN buffer and Lane 8: Dimerization buffer with 5 mM MgCl 2 .
Figure Legend Snippet: Mobility of HIV-1 leader RNA (447nts) in different buffers. The RNA transcripts were incubated in different buffer conditions and were analyzed on 4% non-denaturing TBE gel. Lane 1: Formamide buffer, Lane 2: Tris-Cl buffer, Lane 3: TN buffer, Lane 4: TN buffer with 0.1 mM MgCl 2 , Lane 5: TN buffer with 1.0 mM MgCl 2 , Lane 6: TN buffer with 5 mM MgCl 2 , Lane 7: TEN buffer and Lane 8: Dimerization buffer with 5 mM MgCl 2 .

Techniques Used: Incubation

6) Product Images from "Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism"

Article Title: Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism

Journal: PLoS ONE

doi: 10.1371/journal.pone.0078596

Ability of mutant RHAs to promote the annealing of tRNA Lys3 to viral RNA. 293T cells were first treated with siRNA Con or siRNA RHA , and 16 hours later, were cotransfected with SVC21.BH10 and a plasmid expressing either 6×His tag, or His-tagged wild-type or mutant RHAs. 48 hours later, extracellular viruses were purified and cells were lysed. (A) Western blots of cell lysates probed with antibodies to RHA, His tag, or β-actin. (B) Western blots of viral lysates, containing equal amount of CAp24, probed with antibodies to RHA, His tag, CAp24, or RTp66/p51. (C) One nucleotide extension assay (+1 nt extension). Total viral RNA was isolated from purified HIV-1 particles, and tRNA Lys3 annealed to viral RNA in vivo was extended by 1 nt ([ 32 P]-dCTP), using HIV-1 reverse transcriptase. The extended tRNA Lys3 products are resolved by denaturing 1D PAGE, and visualized using a PhosphorImager. The control gel represents the +1 nt extension of a DNA primer annealed in vitro to viral RNA downstream of the tRNA Lys3 binding site, and is used to show that approximately equal amounts of viral RNA were used in each extension reaction. (D) The values of the +1 nt extended tRNA Lys3 products were quantitated using a PhosphorImager, normalized to the values obtained with virions produced from siRNA Con -treated cells (lane 1), and are presented graphically as a percentage. Shown are the mean values ± standard deviations of 3 independent experiments. *, P
Figure Legend Snippet: Ability of mutant RHAs to promote the annealing of tRNA Lys3 to viral RNA. 293T cells were first treated with siRNA Con or siRNA RHA , and 16 hours later, were cotransfected with SVC21.BH10 and a plasmid expressing either 6×His tag, or His-tagged wild-type or mutant RHAs. 48 hours later, extracellular viruses were purified and cells were lysed. (A) Western blots of cell lysates probed with antibodies to RHA, His tag, or β-actin. (B) Western blots of viral lysates, containing equal amount of CAp24, probed with antibodies to RHA, His tag, CAp24, or RTp66/p51. (C) One nucleotide extension assay (+1 nt extension). Total viral RNA was isolated from purified HIV-1 particles, and tRNA Lys3 annealed to viral RNA in vivo was extended by 1 nt ([ 32 P]-dCTP), using HIV-1 reverse transcriptase. The extended tRNA Lys3 products are resolved by denaturing 1D PAGE, and visualized using a PhosphorImager. The control gel represents the +1 nt extension of a DNA primer annealed in vitro to viral RNA downstream of the tRNA Lys3 binding site, and is used to show that approximately equal amounts of viral RNA were used in each extension reaction. (D) The values of the +1 nt extended tRNA Lys3 products were quantitated using a PhosphorImager, normalized to the values obtained with virions produced from siRNA Con -treated cells (lane 1), and are presented graphically as a percentage. Shown are the mean values ± standard deviations of 3 independent experiments. *, P

Techniques Used: Mutagenesis, Plasmid Preparation, Expressing, Purification, Western Blot, Isolation, In Vivo, Polyacrylamide Gel Electrophoresis, In Vitro, Binding Assay, Produced

RT-PCR analysis of singly (∼ 4.0 kb) and multiply (∼ 1.8 kb) spliced RNA species. Total cellular RNAs analyzed in Figure 4 were subjected to semiquantitative RT-PCR. 4-16 µl of PCR products were heat-denatured, separated in 6% denaturing polyacrylamide gel, transferred onto GeneScreen Plus membrane, and then probed with [ 32 P]-labeled DNA oligonucleotide P131 that can recognize all HIV-1 RNA transcripts. The radioactive signals were visualized using a PhosphorImager. (A) Diagram showing the organization of major splice donor (SD1-5) and acceptor (SA1-8) sites, and the locations of viral exons and oligonucleotide primers on the HIV-1 genomic RNA. Filled boxes represent the exons detected in this study. The viral nucleotide numbers between 1 and 224 correspond to that of human immunodeficiency virus 1 (GenBank accession no. NC_001802). The viral nucleotide numbers between 225 and 9156 correspond to that between 1 and 8932 of human immunodeficiency virus type 1, isolate BH10 genome (GenBank accession no. M15654 K02008 K02009 K02010). (B) Analysis of ∼ 4.0 kb HIV-1 RNA species using primer pair Odp.045/KPNA. (C) Analysis of ∼ 1.8 kb HIV-1 RNA species using primer pair Odp.045/SJ4.7A. (D) Analysis of exon 6D-containing HIV-1 RNA species using primer pair Odp.045/3311A. Shown is a representative of 3 independent experiments.
Figure Legend Snippet: RT-PCR analysis of singly (∼ 4.0 kb) and multiply (∼ 1.8 kb) spliced RNA species. Total cellular RNAs analyzed in Figure 4 were subjected to semiquantitative RT-PCR. 4-16 µl of PCR products were heat-denatured, separated in 6% denaturing polyacrylamide gel, transferred onto GeneScreen Plus membrane, and then probed with [ 32 P]-labeled DNA oligonucleotide P131 that can recognize all HIV-1 RNA transcripts. The radioactive signals were visualized using a PhosphorImager. (A) Diagram showing the organization of major splice donor (SD1-5) and acceptor (SA1-8) sites, and the locations of viral exons and oligonucleotide primers on the HIV-1 genomic RNA. Filled boxes represent the exons detected in this study. The viral nucleotide numbers between 1 and 224 correspond to that of human immunodeficiency virus 1 (GenBank accession no. NC_001802). The viral nucleotide numbers between 225 and 9156 correspond to that between 1 and 8932 of human immunodeficiency virus type 1, isolate BH10 genome (GenBank accession no. M15654 K02008 K02009 K02010). (B) Analysis of ∼ 4.0 kb HIV-1 RNA species using primer pair Odp.045/KPNA. (C) Analysis of ∼ 1.8 kb HIV-1 RNA species using primer pair Odp.045/SJ4.7A. (D) Analysis of exon 6D-containing HIV-1 RNA species using primer pair Odp.045/3311A. Shown is a representative of 3 independent experiments.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Labeling

Ability of mutant RHAs to stimulate the synthesis of HIV-1 mRNAs. 293T cells were cotransfected with SVC21.BH10 and either a plasmid expressing His-tagged wild-type or mutant RHA, or only the 6×His tag. 24 hours later, cell lysates and total cellular RNA were prepared and subjected to Western blotting and Northern blotting analysis respectively. (A) Western blots of cell lysates probed with anti-RHA, anti-His, or anti-β-actin. (B) Northern blotting. The total cellular RNA was resolved by electrophoresis on a denaturing 1% agarose gel, and blotted onto GeneScreen Plus membrane. The membrane was probed with the [32P]-labeled DNAs that are complimentary to HIV-1 5'-UTR. Ethidium bromide-stained rRNAs (18S and 28S) are included as an RNA loading control. Unspliced (US) ∼ 9.2 kb, singly spliced (SS) ∼ 4.0 kb, and multiply spliced (MS) ∼ 1.8 kb RNAs are indicated. (C) The intensity of RNA bands in panel B representing US, SS, or MS RNAs was quantitated using a PhosphorImager instrument, and are presented graphically. Shown is a representative of 3 independent experiments. (D) The ratio of US RNA to SS+MS RNA in panel B was determined. Shown are the mean values ± standard deviations of 3 independent experiments. *, P
Figure Legend Snippet: Ability of mutant RHAs to stimulate the synthesis of HIV-1 mRNAs. 293T cells were cotransfected with SVC21.BH10 and either a plasmid expressing His-tagged wild-type or mutant RHA, or only the 6×His tag. 24 hours later, cell lysates and total cellular RNA were prepared and subjected to Western blotting and Northern blotting analysis respectively. (A) Western blots of cell lysates probed with anti-RHA, anti-His, or anti-β-actin. (B) Northern blotting. The total cellular RNA was resolved by electrophoresis on a denaturing 1% agarose gel, and blotted onto GeneScreen Plus membrane. The membrane was probed with the [32P]-labeled DNAs that are complimentary to HIV-1 5'-UTR. Ethidium bromide-stained rRNAs (18S and 28S) are included as an RNA loading control. Unspliced (US) ∼ 9.2 kb, singly spliced (SS) ∼ 4.0 kb, and multiply spliced (MS) ∼ 1.8 kb RNAs are indicated. (C) The intensity of RNA bands in panel B representing US, SS, or MS RNAs was quantitated using a PhosphorImager instrument, and are presented graphically. Shown is a representative of 3 independent experiments. (D) The ratio of US RNA to SS+MS RNA in panel B was determined. Shown are the mean values ± standard deviations of 3 independent experiments. *, P

Techniques Used: Mutagenesis, Plasmid Preparation, Expressing, Western Blot, Northern Blot, Electrophoresis, Agarose Gel Electrophoresis, Labeling, Staining, Mass Spectrometry

Ability of mutant RHAs to interact with HIV-1 RNA in the cell. 293T cells were transfected with SVC21.BH10 and a plasmid expressing either His-tagged wild-type or mutant RHA, or only the 6×His tag. 24 hours later, cells were cross-linked, lysed, and sonicated. The cell lysates were incubated with Ni-NTA agarose to capture His-tagged protein. RNAs isolated from cell lysates (input) or from nucleoprotein bound to Ni-NTA agarose (precipitate) were subjected to RT-PCR analysis. (A) Western blot of cell lysates was probed with anti-His to detect expression of His-tagged RHA in transfected cells. The expressed 6×His tag peptide alone was not detectable in the Western blot. (B) Western blot of the precipitates was probed with anti-RHA. (C) The input RNA and RNA that was coprecipitated with His-tagged proteins were analyzed by RT-PCR, using primer pair P1-F/R [19] specific to HIV-1 RNA. RT-PCR was performed in the presence (+) or absence (–) of reverse transcriptase.
Figure Legend Snippet: Ability of mutant RHAs to interact with HIV-1 RNA in the cell. 293T cells were transfected with SVC21.BH10 and a plasmid expressing either His-tagged wild-type or mutant RHA, or only the 6×His tag. 24 hours later, cells were cross-linked, lysed, and sonicated. The cell lysates were incubated with Ni-NTA agarose to capture His-tagged protein. RNAs isolated from cell lysates (input) or from nucleoprotein bound to Ni-NTA agarose (precipitate) were subjected to RT-PCR analysis. (A) Western blot of cell lysates was probed with anti-His to detect expression of His-tagged RHA in transfected cells. The expressed 6×His tag peptide alone was not detectable in the Western blot. (B) Western blot of the precipitates was probed with anti-RHA. (C) The input RNA and RNA that was coprecipitated with His-tagged proteins were analyzed by RT-PCR, using primer pair P1-F/R [19] specific to HIV-1 RNA. RT-PCR was performed in the presence (+) or absence (–) of reverse transcriptase.

Techniques Used: Mutagenesis, Transfection, Plasmid Preparation, Expressing, Sonication, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

7) Product Images from "Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism"

Article Title: Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Primer rescue by HIV-1 RT. 5′- 32 P-labeled, ddAMP-terminated L32 primer annealed to WL50 template was incubated with excess HIV-1 RT and the indicated concentrations of PPase-treated ATP (lanes 1–8) or GTP (lanes 9–16) at 37°C for 10 min. After heat inactivation of the RT, the primer was extended by incubation with exonuclease-free Klenow polymerase and dNTPs for 10 min at 37°C. The products were separated by electrophoresis through a 20% denaturing polyacrylamide gel. The original L32-ddAMP primer is indicated as primer and extended primer products are indicated as ext. primer.
Figure Legend Snippet: Primer rescue by HIV-1 RT. 5′- 32 P-labeled, ddAMP-terminated L32 primer annealed to WL50 template was incubated with excess HIV-1 RT and the indicated concentrations of PPase-treated ATP (lanes 1–8) or GTP (lanes 9–16) at 37°C for 10 min. After heat inactivation of the RT, the primer was extended by incubation with exonuclease-free Klenow polymerase and dNTPs for 10 min at 37°C. The products were separated by electrophoresis through a 20% denaturing polyacrylamide gel. The original L32-ddAMP primer is indicated as primer and extended primer products are indicated as ext. primer.

Techniques Used: Labeling, Incubation, Electrophoresis

8) Product Images from "Sargassum fusiforme fraction is a potent and specific inhibitor of HIV-1 fusion and reverse transcriptase"

Article Title: Sargassum fusiforme fraction is a potent and specific inhibitor of HIV-1 fusion and reverse transcriptase

Journal: Virology Journal

doi: 10.1186/1743-422X-5-8

Inhibition of X4 and R5-tropic HIV-1 . GHOST X4/R5 and GFP expressing cells were plate at 1 × 10 5 /well in 12-well plates and incubated at 37°C in CO 2 atmosphere with increasing concentrations of SP4-2, as indicated, then infected with either X4-tropic NL4-3 (panel A, a-d) or with R5-tropic 81A (panel B, e-h), at 0.3 moi, in replicates (n = 4). 48 h after infection cells were quantified by FACS, and % infected cells is shown on each panel. Uninfected and untreated control (mock) is superimposed over each graph in dotted line. Representative of 4 experiments.
Figure Legend Snippet: Inhibition of X4 and R5-tropic HIV-1 . GHOST X4/R5 and GFP expressing cells were plate at 1 × 10 5 /well in 12-well plates and incubated at 37°C in CO 2 atmosphere with increasing concentrations of SP4-2, as indicated, then infected with either X4-tropic NL4-3 (panel A, a-d) or with R5-tropic 81A (panel B, e-h), at 0.3 moi, in replicates (n = 4). 48 h after infection cells were quantified by FACS, and % infected cells is shown on each panel. Uninfected and untreated control (mock) is superimposed over each graph in dotted line. Representative of 4 experiments.

Techniques Used: Inhibition, Expressing, Incubation, Infection, FACS

Inhibition of post entry HIV-1 replication . (A) SupT1 cells were infected for 1.5 hours in the absence of any treatment, with HIV-1 chimera NL4-3 Env - Luc + /VSV-G pseudotype, washed 3 times, and then treated with increasing concentrations of SP4-2, for 24 h. Intracellular luciferase gene marker expression was quantified from cell lysates that were normalized to the same number of viable cells by the MTT assay, and percent inhibition of HIV-1 replication was calculated from a control cell culture of infected but untreated cells, and plotted on the y-axis. (B) Standard cell free fluorescent RT assay was performed in the presence of 2 units recombinant HIV-1 RT/reaction with the indicated concentrations of SP4-2. Percent inhibition was calculated comparative to assay performed in absence of treatment, 100% RT activity. Data are mean ± SD of three separate experiments.
Figure Legend Snippet: Inhibition of post entry HIV-1 replication . (A) SupT1 cells were infected for 1.5 hours in the absence of any treatment, with HIV-1 chimera NL4-3 Env - Luc + /VSV-G pseudotype, washed 3 times, and then treated with increasing concentrations of SP4-2, for 24 h. Intracellular luciferase gene marker expression was quantified from cell lysates that were normalized to the same number of viable cells by the MTT assay, and percent inhibition of HIV-1 replication was calculated from a control cell culture of infected but untreated cells, and plotted on the y-axis. (B) Standard cell free fluorescent RT assay was performed in the presence of 2 units recombinant HIV-1 RT/reaction with the indicated concentrations of SP4-2. Percent inhibition was calculated comparative to assay performed in absence of treatment, 100% RT activity. Data are mean ± SD of three separate experiments.

Techniques Used: Inhibition, Infection, Luciferase, Marker, Expressing, MTT Assay, Cell Culture, Recombinant, Activity Assay

Inhibition of HIV-1 infection . 1G5 T cells were pretreated for 24 h with increasing concentrations of SP4-2, or with 10 -6 M ddC, or mock treated (0 μg SP4-2), as indicated. Then, cells were infected with HIV-1 (NL4-3) at multiplicity of infection (moi) of 0.01 for 1.5 h, washed 3 times, and returned to culture with the same concentration of each treatment, for the duration of the experiment. (A) On day 3 after infection, HIV-1 infection was quantified by luciferase gene marker expression from cell lysates that were normalized to the same number of viable cells, and expressed as relative light units (RLU) on the y-axis. (B) Viability for each cell culture treatment was quantified by MTT uptake. (C) Percent inhibition of HIV-1 was calculated from raw data in (A), utilizing the formula in the Methods, and plotted on the Y-axis as % HIV-1 Inhibition. Data are mean ± SD of three separate experiments.
Figure Legend Snippet: Inhibition of HIV-1 infection . 1G5 T cells were pretreated for 24 h with increasing concentrations of SP4-2, or with 10 -6 M ddC, or mock treated (0 μg SP4-2), as indicated. Then, cells were infected with HIV-1 (NL4-3) at multiplicity of infection (moi) of 0.01 for 1.5 h, washed 3 times, and returned to culture with the same concentration of each treatment, for the duration of the experiment. (A) On day 3 after infection, HIV-1 infection was quantified by luciferase gene marker expression from cell lysates that were normalized to the same number of viable cells, and expressed as relative light units (RLU) on the y-axis. (B) Viability for each cell culture treatment was quantified by MTT uptake. (C) Percent inhibition of HIV-1 was calculated from raw data in (A), utilizing the formula in the Methods, and plotted on the Y-axis as % HIV-1 Inhibition. Data are mean ± SD of three separate experiments.

Techniques Used: Inhibition, Infection, Concentration Assay, Luciferase, Marker, Expressing, Cell Culture, MTT Assay

Inhibition of HIV-1 fusion . SupT1 cells (1 × 10 6 ) were (A) mock infected, (B) infected for 2 h at 0.5 moi with BlaM-Vpr-X4-tropic NL4-3, or (C) infected in the presence of 10 μg/ml SP4-2, or (D) infected in the presence of 250 nM AMD3100. In a parallel experiment, SupT1 cells (1 × 10 6 ) were either (E) mock infected, or (F) infected for 2 h at 0.5 moi with BlaM-Vpr-X4-tropic NL4-3, or (G) infected in the presence of 20 ng/ml sCD4, or (H) infected in the presence of 20 ng/ml sCD4 together with 16 μg/ml SP4-2. Cells were loaded with CCF2/AM dye and fusion was analyzed by multiparameter flow cytometry using a violet laser for excitation of CCF, and gated from 10,000 cells. Percentages in each panel are of cells displaying blue fluorescence (virus fusion positive cells). Representative of 3 separate experiments.
Figure Legend Snippet: Inhibition of HIV-1 fusion . SupT1 cells (1 × 10 6 ) were (A) mock infected, (B) infected for 2 h at 0.5 moi with BlaM-Vpr-X4-tropic NL4-3, or (C) infected in the presence of 10 μg/ml SP4-2, or (D) infected in the presence of 250 nM AMD3100. In a parallel experiment, SupT1 cells (1 × 10 6 ) were either (E) mock infected, or (F) infected for 2 h at 0.5 moi with BlaM-Vpr-X4-tropic NL4-3, or (G) infected in the presence of 20 ng/ml sCD4, or (H) infected in the presence of 20 ng/ml sCD4 together with 16 μg/ml SP4-2. Cells were loaded with CCF2/AM dye and fusion was analyzed by multiparameter flow cytometry using a violet laser for excitation of CCF, and gated from 10,000 cells. Percentages in each panel are of cells displaying blue fluorescence (virus fusion positive cells). Representative of 3 separate experiments.

Techniques Used: Inhibition, Infection, Flow Cytometry, Cytometry, Fluorescence

Inhibition of HIV-1 binding and replication . GHOST cells were plate at 1 × 10 5 /well in 12-well plates and incubated at 37°C in CO 2 atmosphere with increasing concentrations of SP4-2 for 1.5 hours prior to infection. Treatment was washed off 3 times with warm media and plates were transferred to 4°C for 2 h to cool. Then the cells were infected at 4°C with NL4-3 at 0.1 moi for 2 hours. (A) Unbound virus was removed by washing with cold PBS, and viral particles remaining bound to the cells were quantified by p24 ELISA. (B) In a parallel experiment, 4°C infected plates were returned to 37°C for 48 hours, and virus replication was quantified by p24 ELISA. Data are mean ± SD of 6 replicates.
Figure Legend Snippet: Inhibition of HIV-1 binding and replication . GHOST cells were plate at 1 × 10 5 /well in 12-well plates and incubated at 37°C in CO 2 atmosphere with increasing concentrations of SP4-2 for 1.5 hours prior to infection. Treatment was washed off 3 times with warm media and plates were transferred to 4°C for 2 h to cool. Then the cells were infected at 4°C with NL4-3 at 0.1 moi for 2 hours. (A) Unbound virus was removed by washing with cold PBS, and viral particles remaining bound to the cells were quantified by p24 ELISA. (B) In a parallel experiment, 4°C infected plates were returned to 37°C for 48 hours, and virus replication was quantified by p24 ELISA. Data are mean ± SD of 6 replicates.

Techniques Used: Inhibition, Binding Assay, Incubation, Infection, Enzyme-linked Immunosorbent Assay

9) Product Images from "Inability of Human Immunodeficiency Virus Type 1 Produced in Murine Cells To Selectively Incorporate Primer \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{3}^{{\mathrm{Lys}}}\end{equation*}\end{document} ▿ ▿ †"

Article Title: Inability of Human Immunodeficiency Virus Type 1 Produced in Murine Cells To Selectively Incorporate Primer \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{tRNA}}_{3}^{{\mathrm{Lys}}}\end{equation*}\end{document} ▿ ▿ †

Journal: Journal of Virology

doi: 10.1128/JVI.01744-08

Incorporation of LysRS, ProRS, and GagPol into HIV-1 made in murine cells. (A) Viral incorporation of human or mouse LysRS. GM11686-cycT1 cells were cotransfected with BH10 DNA and DNA coding for either human LysRS (hLysRS) or murine LysRS (mLysRS), both of which were tagged with V5. Forty-eight hours posttransfection, viruses were collected from the supernatant, and cells and viruses were lysed. Western blots of cell and viral lysates were probed with anti-V5 and anti-β-actin or with anti-V5 and anti-CAp24, respectively. (B) Fluorescence anisotropy experiment, showing binding of mouse LysRS (mLysRS) to FITC-CAp24. Only a representative data set is shown, but measurements were carried out at least three times. The equilibrium dissociation constant (K d ) is shown. (C) Incorporation of ProRS. GM11686-cycT1 cells were transfected with BH10, and 48 h posttransfection, viruses were collected from the supernatant, and cells and viruses were lysed. Western blots of cell lysate were probed with anti-ProRS, and Western blots of viral lysates were probed with anti-ProRS and anti-CAp24. (D) Incorporation of GagPol. GM11686-cycT1 cells and 293T cells were transfected with BH10. Forty-eight hours posttransfection, viruses were collected from the supernatant and lysed. Western blots of viral lysates were probed with anti-CAp24 and anti-RT.
Figure Legend Snippet: Incorporation of LysRS, ProRS, and GagPol into HIV-1 made in murine cells. (A) Viral incorporation of human or mouse LysRS. GM11686-cycT1 cells were cotransfected with BH10 DNA and DNA coding for either human LysRS (hLysRS) or murine LysRS (mLysRS), both of which were tagged with V5. Forty-eight hours posttransfection, viruses were collected from the supernatant, and cells and viruses were lysed. Western blots of cell and viral lysates were probed with anti-V5 and anti-β-actin or with anti-V5 and anti-CAp24, respectively. (B) Fluorescence anisotropy experiment, showing binding of mouse LysRS (mLysRS) to FITC-CAp24. Only a representative data set is shown, but measurements were carried out at least three times. The equilibrium dissociation constant (K d ) is shown. (C) Incorporation of ProRS. GM11686-cycT1 cells were transfected with BH10, and 48 h posttransfection, viruses were collected from the supernatant, and cells and viruses were lysed. Western blots of cell lysate were probed with anti-ProRS, and Western blots of viral lysates were probed with anti-ProRS and anti-CAp24. (D) Incorporation of GagPol. GM11686-cycT1 cells and 293T cells were transfected with BH10. Forty-eight hours posttransfection, viruses were collected from the supernatant and lysed. Western blots of viral lysates were probed with anti-CAp24 and anti-RT.

Techniques Used: Western Blot, Fluorescence, Binding Assay, Transfection

tRNA Pro incorporation into HIV-1 VLPs produced in murine cells. (A) 2D PAGE resolution of tRNAs in VLPs produced from GM11686 cells. VLPs are produced from vGag-4CTE (panel 1), vGag/GagPol-4CTE (panel 2), hGag (panel 3), and hGag/hGagPol (panel 4). (B, left) Western blots of the expression in GM11686 cells and the extracellular production of hGag and C-terminally deleted hGag (Δ378-500). (Right) 2D PAGE of tRNA extracted from hGag and hGag (Δ378-500) VLPs produced from GM11686 cells. (C) 2D PAGE resolution of tRNAs in HIV-1 hGag/hGagPol VLPs and in MuLV produced in transiently transfected murine A9 cells, the parent cell of GM11686, and lacking human chromosome 2. VLPs are produced from hGag (panel 7), hGag/hGagPol (panel 8), and hGag (Δ378-500) (panel 9), which is expressed in the cell but does not form extracellular VLPs (panel 7). The 2D PAGE pattern of tRNAs in MuLV (panel 10) is also shown.
Figure Legend Snippet: tRNA Pro incorporation into HIV-1 VLPs produced in murine cells. (A) 2D PAGE resolution of tRNAs in VLPs produced from GM11686 cells. VLPs are produced from vGag-4CTE (panel 1), vGag/GagPol-4CTE (panel 2), hGag (panel 3), and hGag/hGagPol (panel 4). (B, left) Western blots of the expression in GM11686 cells and the extracellular production of hGag and C-terminally deleted hGag (Δ378-500). (Right) 2D PAGE of tRNA extracted from hGag and hGag (Δ378-500) VLPs produced from GM11686 cells. (C) 2D PAGE resolution of tRNAs in HIV-1 hGag/hGagPol VLPs and in MuLV produced in transiently transfected murine A9 cells, the parent cell of GM11686, and lacking human chromosome 2. VLPs are produced from hGag (panel 7), hGag/hGagPol (panel 8), and hGag (Δ378-500) (panel 9), which is expressed in the cell but does not form extracellular VLPs (panel 7). The 2D PAGE pattern of tRNAs in MuLV (panel 10) is also shown.

Techniques Used: Produced, Polyacrylamide Gel Electrophoresis, Western Blot, Expressing, Transfection

2D PAGE patterns of viral tRNA. (A) Viral tRNA patterns in HIV-1 produced from transfected 293T cells or GM11686-cycT1 cells. (B) Viral tRNA patterns from HIV-1 produced in 293T cells (left panel) and from mouse mammary tumor virus produced in the stably transfected murine cell line, MM5MT (middle panel). The right panel shows the 2D PAGE pattern when RNA from both viruses are mixed.
Figure Legend Snippet: 2D PAGE patterns of viral tRNA. (A) Viral tRNA patterns in HIV-1 produced from transfected 293T cells or GM11686-cycT1 cells. (B) Viral tRNA patterns from HIV-1 produced in 293T cells (left panel) and from mouse mammary tumor virus produced in the stably transfected murine cell line, MM5MT (middle panel). The right panel shows the 2D PAGE pattern when RNA from both viruses are mixed.

Techniques Used: Polyacrylamide Gel Electrophoresis, Produced, Transfection, Stable Transfection

HIV-1 produced in murine cells show reduced (tRNA Lys3 ) packaging, −SS DNA synthesis, and infectivity. 293T and GM11686-cycT1 cells were spin transfected with HIV-1, and the resulting virions were analyzed. (A) Viral production and packaging. (Left) Extracellular amount of CAp24 per milliliter of culture medium. (Right) Total viral RNA was isolated and analyzed by dot blot hybridization using DNA probes specific for and viral genomic RNA. (B) −SS DNA synthesis. SupT1 cells were infected with equal amounts of HIV-1 produced from either 293T cells or GM11686-cyc-T1 cells. DNA was extracted at different times postinfection. Early (R-U5) minus-strand cDNA production was monitored by real-time PCR as described in Materials and Methods. (Left) Time course of DNA production. Symbols: □, HIV-1 produced in 293T cells; ♦, HIV-1 produced in GM11686-cycT1 cells. (Right) Relative (rel.) production of −SS DNA at 8 h. (C) Viral infectivity. Equal amounts of viral CAp24 were used to challenge TZM-bl indicator cells, and productive infection was measured as the induction of luciferase activity. (Left) Time course of infection. Symbols: □, HIV-1 produced in 293T cells; ♦, HIV-1 produced in GM11686-cycT1 cells. (Right) Relative (rel.) infectivity at 28 h postinfection, normalized to HIV-1 produced in GM11686-cycT1 cells.
Figure Legend Snippet: HIV-1 produced in murine cells show reduced (tRNA Lys3 ) packaging, −SS DNA synthesis, and infectivity. 293T and GM11686-cycT1 cells were spin transfected with HIV-1, and the resulting virions were analyzed. (A) Viral production and packaging. (Left) Extracellular amount of CAp24 per milliliter of culture medium. (Right) Total viral RNA was isolated and analyzed by dot blot hybridization using DNA probes specific for and viral genomic RNA. (B) −SS DNA synthesis. SupT1 cells were infected with equal amounts of HIV-1 produced from either 293T cells or GM11686-cyc-T1 cells. DNA was extracted at different times postinfection. Early (R-U5) minus-strand cDNA production was monitored by real-time PCR as described in Materials and Methods. (Left) Time course of DNA production. Symbols: □, HIV-1 produced in 293T cells; ♦, HIV-1 produced in GM11686-cycT1 cells. (Right) Relative (rel.) production of −SS DNA at 8 h. (C) Viral infectivity. Equal amounts of viral CAp24 were used to challenge TZM-bl indicator cells, and productive infection was measured as the induction of luciferase activity. (Left) Time course of infection. Symbols: □, HIV-1 produced in 293T cells; ♦, HIV-1 produced in GM11686-cycT1 cells. (Right) Relative (rel.) infectivity at 28 h postinfection, normalized to HIV-1 produced in GM11686-cycT1 cells.

Techniques Used: Produced, DNA Synthesis, Infection, Transfection, Isolation, Dot Blot, Hybridization, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay

Viral tRNA in HIV-1 and Gag VLPs. 293T cells were transfected with plasmids coding for HIV-1 or HIV-1 Gag/GagPol VLPs. The RNA from the resulting virus or Gag VLPs was extracted and analyzed by 2D PAGE. (A) 2D PAGE patterns of viral tRNA extracted from VLPs produced from viral Gag/GagPol-RRE (panel 2), viral Gag/GagPol-4CTE (panel 3), or hGag/hGagPol (panel 4). The viral tRNA patterns in HIV-1 (panel 5) and in the cytoplasm of 293T cells (panel 1) are also shown. (B) (tRNA Lys3 )/tRNA His ratios in HIV-1 and VLPs, normalized to that of the cell cytoplasm.
Figure Legend Snippet: Viral tRNA in HIV-1 and Gag VLPs. 293T cells were transfected with plasmids coding for HIV-1 or HIV-1 Gag/GagPol VLPs. The RNA from the resulting virus or Gag VLPs was extracted and analyzed by 2D PAGE. (A) 2D PAGE patterns of viral tRNA extracted from VLPs produced from viral Gag/GagPol-RRE (panel 2), viral Gag/GagPol-4CTE (panel 3), or hGag/hGagPol (panel 4). The viral tRNA patterns in HIV-1 (panel 5) and in the cytoplasm of 293T cells (panel 1) are also shown. (B) (tRNA Lys3 )/tRNA His ratios in HIV-1 and VLPs, normalized to that of the cell cytoplasm.

Techniques Used: Transfection, Polyacrylamide Gel Electrophoresis, Produced

10) Product Images from "Circularization of the HIV-1 genome facilitates strand transfer during reverse transcription"

Article Title: Circularization of the HIV-1 genome facilitates strand transfer during reverse transcription

Journal: RNA

doi: 10.1261/rna.2039610

( A ) Schematic representation of the HIV-1 viral RNA genome. The positions of the open reading frames are indicated. The coding region is flanked at the 5′ and 3′ ends of the genome by untranslated leader regions (UTR). The 5′ UTR
Figure Legend Snippet: ( A ) Schematic representation of the HIV-1 viral RNA genome. The positions of the open reading frames are indicated. The coding region is flanked at the 5′ and 3′ ends of the genome by untranslated leader regions (UTR). The 5′ UTR

Techniques Used:

Initiation of reverse transcription on the 5′ transcript in the presence or absence of the wt3, m3.4, or m3.6 transcript. Reverse transcription was initiated using a DNA primer complementary to the PBS ( A ) or tRNAlys3 ( B ), HIV-1 RT enzyme and
Figure Legend Snippet: Initiation of reverse transcription on the 5′ transcript in the presence or absence of the wt3, m3.4, or m3.6 transcript. Reverse transcription was initiated using a DNA primer complementary to the PBS ( A ) or tRNAlys3 ( B ), HIV-1 RT enzyme and

Techniques Used:

11) Product Images from "Characterization of the Polymerase and RNase H Activities of Human Foamy Virus Reverse Transcriptase"

Article Title: Characterization of the Polymerase and RNase H Activities of Human Foamy Virus Reverse Transcriptase

Journal: Journal of Virology

doi: 10.1128/JVI.78.12.6112-6121.2004

), and a brief description is given in Materials and Methods. The RNA template has the same sequence as the HIV-1 RNA genome and contains sequences from PPT, U3, R, U5, and PBS. A DNA primer complementary to the PBS was 5′-end labeled and annealed to the RNA. The primer was extended as described in Materials and Methods. NC was added to give the concentrations indicated before RT was added to the reaction mixture. The 3′ end of the poly(A) hairpin is approximately 95 nt from the 5′ end of the primer, while the 3′ end of the TAR hairpin is approximately 142 nt from the 5′ end of the primer.
Figure Legend Snippet: ), and a brief description is given in Materials and Methods. The RNA template has the same sequence as the HIV-1 RNA genome and contains sequences from PPT, U3, R, U5, and PBS. A DNA primer complementary to the PBS was 5′-end labeled and annealed to the RNA. The primer was extended as described in Materials and Methods. NC was added to give the concentrations indicated before RT was added to the reaction mixture. The 3′ end of the poly(A) hairpin is approximately 95 nt from the 5′ end of the primer, while the 3′ end of the TAR hairpin is approximately 142 nt from the 5′ end of the primer.

Techniques Used: Sequencing, Labeling

DNA-dependent DNA polymerase activity of FV RT and HIV-1 RT. A 5′-end-labeled DNA primer was annealed to a single-stranded M13mp18 DNA template and extended with either HIV-1 RT or FV RT for the lengths of time indicated. Samples were precipitated with ethanol and then fractionated on a 2.0% alkaline agarose gel. After electrophoresis, the gel was neutralized and dried as described in Materials and Methods. The products were visualized by autoradiography. The size marker was 5′-end-labeled 1.0-kb marker from New England Biolabs.
Figure Legend Snippet: DNA-dependent DNA polymerase activity of FV RT and HIV-1 RT. A 5′-end-labeled DNA primer was annealed to a single-stranded M13mp18 DNA template and extended with either HIV-1 RT or FV RT for the lengths of time indicated. Samples were precipitated with ethanol and then fractionated on a 2.0% alkaline agarose gel. After electrophoresis, the gel was neutralized and dried as described in Materials and Methods. The products were visualized by autoradiography. The size marker was 5′-end-labeled 1.0-kb marker from New England Biolabs.

Techniques Used: Activity Assay, Labeling, Agarose Gel Electrophoresis, Electrophoresis, Autoradiography, Marker

NaPP i - and ATP-dependent pyrophosphorylysis. As described in Materials and Methods, a DNA primer was 5′-end labeled and then annealed to a DNA template. AZTMP was incorporated at the 3′ end of the primer by polymerization with HIV-1 RT in the presence of AZT triphosphate. After purification, the T/P was incubated with either HIV-1 RT or FV RT in the presence of dNTPs and either ATP or NaPP i as the pyrophosphate donor. Pyrophosphorylysis was measured as the ability of the RT to excise the AZT group blocking the primer and extend the primer to the end of the template strand. The amount of total label in the sample and the amount of label in the full-length product were determined with a phosphorimager, and the data are expressed as a percentage of the full-length product. Experiments were done in duplicate, and the results were quite reproducible. Data from a representative experiment are shown.
Figure Legend Snippet: NaPP i - and ATP-dependent pyrophosphorylysis. As described in Materials and Methods, a DNA primer was 5′-end labeled and then annealed to a DNA template. AZTMP was incorporated at the 3′ end of the primer by polymerization with HIV-1 RT in the presence of AZT triphosphate. After purification, the T/P was incubated with either HIV-1 RT or FV RT in the presence of dNTPs and either ATP or NaPP i as the pyrophosphate donor. Pyrophosphorylysis was measured as the ability of the RT to excise the AZT group blocking the primer and extend the primer to the end of the template strand. The amount of total label in the sample and the amount of label in the full-length product were determined with a phosphorimager, and the data are expressed as a percentage of the full-length product. Experiments were done in duplicate, and the results were quite reproducible. Data from a representative experiment are shown.

Techniques Used: Labeling, Purification, Incubation, Blocking Assay

Strand transfer by FV RT and HIV-1 RT. (A) Schematic of the strand transfer assay. The 5′-end-labeled DNA primer (22 nt in length) is annealed to an RNA template (wavy line). Extension of the primer by the polymerase to the end of the RNA template will give a full-length product (designated as a) that is 40 nt in length. The RNA strand in the RNA-DNA duplex is then degraded by the RNase H activity of the RT, allowing the transfer of the DNA strand to a new template (thick line). Further extension of the labeled DNA to the end of the second template will give a strand transfer product (designated as b) that is 63 nt in size. (B) Strand transfer assay. As described, a DNA primer was 5′-end labeled and annealed to an RNA template. The primer was extended by either FV RT or HIV-1 RT for the indicated lengths of time in the presence of dNTPs and a second DNA oligonucleotide, designated the acceptor template. Extension of the primer to the end of the RNA template generates the full-length product (labeled a). Extension to the end of the acceptor template yields the strand transfer product (labeled b). The time necessary to generate the full-length product is dependent only on the polymerase activity of the RT, while that for the strand transfer product is dependent on both the polymerase and the RNase H activities of the enzyme.
Figure Legend Snippet: Strand transfer by FV RT and HIV-1 RT. (A) Schematic of the strand transfer assay. The 5′-end-labeled DNA primer (22 nt in length) is annealed to an RNA template (wavy line). Extension of the primer by the polymerase to the end of the RNA template will give a full-length product (designated as a) that is 40 nt in length. The RNA strand in the RNA-DNA duplex is then degraded by the RNase H activity of the RT, allowing the transfer of the DNA strand to a new template (thick line). Further extension of the labeled DNA to the end of the second template will give a strand transfer product (designated as b) that is 63 nt in size. (B) Strand transfer assay. As described, a DNA primer was 5′-end labeled and annealed to an RNA template. The primer was extended by either FV RT or HIV-1 RT for the indicated lengths of time in the presence of dNTPs and a second DNA oligonucleotide, designated the acceptor template. Extension of the primer to the end of the RNA template generates the full-length product (labeled a). Extension to the end of the acceptor template yields the strand transfer product (labeled b). The time necessary to generate the full-length product is dependent only on the polymerase activity of the RT, while that for the strand transfer product is dependent on both the polymerase and the RNase H activities of the enzyme.

Techniques Used: Labeling, Activity Assay

). A catalytically important aspartic acid residue was chosen as the starting point (amino acids [aa] 498 in HIV-1 RT, aa 583 in MLV RT, aa 669 in FV RT, and aa 70 in E. coli RNase H) and is highlighted. The basic loops for MLV RT and E. coli ). (C) The FV RNase H sequence as determined in this study.
Figure Legend Snippet: ). A catalytically important aspartic acid residue was chosen as the starting point (amino acids [aa] 498 in HIV-1 RT, aa 583 in MLV RT, aa 669 in FV RT, and aa 70 in E. coli RNase H) and is highlighted. The basic loops for MLV RT and E. coli ). (C) The FV RNase H sequence as determined in this study.

Techniques Used: Sequencing

12) Product Images from "Design, synthesis and antiviral evaluation of novel heteroarylcarbothioamide derivatives as dual inhibitors of HIV-1 reverse transcriptase-associated RNase H and RDDP functions"

Article Title: Design, synthesis and antiviral evaluation of novel heteroarylcarbothioamide derivatives as dual inhibitors of HIV-1 reverse transcriptase-associated RNase H and RDDP functions

Journal: Pathogens and Disease

doi: 10.1093/femspd/ftx078

Effect of A15 on the thermal stability of p66/p51 HIV-1 RT. The melting temperature of HIV-1 RT was measured by differential scanning fluorimetry in presence of increasing concentrations of (inverted filled triangles) BTP, (filled circles) VU and (open triangles) A15.
Figure Legend Snippet: Effect of A15 on the thermal stability of p66/p51 HIV-1 RT. The melting temperature of HIV-1 RT was measured by differential scanning fluorimetry in presence of increasing concentrations of (inverted filled triangles) BTP, (filled circles) VU and (open triangles) A15.

Techniques Used:

13) Product Images from "Dual anti-HIV mechanism of clofarabine"

Article Title: Dual anti-HIV mechanism of clofarabine

Journal: Retrovirology

doi: 10.1186/s12977-016-0254-0

Model for the anti-HIV-1 dual action mechanisms of clofarabine in macrophages. Clofarabine di- and triphosphate inhibit RNR to reduce dNTP levels, leading to the kinetic suppression of HIV-1 reverse transcription ( blue ). Clofarabine triphosphate is also directly incorporated by HIV-1 RT, inhibiting extension ( red )
Figure Legend Snippet: Model for the anti-HIV-1 dual action mechanisms of clofarabine in macrophages. Clofarabine di- and triphosphate inhibit RNR to reduce dNTP levels, leading to the kinetic suppression of HIV-1 reverse transcription ( blue ). Clofarabine triphosphate is also directly incorporated by HIV-1 RT, inhibiting extension ( red )

Techniques Used:

Biochemical examination of the dual mechanism of clofarabine. a Direct clofarabine-TP incorporation by HIV-1 RT. A 5′ 32 P-labeled 23-mer DNA primer (P) annealed to a 24-mer DNA template with a single T overhang was incubated with HIV-1 RT and increasing concentrations of clofarabine-triphosphate (Clof-TP). E, Extended product; +, 50 μM dATP positive control; −, no dATP control. b Effect of clofarabine-TP incorporation on DNA synthesis. A 5′ 32 P-labeled 17-mer DNA primer (P) annealed to a 38-mer RNA template was extended by HIV-1 RT with fixed dNTP concentrations found in either activated T cells (T cell, 5 μM) or monocyte-derived macrophages (MDM, 50 nM) with increasing concentrations of clofarabine-TP (two-fold dilutions starting at 125 μM). F, Fully extended product; +, 50 μM dNTP positive control; −, no dNTP control. Blue asterisks (*) indicates the clofarabine-TP incorporation site followed by kinetic pauses (]). c Clofarabine inhibition of NRTI-resistant RT mutants. MAGI cells were treated with increasing concentrations of clofarabine for 2 h prior to infection with Vsvg-pseudotyped HIV-1 containing mutations in RT. Flow cytometry analysis for infected cells was conducted at 48–72 h post-infection. IC 50 values and 95 % confidence intervals are shown. NL4-3 MIG: wild-type HIV-1 RT, Q151: A62V, V75I, F77L, F116Y and Q151M, T69: M41L, A62V, T69S, K70R, T215Y and serine–serine insertion between 69 and 70. d Biochemical simulation of HIV-1 RT activity at dNTP concentrations found in cells with and without clofarabine treatment. A 5′ 32 P-labeled 17-mer DNA primer (P) annealed to a 38-mer RNA template (shown in box ) was extended using an equal amount of purified HIV-1 RT protein with dNTP concentrations found in T cells (T, 5 μM), macrophages (M, 50 nM) or macrophages treated with 300 nM clofarabine (M/C, 10 nM dATP, 28 nM dCTP, 28 nM dGTP, 40 nM TTP). Blue stars (*) indicate kinetic pause sites, F, fully extended 38 bp product; −, no dNTP control
Figure Legend Snippet: Biochemical examination of the dual mechanism of clofarabine. a Direct clofarabine-TP incorporation by HIV-1 RT. A 5′ 32 P-labeled 23-mer DNA primer (P) annealed to a 24-mer DNA template with a single T overhang was incubated with HIV-1 RT and increasing concentrations of clofarabine-triphosphate (Clof-TP). E, Extended product; +, 50 μM dATP positive control; −, no dATP control. b Effect of clofarabine-TP incorporation on DNA synthesis. A 5′ 32 P-labeled 17-mer DNA primer (P) annealed to a 38-mer RNA template was extended by HIV-1 RT with fixed dNTP concentrations found in either activated T cells (T cell, 5 μM) or monocyte-derived macrophages (MDM, 50 nM) with increasing concentrations of clofarabine-TP (two-fold dilutions starting at 125 μM). F, Fully extended product; +, 50 μM dNTP positive control; −, no dNTP control. Blue asterisks (*) indicates the clofarabine-TP incorporation site followed by kinetic pauses (]). c Clofarabine inhibition of NRTI-resistant RT mutants. MAGI cells were treated with increasing concentrations of clofarabine for 2 h prior to infection with Vsvg-pseudotyped HIV-1 containing mutations in RT. Flow cytometry analysis for infected cells was conducted at 48–72 h post-infection. IC 50 values and 95 % confidence intervals are shown. NL4-3 MIG: wild-type HIV-1 RT, Q151: A62V, V75I, F77L, F116Y and Q151M, T69: M41L, A62V, T69S, K70R, T215Y and serine–serine insertion between 69 and 70. d Biochemical simulation of HIV-1 RT activity at dNTP concentrations found in cells with and without clofarabine treatment. A 5′ 32 P-labeled 17-mer DNA primer (P) annealed to a 38-mer RNA template (shown in box ) was extended using an equal amount of purified HIV-1 RT protein with dNTP concentrations found in T cells (T, 5 μM), macrophages (M, 50 nM) or macrophages treated with 300 nM clofarabine (M/C, 10 nM dATP, 28 nM dCTP, 28 nM dGTP, 40 nM TTP). Blue stars (*) indicate kinetic pause sites, F, fully extended 38 bp product; −, no dNTP control

Techniques Used: Labeling, Incubation, Positive Control, DNA Synthesis, Derivative Assay, Inhibition, Infection, Flow Cytometry, Cytometry, Activity Assay, Purification

Anti-HIV-1 activity of clofarabine in primary human activated CD4 + T cells and monocyte derived macrophages. a The structure of clofarabine. b Clofarabine inhibition ( blue lines ) and cytotoxicity ( red lines ) on activated CD4 + T cells of 5 healthy donors. Cells were treated with increasing concentrations of clofarabine for 8 h, washed with PBS, and then infected with pseudotyped HIV-1, (inhibition) or cultured with media (cytotoxicity). Analysis was conducted at 72 h post-infection via flow cytometry (inhibition) or XTT assay (cytotoxicity). The IC 50 is 60.3 nM with a 95 % confidence interval (95 % CI) of 24.1–96.5 nM; the CC 50 is 854.2 nM with a 95 % CI of 712.6–995.8 nM. c The clofarabine inhibition ( blue lines ) and cytotoxicity curves ( red lines ) for monocyte-derived macrophages of 5 healthy donors. Macrophages were treated as described for T cells except analysis was at 5 days post infection. IC 50 = 21.6 nM (95 % CI 17.4–25.8 nM): CC 50 = 6.8 μM (95 % CI 3.2–9.4 μM). d Selectivity Index (SI) difference between activated CD4 + T cells and macrophages. SI values were determined by dividing the average CC 50 of five donors by the average IC 50 of five donors
Figure Legend Snippet: Anti-HIV-1 activity of clofarabine in primary human activated CD4 + T cells and monocyte derived macrophages. a The structure of clofarabine. b Clofarabine inhibition ( blue lines ) and cytotoxicity ( red lines ) on activated CD4 + T cells of 5 healthy donors. Cells were treated with increasing concentrations of clofarabine for 8 h, washed with PBS, and then infected with pseudotyped HIV-1, (inhibition) or cultured with media (cytotoxicity). Analysis was conducted at 72 h post-infection via flow cytometry (inhibition) or XTT assay (cytotoxicity). The IC 50 is 60.3 nM with a 95 % confidence interval (95 % CI) of 24.1–96.5 nM; the CC 50 is 854.2 nM with a 95 % CI of 712.6–995.8 nM. c The clofarabine inhibition ( blue lines ) and cytotoxicity curves ( red lines ) for monocyte-derived macrophages of 5 healthy donors. Macrophages were treated as described for T cells except analysis was at 5 days post infection. IC 50 = 21.6 nM (95 % CI 17.4–25.8 nM): CC 50 = 6.8 μM (95 % CI 3.2–9.4 μM). d Selectivity Index (SI) difference between activated CD4 + T cells and macrophages. SI values were determined by dividing the average CC 50 of five donors by the average IC 50 of five donors

Techniques Used: Activity Assay, Derivative Assay, Inhibition, Infection, Cell Culture, Flow Cytometry, Cytometry, XTT Assay

14) Product Images from "Exploiting Drug-Resistant Enzymes as Tools to Identify Thienopyrimidinone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H"

Article Title: Exploiting Drug-Resistant Enzymes as Tools to Identify Thienopyrimidinone Inhibitors of Human Immunodeficiency Virus Reverse Transcriptase-Associated Ribonuclease H

Journal: Journal of medicinal chemistry

doi: 10.1021/jm400405z

Screening with Engineered HIV-1 RT Mutants Identifies Thienopyrimidinones with Broad Spectrum Activity
Figure Legend Snippet: Screening with Engineered HIV-1 RT Mutants Identifies Thienopyrimidinones with Broad Spectrum Activity

Techniques Used: Activity Assay

Yonetani-Theorell plot for inhibition of HIV-1 RT-RNase H activity in the presence of β-thujaplicinol and (a) Compound 9 or (b) Compound 29 . The inhibitory activity (defined as V 0 /V i ) for each concentration of β-thujaplicinol (β-TP:
Figure Legend Snippet: Yonetani-Theorell plot for inhibition of HIV-1 RT-RNase H activity in the presence of β-thujaplicinol and (a) Compound 9 or (b) Compound 29 . The inhibitory activity (defined as V 0 /V i ) for each concentration of β-thujaplicinol (β-TP:

Techniques Used: Inhibition, Activity Assay, Concentration Assay

Effect of thienopyrimidinone RNase H inhibitors on the thermal stability of p66/p51 HIV-1 RT. β-TP, RNase H active site inhibitor β-thujaplicinol. T m values are the average of triplicate analysis.
Figure Legend Snippet: Effect of thienopyrimidinone RNase H inhibitors on the thermal stability of p66/p51 HIV-1 RT. β-TP, RNase H active site inhibitor β-thujaplicinol. T m values are the average of triplicate analysis.

Techniques Used:

Evaluation of inhibitory activity of 3′,4′-dihydroxyphenyl-containing thienopyrimidinones against HIV-1 DNA polymerase activities (a) and HIV-1 integrase (b) in the presence of 50 μM 3′,4′-dihydroxyphenyl-containing
Figure Legend Snippet: Evaluation of inhibitory activity of 3′,4′-dihydroxyphenyl-containing thienopyrimidinones against HIV-1 DNA polymerase activities (a) and HIV-1 integrase (b) in the presence of 50 μM 3′,4′-dihydroxyphenyl-containing

Techniques Used: Activity Assay

Model of the interface between the p66 RNase H domain (gold) and p51 thumb subdomain (green) of HIV-1 RT. Active site carboxylates (D 443 , E 478 , D 498 , D 549 ) and the conserved His 539 of the RNase H domain are indicated in white. Residues of the p51 thumb
Figure Legend Snippet: Model of the interface between the p66 RNase H domain (gold) and p51 thumb subdomain (green) of HIV-1 RT. Active site carboxylates (D 443 , E 478 , D 498 , D 549 ) and the conserved His 539 of the RNase H domain are indicated in white. Residues of the p51 thumb

Techniques Used:

15) Product Images from "Identification of a 3-aminoimidazo[1,2-a]pyridine inhibitor of HIV-1 reverse transcriptase"

Article Title: Identification of a 3-aminoimidazo[1,2-a]pyridine inhibitor of HIV-1 reverse transcriptase

Journal: Virology Journal

doi: 10.1186/1743-422X-9-305

The F2 compound inhibits HIV-1 reverse transcriptase. A . Effects of F2 (5 μM) treatment on the synthesis of early and late viral DNA in human 293T cells challenged with the VSVg-pseudotyped HIV-1 vector, measured at 24 hours post-infection. AZT (5 μM) was used as a reference compound. The values represent amounts of DNA relative to control, untreated cell populations, with error bars showing standard deviations from three independent real-time quantitative PCR assays. B . Effect of compound F2 on HIV-1 RT activity in vitro , determined by measuring the [alpha32P]-dTTP incorporation. The experiment shown, performed with duplicate samples, is representative of two independent experiments. with error bars representing standard errors of the mean.
Figure Legend Snippet: The F2 compound inhibits HIV-1 reverse transcriptase. A . Effects of F2 (5 μM) treatment on the synthesis of early and late viral DNA in human 293T cells challenged with the VSVg-pseudotyped HIV-1 vector, measured at 24 hours post-infection. AZT (5 μM) was used as a reference compound. The values represent amounts of DNA relative to control, untreated cell populations, with error bars showing standard deviations from three independent real-time quantitative PCR assays. B . Effect of compound F2 on HIV-1 RT activity in vitro , determined by measuring the [alpha32P]-dTTP incorporation. The experiment shown, performed with duplicate samples, is representative of two independent experiments. with error bars representing standard errors of the mean.

Techniques Used: Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction, Activity Assay, In Vitro

The F2 compound blocks an early step of HIV-1 replication. A . Structure of compound F2 . B .Dose–response curve of F2 in 293T cells challenged with the VSVg pseudotyped pNL4-3lucR+E- vector. The effect of the compound on infection was determined by measurement of virus-encoded firefly luciferase activity. The experiment shown, performed with triplicate samples, is representative of six independent experiments. C . Dose–response curve of F2 in CEM-GFP cells challenged with the pLai3Luc2 HIV-1 vector [ 10 ].The numbers of GFP positive cells at 2 days post-infection were determined by flow cytometry and the experiment shown, performed with duplicate samples, is representative of two independent experiments. D . Dose–response curve of F2 in human PBMCs challenged with the NL4-3 Nef+ IRES rluc vector encoding renilla luciferase activity, measured at 5 days post-infection. The experiment shown was performed with eight replicate samples. The error bars (panels B-D) represent the standard errors of the mean.
Figure Legend Snippet: The F2 compound blocks an early step of HIV-1 replication. A . Structure of compound F2 . B .Dose–response curve of F2 in 293T cells challenged with the VSVg pseudotyped pNL4-3lucR+E- vector. The effect of the compound on infection was determined by measurement of virus-encoded firefly luciferase activity. The experiment shown, performed with triplicate samples, is representative of six independent experiments. C . Dose–response curve of F2 in CEM-GFP cells challenged with the pLai3Luc2 HIV-1 vector [ 10 ].The numbers of GFP positive cells at 2 days post-infection were determined by flow cytometry and the experiment shown, performed with duplicate samples, is representative of two independent experiments. D . Dose–response curve of F2 in human PBMCs challenged with the NL4-3 Nef+ IRES rluc vector encoding renilla luciferase activity, measured at 5 days post-infection. The experiment shown was performed with eight replicate samples. The error bars (panels B-D) represent the standard errors of the mean.

Techniques Used: Plasmid Preparation, Infection, Luciferase, Activity Assay, Flow Cytometry, Cytometry

The F2 compound is a NNRTI. A . Isobologram analysis of the effect of combining F2 with either AZT or NVP in human 293T cells challenged with the VSVg-pseudotyped HIV-1 vector. Depicted are isobologram plots for the 90% inhibitory level (EC 90 ). The dashed line indicates the values expected for an additive effect. Values below and to the left of the line indicate synergistic effects. FIC, fractional inhibitory concentration. B . Structure and synthesis of compound F2 compared to THR-50 [ 15 ]. Reagents: (a) 0.1 M in 1,2-dichloroethane – 2,2,2-trifluoroethanol (1:1), 5.0 mol% Sc(OTf) 3 , rt for 96h or microwaved at 140°C for 5min, > 90% yield; (b) 2,6-F 2 BzCl, THF/pyridine (5:1), rt for 2h, 55% yield; (c) Fe, AcOH, reflux, 97% yield; (d) 2,6-F 2 BnBr, NaH, THF, rt overnight, 75% yield.
Figure Legend Snippet: The F2 compound is a NNRTI. A . Isobologram analysis of the effect of combining F2 with either AZT or NVP in human 293T cells challenged with the VSVg-pseudotyped HIV-1 vector. Depicted are isobologram plots for the 90% inhibitory level (EC 90 ). The dashed line indicates the values expected for an additive effect. Values below and to the left of the line indicate synergistic effects. FIC, fractional inhibitory concentration. B . Structure and synthesis of compound F2 compared to THR-50 [ 15 ]. Reagents: (a) 0.1 M in 1,2-dichloroethane – 2,2,2-trifluoroethanol (1:1), 5.0 mol% Sc(OTf) 3 , rt for 96h or microwaved at 140°C for 5min, > 90% yield; (b) 2,6-F 2 BzCl, THF/pyridine (5:1), rt for 2h, 55% yield; (c) Fe, AcOH, reflux, 97% yield; (d) 2,6-F 2 BnBr, NaH, THF, rt overnight, 75% yield.

Techniques Used: Plasmid Preparation, Concentration Assay, Reflux

16) Product Images from "Ribonuclease H/DNA Polymerase HIV-1 Reverse Transcriptase Dual Inhibitor: Mechanistic Studies on the Allosteric Mode of Action of Isatin-Based Compound RMNC6"

Article Title: Ribonuclease H/DNA Polymerase HIV-1 Reverse Transcriptase Dual Inhibitor: Mechanistic Studies on the Allosteric Mode of Action of Isatin-Based Compound RMNC6

Journal: PLoS ONE

doi: 10.1371/journal.pone.0147225

Binding sites of RMNC6 individuated after blind docking experiments on the whole wt HIV-1 RT structure.
Figure Legend Snippet: Binding sites of RMNC6 individuated after blind docking experiments on the whole wt HIV-1 RT structure.

Techniques Used: Binding Assay

Effect of RT inhibitors on the thermal stability of p66/p51 HIV-1 RT. (A). The melting temperature of HIV-1 RT was measured by differential scanning fluorimetry in the presence of increasing concentrations of different inhibitors: (▼) Efavirenz (EFV), (○) β-thujaplicinol (BTP), (Δ) 2-(3, 4-dihydroxyphenyl)-5, 6-dimethylthieno[2, 3-d]pyrimidin-4(3H)-one (VU) and (●) RMNC6. (B). Maximum HIV-1 RT thermal shift (ΔTm) observed in the presence of 50 μM concentration of compounds. ΔTm values are the average of triplicate analysis, standard deviations are indicated as bars.
Figure Legend Snippet: Effect of RT inhibitors on the thermal stability of p66/p51 HIV-1 RT. (A). The melting temperature of HIV-1 RT was measured by differential scanning fluorimetry in the presence of increasing concentrations of different inhibitors: (▼) Efavirenz (EFV), (○) β-thujaplicinol (BTP), (Δ) 2-(3, 4-dihydroxyphenyl)-5, 6-dimethylthieno[2, 3-d]pyrimidin-4(3H)-one (VU) and (●) RMNC6. (B). Maximum HIV-1 RT thermal shift (ΔTm) observed in the presence of 50 μM concentration of compounds. ΔTm values are the average of triplicate analysis, standard deviations are indicated as bars.

Techniques Used: Concentration Assay

Yonetani-Theorell plot of the combination of RMNC6 and EFV on the HIV-1 RT RNA- dependent DNA polymerase activity. HIV-1 RT was incubated in the presence of RMNC6 alone (●) or in presence of different concentrations of EFV: 4 nM (Δ), 8 nM (■) and 16 nM (☐).
Figure Legend Snippet: Yonetani-Theorell plot of the combination of RMNC6 and EFV on the HIV-1 RT RNA- dependent DNA polymerase activity. HIV-1 RT was incubated in the presence of RMNC6 alone (●) or in presence of different concentrations of EFV: 4 nM (Δ), 8 nM (■) and 16 nM (☐).

Techniques Used: Activity Assay, Incubation

17) Product Images from "Increased ability for selection of zidovudine resistance in a distinct class of wild-type HIV-1 from drug-naive persons"

Article Title: Increased ability for selection of zidovudine resistance in a distinct class of wild-type HIV-1 from drug-naive persons

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.241300698

Competitive HIV-1 replication assay among patient-derived recombinant viruses carrying Asp (D), Cys (C), or Ser (S) at codon 215. HIV-1 215D or HIV-1 215C were mixed with HIV-1 215S at known ratios, and the proportion of S at codon 215 was monitored over time. Day 0 represents proportions in the initial virus mixture. ( A ) Mixture of RD03 41L/210W/215S and RD03 41L/210W/215C . ( B ) Mixtures with RD04 210W/215S and RD04 210W/215D .
Figure Legend Snippet: Competitive HIV-1 replication assay among patient-derived recombinant viruses carrying Asp (D), Cys (C), or Ser (S) at codon 215. HIV-1 215D or HIV-1 215C were mixed with HIV-1 215S at known ratios, and the proportion of S at codon 215 was monitored over time. Day 0 represents proportions in the initial virus mixture. ( A ) Mixture of RD03 41L/210W/215S and RD03 41L/210W/215C . ( B ) Mixtures with RD04 210W/215S and RD04 210W/215D .

Techniques Used: Derivative Assay, Recombinant

Competitive HIV-1 replication assay among HXB2 viruses carrying Asp (D), Cys (C), or Ser (S) at codon 215. HXB2 215D or HXB2 215C were mixed with HXB2 215S at known ratios, and the proportion of S at codon 215 was monitored over time. Day 0 represents proportions in the initial virus mixture. ( A ) Mixtures of HXB2 215S and HXB2 215C . ( B ) Mixtures of HXB2 215S and HXB2 215D .
Figure Legend Snippet: Competitive HIV-1 replication assay among HXB2 viruses carrying Asp (D), Cys (C), or Ser (S) at codon 215. HXB2 215D or HXB2 215C were mixed with HXB2 215S at known ratios, and the proportion of S at codon 215 was monitored over time. Day 0 represents proportions in the initial virus mixture. ( A ) Mixtures of HXB2 215S and HXB2 215C . ( B ) Mixtures of HXB2 215S and HXB2 215D .

Techniques Used:

Replication kinetics of recombinant viruses carrying the 215C or 215D mutations alone or in association with 41L or 210W, and comparison with viruses having the WT T at codon 215. ( A ) Recombinant viruses carrying patient-derived RTs. ( B ) Recombinant viruses generated with the RT of HIV-1 HXB2 . Mean p24 values from duplicate cultures are shown. Mock, uninfected cells.
Figure Legend Snippet: Replication kinetics of recombinant viruses carrying the 215C or 215D mutations alone or in association with 41L or 210W, and comparison with viruses having the WT T at codon 215. ( A ) Recombinant viruses carrying patient-derived RTs. ( B ) Recombinant viruses generated with the RT of HIV-1 HXB2 . Mean p24 values from duplicate cultures are shown. Mock, uninfected cells.

Techniques Used: Recombinant, Derivative Assay, Generated

18) Product Images from "Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet"

Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

Journal:

doi: 10.1016/j.jmb.2007.03.006

Lambda exonuclease mapping of the upstream borders of HIV-1 RT bound to P/T
Figure Legend Snippet: Lambda exonuclease mapping of the upstream borders of HIV-1 RT bound to P/T

Techniques Used:

Effects of noncomplementary dNTPs on DNase I footprints and stable complex formation by HIV-1 RT
Figure Legend Snippet: Effects of noncomplementary dNTPs on DNase I footprints and stable complex formation by HIV-1 RT

Techniques Used:

Effects of foscarnet on DNase I protection and stable complex formation by HIV-1 RT
Figure Legend Snippet: Effects of foscarnet on DNase I protection and stable complex formation by HIV-1 RT

Techniques Used:

RecJ f exonuclease mapping of the downstream borders of HIV-1 RT bound to chain-terminated P/T
Figure Legend Snippet: RecJ f exonuclease mapping of the downstream borders of HIV-1 RT bound to chain-terminated P/T

Techniques Used:

Effects of the next complementary dNTP on DNase I protection and stable complex formation by HIV-1 RT
Figure Legend Snippet: Effects of the next complementary dNTP on DNase I protection and stable complex formation by HIV-1 RT

Techniques Used:

Related Articles

SYBR Green Assay:

Article Title: Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA
Article Snippet: Quantification of reverse transcribed products was carried out in a CFX96 thermal cycler (Biorad) using Sybr-Green I, hotstart Taq and reaction buffer (Fermentas), and an MS2 primer set as already described [ ]. .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Article Title: Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration
Article Snippet: Virion lysate was then added to a single-step, RT PCR assay with 35 nM MS2 RNA (Roche) as template, 500 nM of each primer (5’-TCCTGCTCAACTTCCTGTCGAG-3’ and 5’-CACAGGTCAAACCTCCTAGGAATG-3’), and hot-start Taq (Promega), all in 20 mM Tris-Cl pH 8.3, 5 mM (NH4 )2 SO4 , 20 mM KCl, 5 mM MgCl2 , 0.1 mg/ml BSA, 1/20,000 SYBR Green I (Sigma), and 200 μM dNTPs. .. The RT step was 42°C for 20 min, and the PCR was programmed for 40 cycles of denaturation at 95°C for 5 s, annealing 55°C for 5 s, extension at 72°C for 20 s and acquisition at 80°C for 5 s. A standard curve was obtained using known concentrations of recombinant HIV-1 RT (Ambion).

Article Title: Nef Can Enhance the Infectivity of Receptor-Pseudotyped Human Immunodeficiency Virus Type 1 Particles ▿
Article Snippet: Quantification of reverse-transcribed products was carried out in a Light Cycler (Roche) using Sybr green I, hot-start Taq and reaction buffer (Fermentas), and the primer set described previously ( ). .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Article Title: Inhibition of HIV-1 infection by TNPO3 depletion is determined by capsid and detectable after viral cDNA enters the nucleus
Article Snippet: Quantification of reverse transcribed products was carried out in a CFX96 thermal cycler (Biorad) using Sybr-Green I, hotstart Taq and reaction buffer (Fermentas), and an MS2 primer set as already described [ ]. .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Article Title: Nef Decreases HIV-1 Sensitivity to Neutralizing Antibodies that Target the Membrane-proximal External Region of TMgp41
Article Snippet: Quantification of reverse transcribed products was carried out in a CFX96 thermal cycler (Biorad) using Sybr-Green I, hotstart Taq and reaction buffer (Fermentas), and a MS2 primer set already described . .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Functional Assay:

Article Title: Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA
Article Snippet: .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion). .. Generation of stable KD cell lines HeLa, Jurkat T and AKATA cells were transduced with pAPM microRNA-based shRNA vectors targeting either control or CypA mRNA.

Article Title: Nef Can Enhance the Infectivity of Receptor-Pseudotyped Human Immunodeficiency Virus Type 1 Particles ▿
Article Snippet: .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion). .. HIV-1 particle concentrations in different virus samples were normalized to 5 U of recombinant HIV-1 RT per ml.

Article Title: Inhibition of HIV-1 infection by TNPO3 depletion is determined by capsid and detectable after viral cDNA enters the nucleus
Article Snippet: .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion). .. Generation of TNPO3 KD cells and rescue of the TNPO3 protein To generate stable microRNA-based shRNA KDs, HeLa cells were transduced with pAPM microRNA-based shRNA vectors [ ] targeting either control or TNPO3 mRNA (ts1, ts69, and ts72).

Article Title: Nef Decreases HIV-1 Sensitivity to Neutralizing Antibodies that Target the Membrane-proximal External Region of TMgp41
Article Snippet: .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion). .. Neutralization assays Sensitivities of the functional env -pseudovirus or replication competent NL4-3 to neutralizing agents were assayed on TZM-bl or GHOST cells, seeded onto 96-well tissue culture plates a day prior to neutralization.

Reverse Transcriptase Assay:

Article Title: Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA
Article Snippet: Paragraph title: Reverse transcriptase assay (SGPERT) ... A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Article Title: Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration
Article Snippet: Paragraph title: Reverse transcriptase assay ... The RT step was 42°C for 20 min, and the PCR was programmed for 40 cycles of denaturation at 95°C for 5 s, annealing 55°C for 5 s, extension at 72°C for 20 s and acquisition at 80°C for 5 s. A standard curve was obtained using known concentrations of recombinant HIV-1 RT (Ambion).

Article Title: Inhibition of HIV-1 infection by TNPO3 depletion is determined by capsid and detectable after viral cDNA enters the nucleus
Article Snippet: Paragraph title: Reverse transcriptase assay (SGPERT) ... A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Article Title: Nef Decreases HIV-1 Sensitivity to Neutralizing Antibodies that Target the Membrane-proximal External Region of TMgp41
Article Snippet: Paragraph title: Reverse transcriptase assay (SGPERT) ... A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

In Vitro:

Article Title: A cell-intrinsic inhibitor of HIV-1 reverse transcription in CD4+ T cells from elite controllers
Article Snippet: Paragraph title: In vitro kinase assays ... 5μM HIV-1 RT peptides or RT proteins were incubated with 2 mM ATP and 550nM of indicated recombinant CDK (Life Technologies) in NE Buffer.

Polymerase Chain Reaction:

Article Title: Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration
Article Snippet: .. The RT step was 42°C for 20 min, and the PCR was programmed for 40 cycles of denaturation at 95°C for 5 s, annealing 55°C for 5 s, extension at 72°C for 20 s and acquisition at 80°C for 5 s. A standard curve was obtained using known concentrations of recombinant HIV-1 RT (Ambion). .. Quantitation of viral cDNA Cell-free virions were normalized by RT-activity and incubated with CRFK, Hela or Jurkat cells in 6-well plates for 12 hrs, for full-length linear cDNA and 2-LTR circles, or 48 hrs, for Alu PCR.

Centrifugation:

Article Title: Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration
Article Snippet: Reverse transcriptase assay Virus-containing supernatant was harvested 48 hr post-transfection, clarified by low-speed centrifugation, and filtered through 0.45 μm pore filters (Sarstedt). .. The RT step was 42°C for 20 min, and the PCR was programmed for 40 cycles of denaturation at 95°C for 5 s, annealing 55°C for 5 s, extension at 72°C for 20 s and acquisition at 80°C for 5 s. A standard curve was obtained using known concentrations of recombinant HIV-1 RT (Ambion).

Article Title: Nef Can Enhance the Infectivity of Receptor-Pseudotyped Human Immunodeficiency Virus Type 1 Particles ▿
Article Snippet: Virus-containing supernatants were harvested at 48 h posttransfection, clarified by low-speed centrifugation, and filtered through 0.45-μm-pore filters. .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Real-time Polymerase Chain Reaction:

Article Title: Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA
Article Snippet: Reverse transcriptase assay (SGPERT) Reverse transcriptase activity in the supernatants was quantified using a Sybr green I-based real-time PCR enhanced reverse transcriptase assay (SGPERT) that possesses both high sensitivity and an extraordinary dynamic range [ ]. .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Article Title: Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration
Article Snippet: Reverse transcriptase (RT) activity in the supernatant was quantified using a modified Sybr green I-based, real-time PCR, enhanced RT assay [ , ]. .. The RT step was 42°C for 20 min, and the PCR was programmed for 40 cycles of denaturation at 95°C for 5 s, annealing 55°C for 5 s, extension at 72°C for 20 s and acquisition at 80°C for 5 s. A standard curve was obtained using known concentrations of recombinant HIV-1 RT (Ambion).

Article Title: Nef Can Enhance the Infectivity of Receptor-Pseudotyped Human Immunodeficiency Virus Type 1 Particles ▿
Article Snippet: Reverse transcriptase (RT) activity in the supernatants was quantified using a Sybr green I-based real-time PCR enhanced RT assay that possess both high sensitivity and an extraordinary dynamic range ( ). .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Article Title: Inhibition of HIV-1 infection by TNPO3 depletion is determined by capsid and detectable after viral cDNA enters the nucleus
Article Snippet: Reverse transcriptase assay (SGPERT) Reverse transcriptase (RT) activity in the supernatants was quantified using a Sybr green I-based real-time PCR enhanced RT assay (SGPER) that possesses both high sensitivity and an extraordinary dynamic range. .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Article Title: Nef Decreases HIV-1 Sensitivity to Neutralizing Antibodies that Target the Membrane-proximal External Region of TMgp41
Article Snippet: Reverse transcriptase assay (SGPERT) Reverse transcriptase (RT) activity in the supernatants was quantified using a Sybr green I-based real-time PCR enhanced RT assay (SGPER) that possesses both high sensitivity and an extraordinary dynamic range. .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration
Article Snippet: Virion lysate was then added to a single-step, RT PCR assay with 35 nM MS2 RNA (Roche) as template, 500 nM of each primer (5’-TCCTGCTCAACTTCCTGTCGAG-3’ and 5’-CACAGGTCAAACCTCCTAGGAATG-3’), and hot-start Taq (Promega), all in 20 mM Tris-Cl pH 8.3, 5 mM (NH4 )2 SO4 , 20 mM KCl, 5 mM MgCl2 , 0.1 mg/ml BSA, 1/20,000 SYBR Green I (Sigma), and 200 μM dNTPs. .. The RT step was 42°C for 20 min, and the PCR was programmed for 40 cycles of denaturation at 95°C for 5 s, annealing 55°C for 5 s, extension at 72°C for 20 s and acquisition at 80°C for 5 s. A standard curve was obtained using known concentrations of recombinant HIV-1 RT (Ambion).

Incubation:

Article Title: A cell-intrinsic inhibitor of HIV-1 reverse transcription in CD4+ T cells from elite controllers
Article Snippet: .. 5μM HIV-1 RT peptides or RT proteins were incubated with 2 mM ATP and 550nM of indicated recombinant CDK (Life Technologies) in NE Buffer. .. The kinase reaction was stopped by addition of SDS-PAGE loading buffer.

Article Title: In Vitro Characterization of a Simian Immunodeficiency Virus-Human Immunodeficiency Virus (HIV) Chimera Expressing HIV Type 1 Reverse Transcriptase To Study Antiviral Resistance in Pigtail Macaques
Article Snippet: Recombinant HIV-1 RT protein (a kind gift from P. Boyer) and sucrose cushion-isolated HIV-1 and RT-SHIVmne were solubilized and run on a 4-to-12% gradient NU-PAGE bis-Tris gel (Invitrogen). .. Blots were incubated overnight at 4°C with a mixture of six anti-HIV-1 RT monoclonal antibodies (kind gifts from M. Parniak [ ]) and then for 2 h with anti-mouse immunoglobulin G-horseradish peroxidase (Amersham Pharmacia, Piscataway, N.J.).

Activity Assay:

Article Title: Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA
Article Snippet: Reverse transcriptase assay (SGPERT) Reverse transcriptase activity in the supernatants was quantified using a Sybr green I-based real-time PCR enhanced reverse transcriptase assay (SGPERT) that possesses both high sensitivity and an extraordinary dynamic range [ ]. .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Article Title: Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration
Article Snippet: Reverse transcriptase (RT) activity in the supernatant was quantified using a modified Sybr green I-based, real-time PCR, enhanced RT assay [ , ]. .. The RT step was 42°C for 20 min, and the PCR was programmed for 40 cycles of denaturation at 95°C for 5 s, annealing 55°C for 5 s, extension at 72°C for 20 s and acquisition at 80°C for 5 s. A standard curve was obtained using known concentrations of recombinant HIV-1 RT (Ambion).

Article Title: Nef Can Enhance the Infectivity of Receptor-Pseudotyped Human Immunodeficiency Virus Type 1 Particles ▿
Article Snippet: Reverse transcriptase (RT) activity in the supernatants was quantified using a Sybr green I-based real-time PCR enhanced RT assay that possess both high sensitivity and an extraordinary dynamic range ( ). .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Article Title: Inhibition of HIV-1 infection by TNPO3 depletion is determined by capsid and detectable after viral cDNA enters the nucleus
Article Snippet: Reverse transcriptase assay (SGPERT) Reverse transcriptase (RT) activity in the supernatants was quantified using a Sybr green I-based real-time PCR enhanced RT assay (SGPER) that possesses both high sensitivity and an extraordinary dynamic range. .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Article Title: Nef Decreases HIV-1 Sensitivity to Neutralizing Antibodies that Target the Membrane-proximal External Region of TMgp41
Article Snippet: Reverse transcriptase assay (SGPERT) Reverse transcriptase (RT) activity in the supernatants was quantified using a Sybr green I-based real-time PCR enhanced RT assay (SGPER) that possesses both high sensitivity and an extraordinary dynamic range. .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Lysis:

Article Title: Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA
Article Snippet: Briefly, virions in cell-free supernatant were disrupted by adding an equal volume of SGPERT lysis buffer containing 0.25% Triton X-100, 50 mM KCl, 100 mM TrisHCl pH7.4, 0.4 U/μl RNase inhibitor (RiboLock, MBI Fermentas). .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Article Title: Inhibition of HIV-1 infection by TNPO3 depletion is determined by capsid and detectable after viral cDNA enters the nucleus
Article Snippet: Briefly, virions in cell-free supernatant were disrupted by adding an equal volume of SGPERT lysis buffer containing 0.25% Triton X-100, 50 mM KCl, 100 mM TrisHCl pH7.4, 0.4 U/μl RNase inhibitor (RiboLock, MBI Fermentas). .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Article Title: Nef Decreases HIV-1 Sensitivity to Neutralizing Antibodies that Target the Membrane-proximal External Region of TMgp41
Article Snippet: Briefly, virions in cell-free supernatants were disrupted by adding an equal volume of SGPERT lysis buffer containing 0.25% Triton X-100, 50 mM KCl, 100 mM TrisHCl pH7.4, 0.4 U/µl RNase inhibitor (RiboLock, MBI Fermentas). .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion).

Modification:

Article Title: Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration
Article Snippet: Reverse transcriptase (RT) activity in the supernatant was quantified using a modified Sybr green I-based, real-time PCR, enhanced RT assay [ , ]. .. The RT step was 42°C for 20 min, and the PCR was programmed for 40 cycles of denaturation at 95°C for 5 s, annealing 55°C for 5 s, extension at 72°C for 20 s and acquisition at 80°C for 5 s. A standard curve was obtained using known concentrations of recombinant HIV-1 RT (Ambion).

Western Blot:

Article Title: In Vitro Characterization of a Simian Immunodeficiency Virus-Human Immunodeficiency Virus (HIV) Chimera Expressing HIV Type 1 Reverse Transcriptase To Study Antiviral Resistance in Pigtail Macaques
Article Snippet: Paragraph title: Western blot analysis. ... Recombinant HIV-1 RT protein (a kind gift from P. Boyer) and sucrose cushion-isolated HIV-1 and RT-SHIVmne were solubilized and run on a 4-to-12% gradient NU-PAGE bis-Tris gel (Invitrogen).

Quantitation Assay:

Article Title: Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration
Article Snippet: All reactions and quantitation of product were carried out with a Biorad CFX96 cycler. .. The RT step was 42°C for 20 min, and the PCR was programmed for 40 cycles of denaturation at 95°C for 5 s, annealing 55°C for 5 s, extension at 72°C for 20 s and acquisition at 80°C for 5 s. A standard curve was obtained using known concentrations of recombinant HIV-1 RT (Ambion).

Recombinant:

Article Title: Cyclophilin A promotes HIV-1 reverse transcription but its effect on transduction correlates best with its effect on nuclear entry of viral cDNA
Article Snippet: .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion). .. Generation of stable KD cell lines HeLa, Jurkat T and AKATA cells were transduced with pAPM microRNA-based shRNA vectors targeting either control or CypA mRNA.

Article Title: Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration
Article Snippet: .. The RT step was 42°C for 20 min, and the PCR was programmed for 40 cycles of denaturation at 95°C for 5 s, annealing 55°C for 5 s, extension at 72°C for 20 s and acquisition at 80°C for 5 s. A standard curve was obtained using known concentrations of recombinant HIV-1 RT (Ambion). .. Quantitation of viral cDNA Cell-free virions were normalized by RT-activity and incubated with CRFK, Hela or Jurkat cells in 6-well plates for 12 hrs, for full-length linear cDNA and 2-LTR circles, or 48 hrs, for Alu PCR.

Article Title: Nef Can Enhance the Infectivity of Receptor-Pseudotyped Human Immunodeficiency Virus Type 1 Particles ▿
Article Snippet: .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion). .. HIV-1 particle concentrations in different virus samples were normalized to 5 U of recombinant HIV-1 RT per ml.

Article Title: Inhibition of HIV-1 infection by TNPO3 depletion is determined by capsid and detectable after viral cDNA enters the nucleus
Article Snippet: .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion). .. Generation of TNPO3 KD cells and rescue of the TNPO3 protein To generate stable microRNA-based shRNA KDs, HeLa cells were transduced with pAPM microRNA-based shRNA vectors [ ] targeting either control or TNPO3 mRNA (ts1, ts69, and ts72).

Article Title: A cell-intrinsic inhibitor of HIV-1 reverse transcription in CD4+ T cells from elite controllers
Article Snippet: .. 5μM HIV-1 RT peptides or RT proteins were incubated with 2 mM ATP and 550nM of indicated recombinant CDK (Life Technologies) in NE Buffer. .. The kinase reaction was stopped by addition of SDS-PAGE loading buffer.

Article Title: Nef Decreases HIV-1 Sensitivity to Neutralizing Antibodies that Target the Membrane-proximal External Region of TMgp41
Article Snippet: .. A standard curve was obtained using known concentrations (expressed in functional units) of recombinant HIV-1 RT (Ambion). .. Neutralization assays Sensitivities of the functional env -pseudovirus or replication competent NL4-3 to neutralizing agents were assayed on TZM-bl or GHOST cells, seeded onto 96-well tissue culture plates a day prior to neutralization.

Article Title: In Vitro Characterization of a Simian Immunodeficiency Virus-Human Immunodeficiency Virus (HIV) Chimera Expressing HIV Type 1 Reverse Transcriptase To Study Antiviral Resistance in Pigtail Macaques
Article Snippet: .. Recombinant HIV-1 RT protein (a kind gift from P. Boyer) and sucrose cushion-isolated HIV-1 and RT-SHIVmne were solubilized and run on a 4-to-12% gradient NU-PAGE bis-Tris gel (Invitrogen). .. The proteins from the gels were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, Mass.).

SDS Page:

Article Title: A cell-intrinsic inhibitor of HIV-1 reverse transcription in CD4+ T cells from elite controllers
Article Snippet: 5μM HIV-1 RT peptides or RT proteins were incubated with 2 mM ATP and 550nM of indicated recombinant CDK (Life Technologies) in NE Buffer. .. The kinase reaction was stopped by addition of SDS-PAGE loading buffer.

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  • 92
    Thermo Fisher hiv 1 transcript levels
    Quantification of <t>HIV-1</t> genomic RNA and miR-155 levels in virions. Standard curves for both miR-155 and the HIV-1 pol gene were generated by TaqMan qRT-PCR. Experimental values for miR-155 and HIV-1 RNA levels in total RNA isolated from purified HIV-1 virions were measured, and the absolute numbers of each molecule were determined. (A) Number of miR-155 copies per HIV-1 genomic RNA in virions produced from CEM-SS T cells infected with HIV-miR-155BT or with the HIV-RAN control. (B) Similar to panel A except that the number of miR-155 copies per HIV-1 genomic RNA were determined in virions produced by 293T cells expressing ectopic miR-155. (C) Inhibition of HIV-1 infectivity by miR-155 packaged into HIV-1 virions. Pseudotyped virions (HIV-RAN and HIV-155BT) were produced in 293T cells expressing ectopic miR-155, and equivalent amounts of virus, as determined by p24 level, were used to infect naive 293T cells. Cells were lysed at 20 h postinfection, and NLuc levels were determined. The data shown are from three independent experiments with standard deviations indicated.
    Hiv 1 Transcript Levels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher hiv 1 transcription factors
    Debio 1143 reverses <t>HIV-1</t> latency in resting CD4+ T cells from ART-treated patients. Purified resting CD4+ T cells from two ART-treated patients were serially diluted fivefold from 1,000,000 cells per well to 1,600 cells per well and seeded into individual wells of 48-well plates (5 replicates at each dilution). Each dilution was then treated with DMSO, anti-CD3/CD28 IgG-coated microbeads or 1 μM of Debio 1143. At day 2, highly permissive MOLT-4-CCR5 cells were added to each dilution to propagate released virions after latency reversal by LRAs. The ratio of target cells added was 10 7 MOLT-4-CCR5 cells to the 1x10 6 resting CD4+ T cell dilution, and 10 6 MOLT-4-CCR5 cells to all other resting CD4+ T cell dilutions. To increase infection, MOLT-4-CCR5 cells and released virions were spinoculated. Supernatants were collected at day 7 and split for HIV-1 RNA quantification ( A ) and HIV-1 infection ( B ) in triplicate. HIV-1 RNA was measured by RT-qPCR. Control experiments showed that > 95% of HIV-1 RNA in supernatants could be pelleted by centrifugation at 24,000 g for 1 h and were resistant to DNase I treatment. Infectivity of released virions was scored using TZM indicator cells after spinoculation. TZM infection was scored after 48 h by β-galactosidase activity in cell lysates. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p
    Hiv 1 Transcription Factors, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher ccr5
    HIV-infected NHBE ALI cultures shows reduced levels of HIV receptors and secrete functional Tat. Panel A : NHBE ALI cultures redifferentiated at the Air-Liquid Interface were infected with HIV BaL strain as described in Fig 2 . Nine days post-infection, total RNA was isolated and expression of CD4 and <t>CCR5</t> was determined by qRT-PCR. HIV BaL infected NHBE ALI cultures show decreased CD4 and CCR5 expression. Panel B : NHBE ALI cultures redifferentiated at the Air-Liquid Interface were infected with HIV IIIB strain as described in Fig 2 . Nine days post-infection, total RNA was isolated and expression of CD4 and CXCR4 was determined by qRT-PCR. HIV IIIB infected NHBE ALI cultures show decreased CD4 and CXCR4 expression. Panel C : Infected NHBE ALI cultures secrete functionally active Tat. Nine day old infected NHBE ALI cultures grown on transwells were placed on top of HEK 293 cells transiently transfected with LTRshGFPpA plasmid reported by us earlier [ 47 ]. In this construct GFP expression is controlled by HIV LTR such that GFP expression is only observed in presence of Tat. HEK 293 cells co-cultured with uninfected NHBE cells were used as controls. 24 hours post- co-culture, HEK 293 cells were visualized by fluorescence microscopy for EGFP expression. HEK-293 cells co-cultured with infected NHBE cells demonstrate EGFP positive cells suggesting that HIV Tat is secreted in the medium and this can transactivate LTR in co-cultured HEK 293 cells.
    Ccr5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher hiv 1 dna
    Postinfection treatment of <t>HIV-1-infected</t> Jurkat cells with NB-ZSG1 VLPs inhibits HIV-1 gene expression. (A) Jurkat cells were infected with HIV-1 and cultured for 30 days. The infected cells were transduced with VLPs conveying NB-ZSG1 or ZSG1 so that > 95% of the cells were positive for ZSG1 or NB-SZG1 (three successive transductions [TD1, TD2, and TD3], as shown). HIV-1 CA in supernatant from HIV-1-infected Jurkat cells or NB-ZSG1-treated or ZSG1-treated, HIV-1-infected Jurkat cells was quantified by ELISA (left y axis, solid lines) or NB-ZSG1 and ZSG1 expression level in cells by flow cytometry (right y axis, dotted lines). An experiment representative of at least three independent experiments with similar results is shown. (B) Western blot analysis of the cell lysates from panel A with anti-Tat and anti-β-tubulin antibodies. The molecular masses of the NB-ZSG1 fusion protein and β-tubulin are indicated. (C) Total cellular RNA was isolated from HIV-1-infected Jurkat; NB-ZSG1-treated or ZSG1-treated, HIV-1-infected Jurkat; or uninfected Jurkat cells. qRT-PCR assays were performed with primers specific for the 5′ UTR or for sequences in env . The relative level of viral 5′ UTR RNA in each sample was normalized to the amount of GAPDH mRNA in the sample. (D) Total cellular <t>DNA</t> was isolated and subjected to Alu -PCR analysis for the cell lines. In panels C and D, mean values and standard deviations from experiments representative of three independent experiments performed in triplicate are shown. A two-tailed Student t test with equal variance was used to calculate the P value. (E) PMA stimulation of HIV-1 gene expression in an NB-ZSG1-treated, HIV-1-infected Jurkat cell population. Uninfected Jurkat cells, HIV-1-infected Jurkat cells treated with ZSG1 or NB-ZSG1, and U1 cells were incubated with 1 nM PMA for 24 h, and the concentration of CA in the supernatant was measured by ELISA. The experiment was performed in triplicate and repeated three times with similar results. The mean values and standard deviations of a representative experiment are shown.
    Hiv 1 Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Quantification of HIV-1 genomic RNA and miR-155 levels in virions. Standard curves for both miR-155 and the HIV-1 pol gene were generated by TaqMan qRT-PCR. Experimental values for miR-155 and HIV-1 RNA levels in total RNA isolated from purified HIV-1 virions were measured, and the absolute numbers of each molecule were determined. (A) Number of miR-155 copies per HIV-1 genomic RNA in virions produced from CEM-SS T cells infected with HIV-miR-155BT or with the HIV-RAN control. (B) Similar to panel A except that the number of miR-155 copies per HIV-1 genomic RNA were determined in virions produced by 293T cells expressing ectopic miR-155. (C) Inhibition of HIV-1 infectivity by miR-155 packaged into HIV-1 virions. Pseudotyped virions (HIV-RAN and HIV-155BT) were produced in 293T cells expressing ectopic miR-155, and equivalent amounts of virus, as determined by p24 level, were used to infect naive 293T cells. Cells were lysed at 20 h postinfection, and NLuc levels were determined. The data shown are from three independent experiments with standard deviations indicated.

    Journal: mBio

    Article Title: Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity

    doi: 10.1128/mBio.02125-16

    Figure Lengend Snippet: Quantification of HIV-1 genomic RNA and miR-155 levels in virions. Standard curves for both miR-155 and the HIV-1 pol gene were generated by TaqMan qRT-PCR. Experimental values for miR-155 and HIV-1 RNA levels in total RNA isolated from purified HIV-1 virions were measured, and the absolute numbers of each molecule were determined. (A) Number of miR-155 copies per HIV-1 genomic RNA in virions produced from CEM-SS T cells infected with HIV-miR-155BT or with the HIV-RAN control. (B) Similar to panel A except that the number of miR-155 copies per HIV-1 genomic RNA were determined in virions produced by 293T cells expressing ectopic miR-155. (C) Inhibition of HIV-1 infectivity by miR-155 packaged into HIV-1 virions. Pseudotyped virions (HIV-RAN and HIV-155BT) were produced in 293T cells expressing ectopic miR-155, and equivalent amounts of virus, as determined by p24 level, were used to infect naive 293T cells. Cells were lysed at 20 h postinfection, and NLuc levels were determined. The data shown are from three independent experiments with standard deviations indicated.

    Article Snippet: To determine HIV-1 transcript levels, 250 ng of total RNA was reverse transcribed with SuperScript IV (catalog no. 18090010; Thermo Fisher) according to the manufacturer’s instructions.

    Techniques: Generated, Quantitative RT-PCR, Isolation, Purification, Produced, Infection, Expressing, Inhibition

    Relative miRNA and HIV-1 RNA levels in purified HIV-1 virions measured by quantitative PCR. Virions were collected at 72 h postinfection. Fold changes in the levels of packaged miR-92a and miR-155, relative to HIV-1 genomic RNA, were determined by the ΔΔ C T method ( 30 ). An HIV-1 clone bearing a randomized miRNA target (RAN) served as the negative control and was set to 1. (A) Western blot assay to assess the purity of HIV-1 virions. (Top) p24 Western blot assay of HIV-1-infected CEM-SS cell lysates. (Bottom) p24 Western blot assay of purified virions. (B) Packaging of miR-92a into HIV-1 virions containing two miR-92a BTs produced by CEM-SS T cells. (C) Packaging of miR-92a into HIV-1 virions containing two miR-92a BTs produced by 293T cells. (D) Packaging of miR-155 into HIV-1 virions containing two miR-155 BTs produced by CEM-SS cells. (E) Packaging of miR-155 into HIV-1 virions containing miR-155 BTs produced by 293T cells expressing ectopic miR-155. The data shown are from three to five independent experiments with standard deviations indicated.

    Journal: mBio

    Article Title: Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity

    doi: 10.1128/mBio.02125-16

    Figure Lengend Snippet: Relative miRNA and HIV-1 RNA levels in purified HIV-1 virions measured by quantitative PCR. Virions were collected at 72 h postinfection. Fold changes in the levels of packaged miR-92a and miR-155, relative to HIV-1 genomic RNA, were determined by the ΔΔ C T method ( 30 ). An HIV-1 clone bearing a randomized miRNA target (RAN) served as the negative control and was set to 1. (A) Western blot assay to assess the purity of HIV-1 virions. (Top) p24 Western blot assay of HIV-1-infected CEM-SS cell lysates. (Bottom) p24 Western blot assay of purified virions. (B) Packaging of miR-92a into HIV-1 virions containing two miR-92a BTs produced by CEM-SS T cells. (C) Packaging of miR-92a into HIV-1 virions containing two miR-92a BTs produced by 293T cells. (D) Packaging of miR-155 into HIV-1 virions containing two miR-155 BTs produced by CEM-SS cells. (E) Packaging of miR-155 into HIV-1 virions containing miR-155 BTs produced by 293T cells expressing ectopic miR-155. The data shown are from three to five independent experiments with standard deviations indicated.

    Article Snippet: To determine HIV-1 transcript levels, 250 ng of total RNA was reverse transcribed with SuperScript IV (catalog no. 18090010; Thermo Fisher) according to the manufacturer’s instructions.

    Techniques: Purification, Real-time Polymerase Chain Reaction, Negative Control, Western Blot, Infection, Produced, Expressing

    A subset of miRNAs is selectively packaged into HIV-1 virions. (A) Representative Western blot assay of HIV-1 capsid (p24) levels in HIV-1-infected CEM-SS T cells and highly purified virions obtained from the same culture. (B) Most miRNAs are packaged into HIV-1 virions in proportion to their cellular expression level. miRNAs that represent ≥0.1% of the total miRNA level in both purified virions and infected CEM-SS T cells are graphed. The values shown are the averages of data from four independent small RNA-seq experiments. The lines delineate a > 2-fold change in relative miRNA levels between virions and cells. Only five cellular miRNAs fall above this line.

    Journal: mBio

    Article Title: Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity

    doi: 10.1128/mBio.02125-16

    Figure Lengend Snippet: A subset of miRNAs is selectively packaged into HIV-1 virions. (A) Representative Western blot assay of HIV-1 capsid (p24) levels in HIV-1-infected CEM-SS T cells and highly purified virions obtained from the same culture. (B) Most miRNAs are packaged into HIV-1 virions in proportion to their cellular expression level. miRNAs that represent ≥0.1% of the total miRNA level in both purified virions and infected CEM-SS T cells are graphed. The values shown are the averages of data from four independent small RNA-seq experiments. The lines delineate a > 2-fold change in relative miRNA levels between virions and cells. Only five cellular miRNAs fall above this line.

    Article Snippet: To determine HIV-1 transcript levels, 250 ng of total RNA was reverse transcribed with SuperScript IV (catalog no. 18090010; Thermo Fisher) according to the manufacturer’s instructions.

    Techniques: Western Blot, Infection, Purification, Expressing, RNA Sequencing Assay

    Effect of inserted miRNA BTs or PTs on HIV-1 replication. (A) Representative p24 Western blot assay of whole-cell lysates derived from 293T cells producing HIV-NLuc-155BT (lane 1), HIV-NLuc-155PT (lane 2), HIV-NLuc-92aBT (lane 3), HIV-NLuc-92aPT (lane 4), or HIV-NLuc-RAN (lane 5) or from uninfected cells (lane 6). (B) Relative p24 levels determined by Gene Tools software (Syngene). (C) β-Actin internal loading control. (D) NanoLuc levels in CEM-SS, 293T+CD4/CXCR4, and 293T+CD4/CXCR4/miR-155 cells infected with an HIV-155BT, HIV-155PT, HIV-92aBT, HIV-92aPT, or HIV-RAN preparation. The NLuc activity induced upon pNL-NLuc-RAN infection was set to 1, and the relative NLuc levels in CEM-SS, 293T+CD4/CXCR4, and 293T+CD4/CXCR4/miR-155 cells infected with HIV-NLuc-155BT, HIV-NLuc-155PT, HIV-NLuc-92aBT, or HIV-NLuc-92aPT were determined. The data shown in panel D are from four independent experiments with standard deviations indicated.

    Journal: mBio

    Article Title: Induced Packaging of Cellular MicroRNAs into HIV-1 Virions Can Inhibit Infectivity

    doi: 10.1128/mBio.02125-16

    Figure Lengend Snippet: Effect of inserted miRNA BTs or PTs on HIV-1 replication. (A) Representative p24 Western blot assay of whole-cell lysates derived from 293T cells producing HIV-NLuc-155BT (lane 1), HIV-NLuc-155PT (lane 2), HIV-NLuc-92aBT (lane 3), HIV-NLuc-92aPT (lane 4), or HIV-NLuc-RAN (lane 5) or from uninfected cells (lane 6). (B) Relative p24 levels determined by Gene Tools software (Syngene). (C) β-Actin internal loading control. (D) NanoLuc levels in CEM-SS, 293T+CD4/CXCR4, and 293T+CD4/CXCR4/miR-155 cells infected with an HIV-155BT, HIV-155PT, HIV-92aBT, HIV-92aPT, or HIV-RAN preparation. The NLuc activity induced upon pNL-NLuc-RAN infection was set to 1, and the relative NLuc levels in CEM-SS, 293T+CD4/CXCR4, and 293T+CD4/CXCR4/miR-155 cells infected with HIV-NLuc-155BT, HIV-NLuc-155PT, HIV-NLuc-92aBT, or HIV-NLuc-92aPT were determined. The data shown in panel D are from four independent experiments with standard deviations indicated.

    Article Snippet: To determine HIV-1 transcript levels, 250 ng of total RNA was reverse transcribed with SuperScript IV (catalog no. 18090010; Thermo Fisher) according to the manufacturer’s instructions.

    Techniques: Western Blot, Derivative Assay, Software, Infection, Activity Assay

    Debio 1143 reverses HIV-1 latency in resting CD4+ T cells from ART-treated patients. Purified resting CD4+ T cells from two ART-treated patients were serially diluted fivefold from 1,000,000 cells per well to 1,600 cells per well and seeded into individual wells of 48-well plates (5 replicates at each dilution). Each dilution was then treated with DMSO, anti-CD3/CD28 IgG-coated microbeads or 1 μM of Debio 1143. At day 2, highly permissive MOLT-4-CCR5 cells were added to each dilution to propagate released virions after latency reversal by LRAs. The ratio of target cells added was 10 7 MOLT-4-CCR5 cells to the 1x10 6 resting CD4+ T cell dilution, and 10 6 MOLT-4-CCR5 cells to all other resting CD4+ T cell dilutions. To increase infection, MOLT-4-CCR5 cells and released virions were spinoculated. Supernatants were collected at day 7 and split for HIV-1 RNA quantification ( A ) and HIV-1 infection ( B ) in triplicate. HIV-1 RNA was measured by RT-qPCR. Control experiments showed that > 95% of HIV-1 RNA in supernatants could be pelleted by centrifugation at 24,000 g for 1 h and were resistant to DNase I treatment. Infectivity of released virions was scored using TZM indicator cells after spinoculation. TZM infection was scored after 48 h by β-galactosidase activity in cell lysates. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p

    Journal: PLoS ONE

    Article Title: The inhibitor apoptosis protein antagonist Debio 1143 Is an attractive HIV-1 latency reversal candidate

    doi: 10.1371/journal.pone.0211746

    Figure Lengend Snippet: Debio 1143 reverses HIV-1 latency in resting CD4+ T cells from ART-treated patients. Purified resting CD4+ T cells from two ART-treated patients were serially diluted fivefold from 1,000,000 cells per well to 1,600 cells per well and seeded into individual wells of 48-well plates (5 replicates at each dilution). Each dilution was then treated with DMSO, anti-CD3/CD28 IgG-coated microbeads or 1 μM of Debio 1143. At day 2, highly permissive MOLT-4-CCR5 cells were added to each dilution to propagate released virions after latency reversal by LRAs. The ratio of target cells added was 10 7 MOLT-4-CCR5 cells to the 1x10 6 resting CD4+ T cell dilution, and 10 6 MOLT-4-CCR5 cells to all other resting CD4+ T cell dilutions. To increase infection, MOLT-4-CCR5 cells and released virions were spinoculated. Supernatants were collected at day 7 and split for HIV-1 RNA quantification ( A ) and HIV-1 infection ( B ) in triplicate. HIV-1 RNA was measured by RT-qPCR. Control experiments showed that > 95% of HIV-1 RNA in supernatants could be pelleted by centrifugation at 24,000 g for 1 h and were resistant to DNase I treatment. Infectivity of released virions was scored using TZM indicator cells after spinoculation. TZM infection was scored after 48 h by β-galactosidase activity in cell lysates. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p

    Article Snippet: For the analysis of the subcellular localization (nuclear versus cytosolic) of selected HIV-1 transcription factors, NE-PER Nuclear and Cytoplasmic Extraction kit from ThermoScientific Fisher (Pierce) was used according to the manufacturer’s instructions. siRNA control and siRNA BIRC2 were obtained from Sigma and used according to the manufacturer’s instructions.

    Techniques: Purification, Infection, Quantitative RT-PCR, Centrifugation, Activity Assay

    Debio 1143 reverses HIV-1 latency. A . Typical FACS graphs for the percentage of GFP+ HIV-1 latent reporter cells. The population on the forward scatter/side scatter dot plot was placed by the automatic scaling feature of the cytometer. Minor variability between reporter cell replicates was observed as shown by representative graphs of 2D8 cells treated with control DMSO or Debio 1143 (1 μM). B. Latently infected JLat 10.6, 2D10 and 5A8 GFP reporter cells (250,000) (duplicate) were treated for 48 h with LCL161 (1 μM), Debio 1143 (1 μM) or in combination with panobinostat (10 nM) and vorinostat (500 nM). GFP expression was quantified by FACS. Data are expressed as % of GFP levels. Two distinct experiments were conducted in triplicate and averaged data are presented. C. Same as B except that the capacity of Debio 1143 at reversing HIV-1 latency in 2D10 cells was compared with that of other IAPa. D. CD4+ T-lymphocytes (250,000) (triplicate) were incubated with the LRAs and cytotoxicity was quantified after 48 h by LDH assay as above ( Fig 2 ). E. 2D10 cells were treated with control (siCTL) and BIRC2 (siBIRC2) siRNA for 24 h, then incubated with DMSO or Debio 1143 (1 μM) and analyzed for GFP expression after 48 h (left panel) and cell lysates analyzed by Western blotting for BIRC2, NIK and CypA expression. F. 2D10 cells (triplicate) were first incubated with or without MG132 (25 μM) for 30 min, subsequently exposed to DMSO or Debio 1143 (1 μM) for 48 h, and analyzed by FACS for GFP content. G. Wild-type or NIK knockout (ΔNIK) 2D10 cells were exposed to DMSO or Debio 1143 (1 μM) for 48 h, and analyzed by FACS for GFP content. Two distinct experiments were conducted in triplicate and averaged data are presented. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p

    Journal: PLoS ONE

    Article Title: The inhibitor apoptosis protein antagonist Debio 1143 Is an attractive HIV-1 latency reversal candidate

    doi: 10.1371/journal.pone.0211746

    Figure Lengend Snippet: Debio 1143 reverses HIV-1 latency. A . Typical FACS graphs for the percentage of GFP+ HIV-1 latent reporter cells. The population on the forward scatter/side scatter dot plot was placed by the automatic scaling feature of the cytometer. Minor variability between reporter cell replicates was observed as shown by representative graphs of 2D8 cells treated with control DMSO or Debio 1143 (1 μM). B. Latently infected JLat 10.6, 2D10 and 5A8 GFP reporter cells (250,000) (duplicate) were treated for 48 h with LCL161 (1 μM), Debio 1143 (1 μM) or in combination with panobinostat (10 nM) and vorinostat (500 nM). GFP expression was quantified by FACS. Data are expressed as % of GFP levels. Two distinct experiments were conducted in triplicate and averaged data are presented. C. Same as B except that the capacity of Debio 1143 at reversing HIV-1 latency in 2D10 cells was compared with that of other IAPa. D. CD4+ T-lymphocytes (250,000) (triplicate) were incubated with the LRAs and cytotoxicity was quantified after 48 h by LDH assay as above ( Fig 2 ). E. 2D10 cells were treated with control (siCTL) and BIRC2 (siBIRC2) siRNA for 24 h, then incubated with DMSO or Debio 1143 (1 μM) and analyzed for GFP expression after 48 h (left panel) and cell lysates analyzed by Western blotting for BIRC2, NIK and CypA expression. F. 2D10 cells (triplicate) were first incubated with or without MG132 (25 μM) for 30 min, subsequently exposed to DMSO or Debio 1143 (1 μM) for 48 h, and analyzed by FACS for GFP content. G. Wild-type or NIK knockout (ΔNIK) 2D10 cells were exposed to DMSO or Debio 1143 (1 μM) for 48 h, and analyzed by FACS for GFP content. Two distinct experiments were conducted in triplicate and averaged data are presented. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p

    Article Snippet: For the analysis of the subcellular localization (nuclear versus cytosolic) of selected HIV-1 transcription factors, NE-PER Nuclear and Cytoplasmic Extraction kit from ThermoScientific Fisher (Pierce) was used according to the manufacturer’s instructions. siRNA control and siRNA BIRC2 were obtained from Sigma and used according to the manufacturer’s instructions.

    Techniques: FACS, Cytometry, Infection, Expressing, Incubation, Lactate Dehydrogenase Assay, Western Blot, Knock-Out

    Debio 1143 reverses ex vivo HIV-1 latency in resting CD4+ T cells isolated from BLT mice. A. Experimental design for the HIV-1 latency model in BLT mice. When ART is interrupted, viral rebound occurs B. Same as A except that ART was maintained until isolation of resting CD4+ T cells from blood, thymic organoid, lung, spleen, bone marrow, lymph nodes and liver of HIV-1-infected BLT mice (5 mice per treatment). Isolated resting CD4+ T cells from each group were pooled, counted, split in triplicate and treated with vehicle, PMA (20 ng/mL) + ionomycin (1 μg/mL), Debio 1143 (1 μM), LCL161 (1 μM), panobinostat (1 μM) or vorinostat (1 μM). De novo released virions from supernatants were purified as above and qua ntified by RT-qPCR. Data are expressed in copies of HIV-1 RNA/mL of supernatant. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p

    Journal: PLoS ONE

    Article Title: The inhibitor apoptosis protein antagonist Debio 1143 Is an attractive HIV-1 latency reversal candidate

    doi: 10.1371/journal.pone.0211746

    Figure Lengend Snippet: Debio 1143 reverses ex vivo HIV-1 latency in resting CD4+ T cells isolated from BLT mice. A. Experimental design for the HIV-1 latency model in BLT mice. When ART is interrupted, viral rebound occurs B. Same as A except that ART was maintained until isolation of resting CD4+ T cells from blood, thymic organoid, lung, spleen, bone marrow, lymph nodes and liver of HIV-1-infected BLT mice (5 mice per treatment). Isolated resting CD4+ T cells from each group were pooled, counted, split in triplicate and treated with vehicle, PMA (20 ng/mL) + ionomycin (1 μg/mL), Debio 1143 (1 μM), LCL161 (1 μM), panobinostat (1 μM) or vorinostat (1 μM). De novo released virions from supernatants were purified as above and qua ntified by RT-qPCR. Data are expressed in copies of HIV-1 RNA/mL of supernatant. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p

    Article Snippet: For the analysis of the subcellular localization (nuclear versus cytosolic) of selected HIV-1 transcription factors, NE-PER Nuclear and Cytoplasmic Extraction kit from ThermoScientific Fisher (Pierce) was used according to the manufacturer’s instructions. siRNA control and siRNA BIRC2 were obtained from Sigma and used according to the manufacturer’s instructions.

    Techniques: Ex Vivo, Isolation, Mouse Assay, Infection, Purification, Quantitative RT-PCR

    Analysis of the mechanisms of action of Debio 1143. CD4+ T-lymphocytes (100,000) (triplicate) were incubated with VSVG-NL4.3-GFP and Debio 1143 (0 to 10 μM) for 24 h and analyzed for infection by FACS ( A ) and for cytotoxicity by LDH assay ( B ). C. NF-κB reporter (Luc)-3T3 cells (BPS Bioscience) (100,000) (triplicate) were treated with Debio 1143 and luciferase activity in cell lysates was quantified after 6 h. D. CD4+ T-lymphocytes (1,000,000) were treated (from 15 min to 2 h) with DMSO, Debio 1143 (1 μM) and TNF alpha (10 ng/mL) and cell lysates analyzed by Western blotting for BIRC2, IkB alpha and CypA expression. E. Same as D except that cells were treated for 15 min with 0, 0.1, 1 and 10 μM of Debio 1143. F. Same as D except that cells were treated for 24 h with DMSO or 1 μM of Debio 1143, and cell lysates analyzed by Western blotting for BIRC2, p100/52, NIK and CypA expression. G. Same as D except that cells were treated for 24 h with DMSO, 1 μM of Debio 1143 or 10 ng/mL of TNF alpha, and cytosolic and nuclear extracts analyzed by Western blotting for the expression of various components of the NF-kB signaling pathways and cytosolic and nuclear markers. H. 2D10 cells (500,000) (triplicate) were treated with 1 μM Debio 1143 for 10 h prior to ChIP analysis using control, anti-RELB or anti-RELA IgG. RELB- and RELA-specific association with the HIV-1 LTR and the IkB alpha gene promoter region, or an intergenic region upstream of the PABPC1 gene not known to contain NF-kB binding sites as negative control, was analyzed by qPCR and is presented as fold enrichment over control IgG. Data from A to F are each representative of two independent experiments. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p

    Journal: PLoS ONE

    Article Title: The inhibitor apoptosis protein antagonist Debio 1143 Is an attractive HIV-1 latency reversal candidate

    doi: 10.1371/journal.pone.0211746

    Figure Lengend Snippet: Analysis of the mechanisms of action of Debio 1143. CD4+ T-lymphocytes (100,000) (triplicate) were incubated with VSVG-NL4.3-GFP and Debio 1143 (0 to 10 μM) for 24 h and analyzed for infection by FACS ( A ) and for cytotoxicity by LDH assay ( B ). C. NF-κB reporter (Luc)-3T3 cells (BPS Bioscience) (100,000) (triplicate) were treated with Debio 1143 and luciferase activity in cell lysates was quantified after 6 h. D. CD4+ T-lymphocytes (1,000,000) were treated (from 15 min to 2 h) with DMSO, Debio 1143 (1 μM) and TNF alpha (10 ng/mL) and cell lysates analyzed by Western blotting for BIRC2, IkB alpha and CypA expression. E. Same as D except that cells were treated for 15 min with 0, 0.1, 1 and 10 μM of Debio 1143. F. Same as D except that cells were treated for 24 h with DMSO or 1 μM of Debio 1143, and cell lysates analyzed by Western blotting for BIRC2, p100/52, NIK and CypA expression. G. Same as D except that cells were treated for 24 h with DMSO, 1 μM of Debio 1143 or 10 ng/mL of TNF alpha, and cytosolic and nuclear extracts analyzed by Western blotting for the expression of various components of the NF-kB signaling pathways and cytosolic and nuclear markers. H. 2D10 cells (500,000) (triplicate) were treated with 1 μM Debio 1143 for 10 h prior to ChIP analysis using control, anti-RELB or anti-RELA IgG. RELB- and RELA-specific association with the HIV-1 LTR and the IkB alpha gene promoter region, or an intergenic region upstream of the PABPC1 gene not known to contain NF-kB binding sites as negative control, was analyzed by qPCR and is presented as fold enrichment over control IgG. Data from A to F are each representative of two independent experiments. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p

    Article Snippet: For the analysis of the subcellular localization (nuclear versus cytosolic) of selected HIV-1 transcription factors, NE-PER Nuclear and Cytoplasmic Extraction kit from ThermoScientific Fisher (Pierce) was used according to the manufacturer’s instructions. siRNA control and siRNA BIRC2 were obtained from Sigma and used according to the manufacturer’s instructions.

    Techniques: Incubation, Infection, FACS, Lactate Dehydrogenase Assay, Luciferase, Activity Assay, Western Blot, Expressing, Chromatin Immunoprecipitation, Binding Assay, Negative Control, Real-time Polymerase Chain Reaction

    Debio 1143 does not stimulate the production of pro-inflammatory cytokines. A. PBMCs from an ART-treated patient were incubated with DMSO, anti-CD3 (200 ng/mL)/CD28 (500 ng/mL) antibodies (mAbs) or Debio 1143 (1 μM). Supernatants were collected after 24 h, and cytokine concentrations were determined by BioPlex analysis. B. Cytokine mRNAs levels from PBMCs were quantified by PCR using β-actin as control. C. Blood of HIV-1-infected BLT mice under ART untreated (n = 10) or treated with Debio 1143 (100 mg/kg, p.o.) (n = 10) was collected 48 h post-drug treatment and human cytokine levels were quantified by BioPlex analysis. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p

    Journal: PLoS ONE

    Article Title: The inhibitor apoptosis protein antagonist Debio 1143 Is an attractive HIV-1 latency reversal candidate

    doi: 10.1371/journal.pone.0211746

    Figure Lengend Snippet: Debio 1143 does not stimulate the production of pro-inflammatory cytokines. A. PBMCs from an ART-treated patient were incubated with DMSO, anti-CD3 (200 ng/mL)/CD28 (500 ng/mL) antibodies (mAbs) or Debio 1143 (1 μM). Supernatants were collected after 24 h, and cytokine concentrations were determined by BioPlex analysis. B. Cytokine mRNAs levels from PBMCs were quantified by PCR using β-actin as control. C. Blood of HIV-1-infected BLT mice under ART untreated (n = 10) or treated with Debio 1143 (100 mg/kg, p.o.) (n = 10) was collected 48 h post-drug treatment and human cytokine levels were quantified by BioPlex analysis. P-values are presented. ANOVA and Bonferroni’s multiple comparison tests were used to compare means of different groups. Bar graphs indicate each sample point, mean, and SEM. Key for adjusted p-values from Bonferroni’s multiple comparison tests on graphs: ns = no significance, * = 0.01≤p

    Article Snippet: For the analysis of the subcellular localization (nuclear versus cytosolic) of selected HIV-1 transcription factors, NE-PER Nuclear and Cytoplasmic Extraction kit from ThermoScientific Fisher (Pierce) was used according to the manufacturer’s instructions. siRNA control and siRNA BIRC2 were obtained from Sigma and used according to the manufacturer’s instructions.

    Techniques: Incubation, Polymerase Chain Reaction, Infection, Mouse Assay

    HIV-infected NHBE ALI cultures shows reduced levels of HIV receptors and secrete functional Tat. Panel A : NHBE ALI cultures redifferentiated at the Air-Liquid Interface were infected with HIV BaL strain as described in Fig 2 . Nine days post-infection, total RNA was isolated and expression of CD4 and CCR5 was determined by qRT-PCR. HIV BaL infected NHBE ALI cultures show decreased CD4 and CCR5 expression. Panel B : NHBE ALI cultures redifferentiated at the Air-Liquid Interface were infected with HIV IIIB strain as described in Fig 2 . Nine days post-infection, total RNA was isolated and expression of CD4 and CXCR4 was determined by qRT-PCR. HIV IIIB infected NHBE ALI cultures show decreased CD4 and CXCR4 expression. Panel C : Infected NHBE ALI cultures secrete functionally active Tat. Nine day old infected NHBE ALI cultures grown on transwells were placed on top of HEK 293 cells transiently transfected with LTRshGFPpA plasmid reported by us earlier [ 47 ]. In this construct GFP expression is controlled by HIV LTR such that GFP expression is only observed in presence of Tat. HEK 293 cells co-cultured with uninfected NHBE cells were used as controls. 24 hours post- co-culture, HEK 293 cells were visualized by fluorescence microscopy for EGFP expression. HEK-293 cells co-cultured with infected NHBE cells demonstrate EGFP positive cells suggesting that HIV Tat is secreted in the medium and this can transactivate LTR in co-cultured HEK 293 cells.

    Journal: PLoS ONE

    Article Title: HIV Infects Bronchial Epithelium and Suppresses Components of the Mucociliary Clearance Apparatus

    doi: 10.1371/journal.pone.0169161

    Figure Lengend Snippet: HIV-infected NHBE ALI cultures shows reduced levels of HIV receptors and secrete functional Tat. Panel A : NHBE ALI cultures redifferentiated at the Air-Liquid Interface were infected with HIV BaL strain as described in Fig 2 . Nine days post-infection, total RNA was isolated and expression of CD4 and CCR5 was determined by qRT-PCR. HIV BaL infected NHBE ALI cultures show decreased CD4 and CCR5 expression. Panel B : NHBE ALI cultures redifferentiated at the Air-Liquid Interface were infected with HIV IIIB strain as described in Fig 2 . Nine days post-infection, total RNA was isolated and expression of CD4 and CXCR4 was determined by qRT-PCR. HIV IIIB infected NHBE ALI cultures show decreased CD4 and CXCR4 expression. Panel C : Infected NHBE ALI cultures secrete functionally active Tat. Nine day old infected NHBE ALI cultures grown on transwells were placed on top of HEK 293 cells transiently transfected with LTRshGFPpA plasmid reported by us earlier [ 47 ]. In this construct GFP expression is controlled by HIV LTR such that GFP expression is only observed in presence of Tat. HEK 293 cells co-cultured with uninfected NHBE cells were used as controls. 24 hours post- co-culture, HEK 293 cells were visualized by fluorescence microscopy for EGFP expression. HEK-293 cells co-cultured with infected NHBE cells demonstrate EGFP positive cells suggesting that HIV Tat is secreted in the medium and this can transactivate LTR in co-cultured HEK 293 cells.

    Article Snippet: Following transfe r, blot was blocked in 5% milk then incubated in CD4 primary antibody (1: 500; Sigma-Aldrich), CXCR4 (1:1000; Thermo scientific) or CCR5 (1:1000; Thermo scientific).

    Techniques: Infection, Functional Assay, Isolation, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Construct, Cell Culture, Co-Culture Assay, Fluorescence, Microscopy

    NHBE redifferentiated at the ALI express HIV receptors and co-receptors and can support both R5 and X4-tropic infection. Panel A : We analyzed expression of CD4, CCR5 and CXCR4 in NHBE by qRT-PCR. Total RNA was isolated from NHBE ALI cultures and expression of CD4 (Applied Biosystems # HS01058407-m1), CXCR4 (Applied Biosystems # HS00607978-s1) and CCR5 (Applied Biosystems # HS99999149-s1) was determined by qRT-PCR using Taqman probes. CFTR (Applied Biosystems # HS00357011-m1) expression was determined for comparison. NHBE ALI cultures express all canonical HIV receptors. Panel B : Western blot analysis from three different lungs demonstrates that NHBE ALI cultures show comparable expression of all HIV receptors. While RNA levels were significantly lower for CCR5 than CD4 and CXCR4, protein levels were comparable with CD4 and CXCR4. This could be due to inherent CCR5 mRNA instability due to a pseudoknot structure in the mRNA that promotes non-sense mediated decay [ 35 ]. Panel C : To determine if bronchial epithelial cells express HIV receptors and co-receptors in vivo, Bronchial brushings were analyzed for expression of CD4, CCR5 and CXCR4 by qRT-PCR. Expression patterns of the three receptors were similar to that observed in our ex vivo model with maximal expression of CD4 followed by CXCR4 and significantly lower CCR5 expression validating the physiological relevance of our ex vivo NHBE ALI culture model. Panel D and E : NHBE ALI cultures can be infected with both R5 and X4-tropic strains of HIV. NHBE cultures redifferentiated at the Air-Liquid Interface were infected apically and basolaterally with either HIV IIIB (X4-tropic) or HIV BaL (R5-tropic) strains. After 16 hours cells were washed apically and basolaterally with PBS four times to remove any residual input virus. The fourth wash was collected for p24 analysis and measured as Day 0 to confirm that all input virus had been removed. No p24 was detected in the 4 th wash (Day 0) confirming that all input virus had been removed. We observe an initial spike in p24 output on Day 3 for both HIV IIIB and HIV BaL infections that declines gradually till Day 9 (panel D). Experiments were terminated and total RNA was isolated and cell associated HIV RNA was quantitated by qRT-PCR using Taqman probe (Applied Biosystems # PA03453409-s1). NHBE cells demonstrate cell associated viral RNA for both R5 and X4-tropic infections (panel E). Panel F : Another set of NHBE ALI cultures similarly infected in presence of with maraviroc (For HIV BaL infection) or AMD3100 (for HIV IIIB infection) which was retained for the remainder of the experiment. Maravoiroc was able to block infection of NHBE ALI cultures by the R5-tropic strain HIV BaL. Likewise, AMD3100 was able to block infection by the X4-tropic strain HIV IIIB. n = NHBE ALI cultures from 3 different lungs * = significant (p

    Journal: PLoS ONE

    Article Title: HIV Infects Bronchial Epithelium and Suppresses Components of the Mucociliary Clearance Apparatus

    doi: 10.1371/journal.pone.0169161

    Figure Lengend Snippet: NHBE redifferentiated at the ALI express HIV receptors and co-receptors and can support both R5 and X4-tropic infection. Panel A : We analyzed expression of CD4, CCR5 and CXCR4 in NHBE by qRT-PCR. Total RNA was isolated from NHBE ALI cultures and expression of CD4 (Applied Biosystems # HS01058407-m1), CXCR4 (Applied Biosystems # HS00607978-s1) and CCR5 (Applied Biosystems # HS99999149-s1) was determined by qRT-PCR using Taqman probes. CFTR (Applied Biosystems # HS00357011-m1) expression was determined for comparison. NHBE ALI cultures express all canonical HIV receptors. Panel B : Western blot analysis from three different lungs demonstrates that NHBE ALI cultures show comparable expression of all HIV receptors. While RNA levels were significantly lower for CCR5 than CD4 and CXCR4, protein levels were comparable with CD4 and CXCR4. This could be due to inherent CCR5 mRNA instability due to a pseudoknot structure in the mRNA that promotes non-sense mediated decay [ 35 ]. Panel C : To determine if bronchial epithelial cells express HIV receptors and co-receptors in vivo, Bronchial brushings were analyzed for expression of CD4, CCR5 and CXCR4 by qRT-PCR. Expression patterns of the three receptors were similar to that observed in our ex vivo model with maximal expression of CD4 followed by CXCR4 and significantly lower CCR5 expression validating the physiological relevance of our ex vivo NHBE ALI culture model. Panel D and E : NHBE ALI cultures can be infected with both R5 and X4-tropic strains of HIV. NHBE cultures redifferentiated at the Air-Liquid Interface were infected apically and basolaterally with either HIV IIIB (X4-tropic) or HIV BaL (R5-tropic) strains. After 16 hours cells were washed apically and basolaterally with PBS four times to remove any residual input virus. The fourth wash was collected for p24 analysis and measured as Day 0 to confirm that all input virus had been removed. No p24 was detected in the 4 th wash (Day 0) confirming that all input virus had been removed. We observe an initial spike in p24 output on Day 3 for both HIV IIIB and HIV BaL infections that declines gradually till Day 9 (panel D). Experiments were terminated and total RNA was isolated and cell associated HIV RNA was quantitated by qRT-PCR using Taqman probe (Applied Biosystems # PA03453409-s1). NHBE cells demonstrate cell associated viral RNA for both R5 and X4-tropic infections (panel E). Panel F : Another set of NHBE ALI cultures similarly infected in presence of with maraviroc (For HIV BaL infection) or AMD3100 (for HIV IIIB infection) which was retained for the remainder of the experiment. Maravoiroc was able to block infection of NHBE ALI cultures by the R5-tropic strain HIV BaL. Likewise, AMD3100 was able to block infection by the X4-tropic strain HIV IIIB. n = NHBE ALI cultures from 3 different lungs * = significant (p

    Article Snippet: Following transfe r, blot was blocked in 5% milk then incubated in CD4 primary antibody (1: 500; Sigma-Aldrich), CXCR4 (1:1000; Thermo scientific) or CCR5 (1:1000; Thermo scientific).

    Techniques: Infection, Expressing, Quantitative RT-PCR, Isolation, Western Blot, In Vivo, Ex Vivo, Blocking Assay

    Postinfection treatment of HIV-1-infected Jurkat cells with NB-ZSG1 VLPs inhibits HIV-1 gene expression. (A) Jurkat cells were infected with HIV-1 and cultured for 30 days. The infected cells were transduced with VLPs conveying NB-ZSG1 or ZSG1 so that > 95% of the cells were positive for ZSG1 or NB-SZG1 (three successive transductions [TD1, TD2, and TD3], as shown). HIV-1 CA in supernatant from HIV-1-infected Jurkat cells or NB-ZSG1-treated or ZSG1-treated, HIV-1-infected Jurkat cells was quantified by ELISA (left y axis, solid lines) or NB-ZSG1 and ZSG1 expression level in cells by flow cytometry (right y axis, dotted lines). An experiment representative of at least three independent experiments with similar results is shown. (B) Western blot analysis of the cell lysates from panel A with anti-Tat and anti-β-tubulin antibodies. The molecular masses of the NB-ZSG1 fusion protein and β-tubulin are indicated. (C) Total cellular RNA was isolated from HIV-1-infected Jurkat; NB-ZSG1-treated or ZSG1-treated, HIV-1-infected Jurkat; or uninfected Jurkat cells. qRT-PCR assays were performed with primers specific for the 5′ UTR or for sequences in env . The relative level of viral 5′ UTR RNA in each sample was normalized to the amount of GAPDH mRNA in the sample. (D) Total cellular DNA was isolated and subjected to Alu -PCR analysis for the cell lines. In panels C and D, mean values and standard deviations from experiments representative of three independent experiments performed in triplicate are shown. A two-tailed Student t test with equal variance was used to calculate the P value. (E) PMA stimulation of HIV-1 gene expression in an NB-ZSG1-treated, HIV-1-infected Jurkat cell population. Uninfected Jurkat cells, HIV-1-infected Jurkat cells treated with ZSG1 or NB-ZSG1, and U1 cells were incubated with 1 nM PMA for 24 h, and the concentration of CA in the supernatant was measured by ELISA. The experiment was performed in triplicate and repeated three times with similar results. The mean values and standard deviations of a representative experiment are shown.

    Journal: mBio

    Article Title: Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein

    doi: 10.1128/mBio.00518-16

    Figure Lengend Snippet: Postinfection treatment of HIV-1-infected Jurkat cells with NB-ZSG1 VLPs inhibits HIV-1 gene expression. (A) Jurkat cells were infected with HIV-1 and cultured for 30 days. The infected cells were transduced with VLPs conveying NB-ZSG1 or ZSG1 so that > 95% of the cells were positive for ZSG1 or NB-SZG1 (three successive transductions [TD1, TD2, and TD3], as shown). HIV-1 CA in supernatant from HIV-1-infected Jurkat cells or NB-ZSG1-treated or ZSG1-treated, HIV-1-infected Jurkat cells was quantified by ELISA (left y axis, solid lines) or NB-ZSG1 and ZSG1 expression level in cells by flow cytometry (right y axis, dotted lines). An experiment representative of at least three independent experiments with similar results is shown. (B) Western blot analysis of the cell lysates from panel A with anti-Tat and anti-β-tubulin antibodies. The molecular masses of the NB-ZSG1 fusion protein and β-tubulin are indicated. (C) Total cellular RNA was isolated from HIV-1-infected Jurkat; NB-ZSG1-treated or ZSG1-treated, HIV-1-infected Jurkat; or uninfected Jurkat cells. qRT-PCR assays were performed with primers specific for the 5′ UTR or for sequences in env . The relative level of viral 5′ UTR RNA in each sample was normalized to the amount of GAPDH mRNA in the sample. (D) Total cellular DNA was isolated and subjected to Alu -PCR analysis for the cell lines. In panels C and D, mean values and standard deviations from experiments representative of three independent experiments performed in triplicate are shown. A two-tailed Student t test with equal variance was used to calculate the P value. (E) PMA stimulation of HIV-1 gene expression in an NB-ZSG1-treated, HIV-1-infected Jurkat cell population. Uninfected Jurkat cells, HIV-1-infected Jurkat cells treated with ZSG1 or NB-ZSG1, and U1 cells were incubated with 1 nM PMA for 24 h, and the concentration of CA in the supernatant was measured by ELISA. The experiment was performed in triplicate and repeated three times with similar results. The mean values and standard deviations of a representative experiment are shown.

    Article Snippet: All RNA samples were treated with Turbo DNase I and used in a PCR to confirm the removal of any contaminating HIV-1 DNA. cDNA was synthesized with 500 ng of total RNA, random hexamer primers, and Superscript III reverse transcriptase (Thermo Fisher, Waltham, MA) in accordance with the manufacturer’s instructions.

    Techniques: Infection, Expressing, Cell Culture, Transduction, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Western Blot, Isolation, Quantitative RT-PCR, Polymerase Chain Reaction, Two Tailed Test, Incubation, Concentration Assay

    ChIP assays for a DNA-protein complex in HIV-1-infected Jurkat cells treated with ZSG1 or NB-ZSG1. (A) Schematic of the HIV-1 LTR promoter from −200 to +200. The binding sites for NF-κB, SP1, and the TATA binding protein (TBP) and the location of nuc-1 are indicated. Viral DNA in the U5 region was detected with primers specific for HIV NL43 (indicated by arrowheads). (B) ChIP assays were performed with an anti-RNAPII or anti-H3K9ac antibody and primers for nuc-1 . The gene for GAPDH was used as a reference. The relative enrichment of viral DNA in the immunoprecipitation reaction was calculated as a signal over the background with the same DNA-protein complex and an isotype-matched anti-GFP antibody. The experiments were performed three times, and the mean values and stand deviations are shown. The P values were calculated with a Student t test.

    Journal: mBio

    Article Title: Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein

    doi: 10.1128/mBio.00518-16

    Figure Lengend Snippet: ChIP assays for a DNA-protein complex in HIV-1-infected Jurkat cells treated with ZSG1 or NB-ZSG1. (A) Schematic of the HIV-1 LTR promoter from −200 to +200. The binding sites for NF-κB, SP1, and the TATA binding protein (TBP) and the location of nuc-1 are indicated. Viral DNA in the U5 region was detected with primers specific for HIV NL43 (indicated by arrowheads). (B) ChIP assays were performed with an anti-RNAPII or anti-H3K9ac antibody and primers for nuc-1 . The gene for GAPDH was used as a reference. The relative enrichment of viral DNA in the immunoprecipitation reaction was calculated as a signal over the background with the same DNA-protein complex and an isotype-matched anti-GFP antibody. The experiments were performed three times, and the mean values and stand deviations are shown. The P values were calculated with a Student t test.

    Article Snippet: All RNA samples were treated with Turbo DNase I and used in a PCR to confirm the removal of any contaminating HIV-1 DNA. cDNA was synthesized with 500 ng of total RNA, random hexamer primers, and Superscript III reverse transcriptase (Thermo Fisher, Waltham, MA) in accordance with the manufacturer’s instructions.

    Techniques: Chromatin Immunoprecipitation, Infection, Binding Assay, Immunoprecipitation

    Jurkat-NB-ZSG1 cells harbor proviral HIV-1 DNA but no detectable viral mRNA. (A) PCR amplification of an HIV-1 env gene segment with total cellular DNA obtained from uninfected Jurkat, Jurkat-ZSG1, and Jurkat-NB-ZSG1 cells (lane 1) or from the same cell lines incubated with heat-inactivated (H.I.) virus (lane 2) or treated with or without nevirapine (NVP) for 2 h and infected with HIV-1 NL4.3 (lanes 3 and 4). A PCR master mix alone was used as a negative (Neg.) control (lane 5) or with proviral DNA added as a positive (Pos.) control (Ctrl.) (lane 6). (B) Total cellular DNA obtained on days 28 and 64 from HIV-1-infected cell lines and assayed by endpoint PCR for env DNA. No viral DNA was detected in uninfected cells processed simultaneously (lanes 4 to 6). Negative and positive controls as in panel A are shown (lanes 7 and 8). (C) Total cellular DNA from day 3 HIV-1-infected or uninfected Jurkat-NB-ZSG1 and Jurkat-ZSG1 cells was assayed by Alu -PCR for integrated proviral DNA. The relative copy number was normalized to the CCR5 gene level in each sample. (D) On the days indicated, 1 nM PMA or DMSO (carrier) was added to Jurkat-NB-ZSG1 and Jurkat-ZSG1 cell cultures and incubated for 24 h. The soluble CA in the culture supernatant was then quantified by ELISA (the dotted line shows the limit of detection). ACH2 cell were used as a control for activation of HIV-1 gene expression by PMA. (E) Coculture of HIV-1-infected Jurkat-NB-ZSG1 or Jurkat-ZSG1 cells with uninfected Jurkat cells at a ratio of 1:1 or 1:100, respectively, for 28 days. The CA level in the supernatant was assayed by ELISA. (F) JLat 6.3, JLat 6.3-ZSG1, and JLat 6.3-NB-ZSG1 cells were incubated with 10 nM PMA for 24 h, and the CA levels in the supernatants were assayed by ELISA (the dotted line shows the limit of detection). The GFP-positive cell population was measured by flow cytometry. The assays in panels E and F were performed three times in triplicate, and the mean values and standard deviations are shown. Experiments representative of at least three independent experiments with similar results are shown. The P value in panel C was calculated with a Student t test.

    Journal: mBio

    Article Title: Shutdown of HIV-1 Transcription in T Cells by Nullbasic, a Mutant Tat Protein

    doi: 10.1128/mBio.00518-16

    Figure Lengend Snippet: Jurkat-NB-ZSG1 cells harbor proviral HIV-1 DNA but no detectable viral mRNA. (A) PCR amplification of an HIV-1 env gene segment with total cellular DNA obtained from uninfected Jurkat, Jurkat-ZSG1, and Jurkat-NB-ZSG1 cells (lane 1) or from the same cell lines incubated with heat-inactivated (H.I.) virus (lane 2) or treated with or without nevirapine (NVP) for 2 h and infected with HIV-1 NL4.3 (lanes 3 and 4). A PCR master mix alone was used as a negative (Neg.) control (lane 5) or with proviral DNA added as a positive (Pos.) control (Ctrl.) (lane 6). (B) Total cellular DNA obtained on days 28 and 64 from HIV-1-infected cell lines and assayed by endpoint PCR for env DNA. No viral DNA was detected in uninfected cells processed simultaneously (lanes 4 to 6). Negative and positive controls as in panel A are shown (lanes 7 and 8). (C) Total cellular DNA from day 3 HIV-1-infected or uninfected Jurkat-NB-ZSG1 and Jurkat-ZSG1 cells was assayed by Alu -PCR for integrated proviral DNA. The relative copy number was normalized to the CCR5 gene level in each sample. (D) On the days indicated, 1 nM PMA or DMSO (carrier) was added to Jurkat-NB-ZSG1 and Jurkat-ZSG1 cell cultures and incubated for 24 h. The soluble CA in the culture supernatant was then quantified by ELISA (the dotted line shows the limit of detection). ACH2 cell were used as a control for activation of HIV-1 gene expression by PMA. (E) Coculture of HIV-1-infected Jurkat-NB-ZSG1 or Jurkat-ZSG1 cells with uninfected Jurkat cells at a ratio of 1:1 or 1:100, respectively, for 28 days. The CA level in the supernatant was assayed by ELISA. (F) JLat 6.3, JLat 6.3-ZSG1, and JLat 6.3-NB-ZSG1 cells were incubated with 10 nM PMA for 24 h, and the CA levels in the supernatants were assayed by ELISA (the dotted line shows the limit of detection). The GFP-positive cell population was measured by flow cytometry. The assays in panels E and F were performed three times in triplicate, and the mean values and standard deviations are shown. Experiments representative of at least three independent experiments with similar results are shown. The P value in panel C was calculated with a Student t test.

    Article Snippet: All RNA samples were treated with Turbo DNase I and used in a PCR to confirm the removal of any contaminating HIV-1 DNA. cDNA was synthesized with 500 ng of total RNA, random hexamer primers, and Superscript III reverse transcriptase (Thermo Fisher, Waltham, MA) in accordance with the manufacturer’s instructions.

    Techniques: Polymerase Chain Reaction, Amplification, Incubation, Infection, Enzyme-linked Immunosorbent Assay, Activation Assay, Expressing, Flow Cytometry, Cytometry