Structured Review

Santa Cruz Biotechnology hsv 1 infection
A (upper panel). Polarized tonsil epithelial cells were treated with active or inactive recombinant HIV tat and gp120 in combination for 5 days, and TER was then measured. A (lower panel). The same cells were used to evaluate paracellular permeability after 5 days of treatment, as determined by leakage of IgG (Fab’) 2 from the apical chamber to the basolateral chamber. OD, optical density. B. The same cells were immunostained for ZO-1 (green). Cell nuclei are stained in blue. C. <t>HSV-1</t> at an MOI of 10 PFU per cell was added to the upper chamber of polarized cells, and culture medium was collected from the basolateral chamber after 1, 2, or 4 h. HSV-1 paracellular spread was confirmed by detection of ICP4 protein in Vero cells (green) 4 h after infection. Cell nuclei were stained with propidium iodide (red). Yellow indicates colocalization of ICP4 with the nuclear marker. D. HSV-1 paracellular spread was quantified by counting of HSV-1-infected Vero cells in 10 random microscopic fields and determining the percentage of cells positive for ICP4. A, D: Error bars indicate SEM (n = 3).
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1) Product Images from "HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread"

Article Title: HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

Journal: PLoS ONE

doi: 10.1371/journal.pone.0088803

A (upper panel). Polarized tonsil epithelial cells were treated with active or inactive recombinant HIV tat and gp120 in combination for 5 days, and TER was then measured. A (lower panel). The same cells were used to evaluate paracellular permeability after 5 days of treatment, as determined by leakage of IgG (Fab’) 2 from the apical chamber to the basolateral chamber. OD, optical density. B. The same cells were immunostained for ZO-1 (green). Cell nuclei are stained in blue. C. HSV-1 at an MOI of 10 PFU per cell was added to the upper chamber of polarized cells, and culture medium was collected from the basolateral chamber after 1, 2, or 4 h. HSV-1 paracellular spread was confirmed by detection of ICP4 protein in Vero cells (green) 4 h after infection. Cell nuclei were stained with propidium iodide (red). Yellow indicates colocalization of ICP4 with the nuclear marker. D. HSV-1 paracellular spread was quantified by counting of HSV-1-infected Vero cells in 10 random microscopic fields and determining the percentage of cells positive for ICP4. A, D: Error bars indicate SEM (n = 3).
Figure Legend Snippet: A (upper panel). Polarized tonsil epithelial cells were treated with active or inactive recombinant HIV tat and gp120 in combination for 5 days, and TER was then measured. A (lower panel). The same cells were used to evaluate paracellular permeability after 5 days of treatment, as determined by leakage of IgG (Fab’) 2 from the apical chamber to the basolateral chamber. OD, optical density. B. The same cells were immunostained for ZO-1 (green). Cell nuclei are stained in blue. C. HSV-1 at an MOI of 10 PFU per cell was added to the upper chamber of polarized cells, and culture medium was collected from the basolateral chamber after 1, 2, or 4 h. HSV-1 paracellular spread was confirmed by detection of ICP4 protein in Vero cells (green) 4 h after infection. Cell nuclei were stained with propidium iodide (red). Yellow indicates colocalization of ICP4 with the nuclear marker. D. HSV-1 paracellular spread was quantified by counting of HSV-1-infected Vero cells in 10 random microscopic fields and determining the percentage of cells positive for ICP4. A, D: Error bars indicate SEM (n = 3).

Techniques Used: Recombinant, Permeability, Staining, Infection, Marker

A. Polarized tonsil epithelial cells were incubated with dual X4- and R5-tropic HIV-1 SF33 for 5 days. One set of cells was exposed to UV-inactivated virions. Culture medium was changed daily to add fresh virus, and TER was measured. B. HSV-1 was added to the apical surface of polarized cells upon complete disruption of TJs at 5 days. HSV paracellular spread at 1, 2, and 4 h after incubation was examined in Vero cells grown in the basolateral chamber of filter inserts by immunostaining of ICP4 protein. HSV-1 paracellular spread was quantified by counting HSV-1-infected Vero cells, and the percentage of cells positive for ICP4 was determined. A, B: Error bars indicate SEM (n = 3).
Figure Legend Snippet: A. Polarized tonsil epithelial cells were incubated with dual X4- and R5-tropic HIV-1 SF33 for 5 days. One set of cells was exposed to UV-inactivated virions. Culture medium was changed daily to add fresh virus, and TER was measured. B. HSV-1 was added to the apical surface of polarized cells upon complete disruption of TJs at 5 days. HSV paracellular spread at 1, 2, and 4 h after incubation was examined in Vero cells grown in the basolateral chamber of filter inserts by immunostaining of ICP4 protein. HSV-1 paracellular spread was quantified by counting HSV-1-infected Vero cells, and the percentage of cells positive for ICP4 was determined. A, B: Error bars indicate SEM (n = 3).

Techniques Used: Incubation, Immunostaining, Infection

A. Polarized tonsil epithelial cells were coimmunostained for nectin-1 and E-cadherin. Yellow in the merged panel indicates colocalization of nectin-1 and E-cadherin. B. Polarized tonsil cells were treated with active or inactive tat and gp120 in combination for 5 days. In parallel experiments, cells were exposed to HIV-1 SF33 for 5 days. Cells were then immunostained for E-cadherin and nectin-1. C. Polarized cells were treated with active or inactive tat and gp120 in combination or with cell-free HIV-1 SF33 for 5 days. Cells were then extracted, and E-cadherin and nectin-1 were detected by Western blot assay. D. Apical or basolateral membranes of polarized epithelial cells were treated with inactive or active HIV tat and labeled with sulfo-NHS-LC-biotin. Nectin-1 was detected in the avidin-precipitated total membrane proteins by Western blot assay. E. HSV-1 gD(306t) at 20 µg/ml was added to apical or basolateral membranes of polarized epithelial cells treated with inactive or active HIV tat/gp120. After 30 min the cell surface was labeled with sulfo-NHS-LC-biotin. Proteins biotinylated at the cell surface were precipitated with streptavidin–agarose beads, and gD was detected by Western blot assay. AP, apical; BL, basolateral.
Figure Legend Snippet: A. Polarized tonsil epithelial cells were coimmunostained for nectin-1 and E-cadherin. Yellow in the merged panel indicates colocalization of nectin-1 and E-cadherin. B. Polarized tonsil cells were treated with active or inactive tat and gp120 in combination for 5 days. In parallel experiments, cells were exposed to HIV-1 SF33 for 5 days. Cells were then immunostained for E-cadherin and nectin-1. C. Polarized cells were treated with active or inactive tat and gp120 in combination or with cell-free HIV-1 SF33 for 5 days. Cells were then extracted, and E-cadherin and nectin-1 were detected by Western blot assay. D. Apical or basolateral membranes of polarized epithelial cells were treated with inactive or active HIV tat and labeled with sulfo-NHS-LC-biotin. Nectin-1 was detected in the avidin-precipitated total membrane proteins by Western blot assay. E. HSV-1 gD(306t) at 20 µg/ml was added to apical or basolateral membranes of polarized epithelial cells treated with inactive or active HIV tat/gp120. After 30 min the cell surface was labeled with sulfo-NHS-LC-biotin. Proteins biotinylated at the cell surface were precipitated with streptavidin–agarose beads, and gD was detected by Western blot assay. AP, apical; BL, basolateral.

Techniques Used: Western Blot, Labeling, Avidin-Biotin Assay

A. Polarized tonsil cells were treated with HIV tat/gp120 or HIV virions for 5 days and infected with HSV-1. After 24 h, cells were fixed and immunostained using anti-HSV-1 gB antibodies (red). Cell nuclei are stained in blue. B. HSV-1 infection was quantitatively evaluated, and the percentage of cells positive for gB was determined. Error bars indicate SEM. C. Cells were incubated with antibodies against nectin-1 for 1 h and then infected with HSV-1. Cells were fixed after 24 h, HSV-1 infection was confirmed by detection of goat anti-HSV-1 immune serum, and the number of infected cells was counted. ab, cells incubated with antibodies. c, control cells without antibodies. Error bars indicate SEM. *P<0.01, **P<0.001, all compared with the control group.
Figure Legend Snippet: A. Polarized tonsil cells were treated with HIV tat/gp120 or HIV virions for 5 days and infected with HSV-1. After 24 h, cells were fixed and immunostained using anti-HSV-1 gB antibodies (red). Cell nuclei are stained in blue. B. HSV-1 infection was quantitatively evaluated, and the percentage of cells positive for gB was determined. Error bars indicate SEM. C. Cells were incubated with antibodies against nectin-1 for 1 h and then infected with HSV-1. Cells were fixed after 24 h, HSV-1 infection was confirmed by detection of goat anti-HSV-1 immune serum, and the number of infected cells was counted. ab, cells incubated with antibodies. c, control cells without antibodies. Error bars indicate SEM. *P<0.01, **P<0.001, all compared with the control group.

Techniques Used: Infection, Staining, Incubation

A. Polarized tonsil cells were treated with active or inactive tat/gp120 and HIV virions for 5 days. Disrupted cells were infected with HSV-1 at 0.01 PFU per cell from basolateral membranes of polarized cells. After 3 days, cells were fixed and immunostained using goat anti-HSV immune serum (green). Cell nuclei are stained in red. Yellow represents colocalization of HSV proteins and nuclei. B. (upper panel) Plaque numbers were counted from 3 independent filter inserts and data are presented as the average number of HSV-infected plaques per insert. (lower panel) Cell-to-cell spread of HSV-1 was quantitatively evaluated by counting HSV-infected cells in the plaques. Results are presented as the average number of HSV-infected cells per plaque. Error bars indicate SEM. C. Polarized cells were infected with HSV-1. After 4 h, antibodies to nectin-1 and gD were added separately and in combination. Cell medium was changed daily to add fresh antibodies. Cells were fixed and immunostained for HSV-1, and the plaque numbers (upper panel) and the number of HSV-1-positive cells in plaques were counted (lower panel). Error bars indicate SEM. *P<0.05, *P<0.01, **P<0.001, all compared with the control group.
Figure Legend Snippet: A. Polarized tonsil cells were treated with active or inactive tat/gp120 and HIV virions for 5 days. Disrupted cells were infected with HSV-1 at 0.01 PFU per cell from basolateral membranes of polarized cells. After 3 days, cells were fixed and immunostained using goat anti-HSV immune serum (green). Cell nuclei are stained in red. Yellow represents colocalization of HSV proteins and nuclei. B. (upper panel) Plaque numbers were counted from 3 independent filter inserts and data are presented as the average number of HSV-infected plaques per insert. (lower panel) Cell-to-cell spread of HSV-1 was quantitatively evaluated by counting HSV-infected cells in the plaques. Results are presented as the average number of HSV-infected cells per plaque. Error bars indicate SEM. C. Polarized cells were infected with HSV-1. After 4 h, antibodies to nectin-1 and gD were added separately and in combination. Cell medium was changed daily to add fresh antibodies. Cells were fixed and immunostained for HSV-1, and the plaque numbers (upper panel) and the number of HSV-1-positive cells in plaques were counted (lower panel). Error bars indicate SEM. *P<0.05, *P<0.01, **P<0.001, all compared with the control group.

Techniques Used: Infection, Staining

The oral mucosal epithelium consists of stratified squamous epithelial cells. Each layer of epithelial cells forms lateral intercellular junctional complexes, including AJs and TJs. HSV-1 gD receptor nectin-1 is sequestered within the intact AJs area of lateral membranes of epithelial cells (left panel). HIV-induced disruption of AJs exposes nectin-1 from its sequestered areas (right panel), which binds to HSV gD and thereby promotes HSV infection and cell-to-cell spread within the oral epithelium. HIV-induced disruption of TJs leads to paracellular spread of HSV virions, which may facilitate penetration of virus from the apical to the basolateral direction into the deeper part of the epithelium, and from the basolateral to the apical direction leading to release of virus into saliva. Thus, HIV-induced disruption of epithelial junctions may facilitate the spread of HSV-1 infection within the mucosal epithelium, leading to the rapid progression of HSV-mediated mucosal lesions and ulcers.
Figure Legend Snippet: The oral mucosal epithelium consists of stratified squamous epithelial cells. Each layer of epithelial cells forms lateral intercellular junctional complexes, including AJs and TJs. HSV-1 gD receptor nectin-1 is sequestered within the intact AJs area of lateral membranes of epithelial cells (left panel). HIV-induced disruption of AJs exposes nectin-1 from its sequestered areas (right panel), which binds to HSV gD and thereby promotes HSV infection and cell-to-cell spread within the oral epithelium. HIV-induced disruption of TJs leads to paracellular spread of HSV virions, which may facilitate penetration of virus from the apical to the basolateral direction into the deeper part of the epithelium, and from the basolateral to the apical direction leading to release of virus into saliva. Thus, HIV-induced disruption of epithelial junctions may facilitate the spread of HSV-1 infection within the mucosal epithelium, leading to the rapid progression of HSV-mediated mucosal lesions and ulcers.

Techniques Used: Infection


Structured Review

Santa Cruz Biotechnology hsv
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Structured Review

Santa Cruz Biotechnology hsv
Herpesvirus ribonucleotide reductases conservation. (A) Amino acid sequences from ribonucleotide reductase large subunits were aligned using multiple-sequence comparison by log expectation (MUSCLE), and phylogeny was constructed using a neighbor-joining tree without distance corrections and scaled for equal branch lengths. Shaded boxes indicate herpesvirus subfamilies, which group closely to established phylogenetic trees. Protein names for human herpesvirus ribonucleotide reductase large and small subunits are shown on the right. (B) Schematic of representative RNR large subunit polypeptides from alpha-, beta-, and gammaherpesviruses with conserved core sequences (colored) and unique N- and C-terminal extensions (gray). The diagram is approximately to scale, with an ∼190-amino-acid (aa) portion of <t>HSV-1</t> ICP6 omitted to fit the figure.
Hsv, supplied by Santa Cruz Biotechnology, 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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Images

1) Product Images from "A Conserved Mechanism of APOBEC3 Relocalization by Herpesviral Ribonucleotide Reductase Large Subunits"

Article Title: A Conserved Mechanism of APOBEC3 Relocalization by Herpesviral Ribonucleotide Reductase Large Subunits

Journal: Journal of Virology

doi: 10.1128/JVI.01539-19

Herpesvirus ribonucleotide reductases conservation. (A) Amino acid sequences from ribonucleotide reductase large subunits were aligned using multiple-sequence comparison by log expectation (MUSCLE), and phylogeny was constructed using a neighbor-joining tree without distance corrections and scaled for equal branch lengths. Shaded boxes indicate herpesvirus subfamilies, which group closely to established phylogenetic trees. Protein names for human herpesvirus ribonucleotide reductase large and small subunits are shown on the right. (B) Schematic of representative RNR large subunit polypeptides from alpha-, beta-, and gammaherpesviruses with conserved core sequences (colored) and unique N- and C-terminal extensions (gray). The diagram is approximately to scale, with an ∼190-amino-acid (aa) portion of HSV-1 ICP6 omitted to fit the figure.
Figure Legend Snippet: Herpesvirus ribonucleotide reductases conservation. (A) Amino acid sequences from ribonucleotide reductase large subunits were aligned using multiple-sequence comparison by log expectation (MUSCLE), and phylogeny was constructed using a neighbor-joining tree without distance corrections and scaled for equal branch lengths. Shaded boxes indicate herpesvirus subfamilies, which group closely to established phylogenetic trees. Protein names for human herpesvirus ribonucleotide reductase large and small subunits are shown on the right. (B) Schematic of representative RNR large subunit polypeptides from alpha-, beta-, and gammaherpesviruses with conserved core sequences (colored) and unique N- and C-terminal extensions (gray). The diagram is approximately to scale, with an ∼190-amino-acid (aa) portion of HSV-1 ICP6 omitted to fit the figure.

Techniques Used: Sequencing, Construct

HSV-1 ICP6 binds and relocalizes A3B and A3A. (A) Coimmunoprecipitation of transfected HSV-1 FLAG-ICP6 with the indicated A3-HA constructs in 293T cells. Cells were lysed at 48 h posttransfection for anti-FLAG pulldown, and the resulting proteins were analyzed by immunoblotting. EBV FLAG-BORF2 transfections with A3B and A3G were used as positive and negative co-IP controls, respectively. (B) Representative images of U2OS cells transfected with either A3-mCherry or FLAG-RNR constructs. Cells were fixed at 48 h posttransfection, permeabilized, and stained with anti-FLAG antibody and Hoechst stain. Cotransfection with A3B-mCherry and EBV FLAG-BORF2 was used as the positive control for relocalization from nuclear to cytoplasmic aggregates. A3 localization was compared in the presence and absence of HSV-1 FLAG-ICP6 cotransfection.
Figure Legend Snippet: HSV-1 ICP6 binds and relocalizes A3B and A3A. (A) Coimmunoprecipitation of transfected HSV-1 FLAG-ICP6 with the indicated A3-HA constructs in 293T cells. Cells were lysed at 48 h posttransfection for anti-FLAG pulldown, and the resulting proteins were analyzed by immunoblotting. EBV FLAG-BORF2 transfections with A3B and A3G were used as positive and negative co-IP controls, respectively. (B) Representative images of U2OS cells transfected with either A3-mCherry or FLAG-RNR constructs. Cells were fixed at 48 h posttransfection, permeabilized, and stained with anti-FLAG antibody and Hoechst stain. Cotransfection with A3B-mCherry and EBV FLAG-BORF2 was used as the positive control for relocalization from nuclear to cytoplasmic aggregates. A3 localization was compared in the presence and absence of HSV-1 FLAG-ICP6 cotransfection.

Techniques Used: Transfection, Construct, Western Blot, Co-Immunoprecipitation Assay, Staining, Cotransfection, Positive Control

HSV-1 infection relocalizes A3B and A3A. (A) Representative images of U2OS cells transfected with A3-mCherry constructs, followed by mock or HSV-1 K26GFP infection at 48 h posttransfection. Cells were fixed at 8 hpi, stained with Hoechst stain, and then imaged directly. The viral capsid protein VP26 is tagged with GFP, which marks infected cells. (B) Quantification of A3 localization patterns in U2OS cells after mock infection or infection with different HSV-1 strains. The mean fluorescence intensity of the nuclear signal was divided by that of the cytoplasmic compartment. Statistical analysis was performed using unpaired Student’s t test between the indicated groups (n.s., not significant [P > 0.01]).
Figure Legend Snippet: HSV-1 infection relocalizes A3B and A3A. (A) Representative images of U2OS cells transfected with A3-mCherry constructs, followed by mock or HSV-1 K26GFP infection at 48 h posttransfection. Cells were fixed at 8 hpi, stained with Hoechst stain, and then imaged directly. The viral capsid protein VP26 is tagged with GFP, which marks infected cells. (B) Quantification of A3 localization patterns in U2OS cells after mock infection or infection with different HSV-1 strains. The mean fluorescence intensity of the nuclear signal was divided by that of the cytoplasmic compartment. Statistical analysis was performed using unpaired Student’s t test between the indicated groups (n.s., not significant [P > 0.01]).

Techniques Used: Infection, Transfection, Construct, Staining, Fluorescence

Time course of HSV-1-mediated relocalization of A3B and A3A. Shown are representative images of U2OS cells transfected with A3-mCherry constructs, followed by mock or HSV-1 KOS1.1 infection at 48 h posttransfection. Cells were fixed at either 3, 6, 9, or 12 hpi and stained with anti-ICP27 antibody to mark infected cells and Hoechst stain to stain the nuclear compartment.
Figure Legend Snippet: Time course of HSV-1-mediated relocalization of A3B and A3A. Shown are representative images of U2OS cells transfected with A3-mCherry constructs, followed by mock or HSV-1 KOS1.1 infection at 48 h posttransfection. Cells were fixed at either 3, 6, 9, or 12 hpi and stained with anti-ICP27 antibody to mark infected cells and Hoechst stain to stain the nuclear compartment.

Techniques Used: Transfection, Construct, Infection, Staining

A3B and A3A relocalization is dependent on HSV-1 ICP6. (A) Representative images of Vero cells transfected with A3-mCherry constructs, followed by mock, wild-type HSV-1 KOS1.1, or HSV-1 KOS1.1ΔICP6 infection at 48 h posttransfection. Cells were fixed 8 h after HSV-1 infection, permeabilized, and stained with anti-ICP27 antibody to mark infected cells and Hoechst stain. (B) Representative images from an experiment similar to that described above for panel A, except using U2OS cells and the mutant virus HSV-1 KOS1.1ΔICP4.
Figure Legend Snippet: A3B and A3A relocalization is dependent on HSV-1 ICP6. (A) Representative images of Vero cells transfected with A3-mCherry constructs, followed by mock, wild-type HSV-1 KOS1.1, or HSV-1 KOS1.1ΔICP6 infection at 48 h posttransfection. Cells were fixed 8 h after HSV-1 infection, permeabilized, and stained with anti-ICP27 antibody to mark infected cells and Hoechst stain. (B) Representative images from an experiment similar to that described above for panel A, except using U2OS cells and the mutant virus HSV-1 KOS1.1ΔICP4.

Techniques Used: Transfection, Construct, Infection, Staining, Mutagenesis

A3B and A3A do not impact HSV-1 replication or plaque formation. (A) Bar plot of HSV-1 titers produced from HFF-1 cells stably transduced with the control vector or the indicated HA-tagged A3 constructs. Cells were infected in triplicate at an MOI of 0.002 PFU/cell with either HSV-1 KOS1.1 or KOSΔICP6. The infected cultures were harvested at 48 hpi, and titers were determined on Vero cells to determine the level of viral progeny production. Statistical analysis was performed using unpaired Student’s t test (n.s., not significant [P > 0.01 for all comparisons]). (B) Bar plot of KOS1.1 or KOSΔICP6 mutant stock titers determined on U2OS or Vero cells stably transduced with the control vector or the indicated HA-tagged A3 constructs. The cells were fixed at 72 hpi and stained with Giemsa stain for counting. WT, wild type.
Figure Legend Snippet: A3B and A3A do not impact HSV-1 replication or plaque formation. (A) Bar plot of HSV-1 titers produced from HFF-1 cells stably transduced with the control vector or the indicated HA-tagged A3 constructs. Cells were infected in triplicate at an MOI of 0.002 PFU/cell with either HSV-1 KOS1.1 or KOSΔICP6. The infected cultures were harvested at 48 hpi, and titers were determined on Vero cells to determine the level of viral progeny production. Statistical analysis was performed using unpaired Student’s t test (n.s., not significant [P > 0.01 for all comparisons]). (B) Bar plot of KOS1.1 or KOSΔICP6 mutant stock titers determined on U2OS or Vero cells stably transduced with the control vector or the indicated HA-tagged A3 constructs. The cells were fixed at 72 hpi and stained with Giemsa stain for counting. WT, wild type.

Techniques Used: Produced, Stable Transfection, Transduction, Plasmid Preparation, Construct, Infection, Mutagenesis, Staining, Giemsa Stain


Structured Review

Santa Cruz Biotechnology hsv
(A) Amino acid sequences from ribonucleotide reductase large subunits were aligned using Multiple Sequence Comparison by Log-Expectation (MUSCLE) and phylogeny was constructed using neighbor-joining tree without distance corrections and scaled for equal branch lengths (scale bar = 1). Shaded boxes indicate herpesvirus subfamilies, which group closely to established phylogenetic trees. Protein names for human herpesvirus ribonucleotide reductase large and small subunits shown on the right. (B) Schematic of representative RNR large subunit polypeptides from α-, β-, and γ-herpesviruses with conserved core sequences (colored) and unique N- and C-terminal extensions (gray). Diagram is approximately to scale with a ∼190 amino acid portion of <t>HSV-1</t> ICP6 omitted to fit the figure. Scale bar is 100 amino acids.
Hsv, supplied by Santa Cruz Biotechnology, 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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1) Product Images from "A Conserved Mechanism of APOBEC3 Relocalization by Herpesviral Ribonucleotide Reductase Large Subunits"

Article Title: A Conserved Mechanism of APOBEC3 Relocalization by Herpesviral Ribonucleotide Reductase Large Subunits

Journal: bioRxiv

doi: 10.1101/765735

(A) Amino acid sequences from ribonucleotide reductase large subunits were aligned using Multiple Sequence Comparison by Log-Expectation (MUSCLE) and phylogeny was constructed using neighbor-joining tree without distance corrections and scaled for equal branch lengths (scale bar = 1). Shaded boxes indicate herpesvirus subfamilies, which group closely to established phylogenetic trees. Protein names for human herpesvirus ribonucleotide reductase large and small subunits shown on the right. (B) Schematic of representative RNR large subunit polypeptides from α-, β-, and γ-herpesviruses with conserved core sequences (colored) and unique N- and C-terminal extensions (gray). Diagram is approximately to scale with a ∼190 amino acid portion of HSV-1 ICP6 omitted to fit the figure. Scale bar is 100 amino acids.
Figure Legend Snippet: (A) Amino acid sequences from ribonucleotide reductase large subunits were aligned using Multiple Sequence Comparison by Log-Expectation (MUSCLE) and phylogeny was constructed using neighbor-joining tree without distance corrections and scaled for equal branch lengths (scale bar = 1). Shaded boxes indicate herpesvirus subfamilies, which group closely to established phylogenetic trees. Protein names for human herpesvirus ribonucleotide reductase large and small subunits shown on the right. (B) Schematic of representative RNR large subunit polypeptides from α-, β-, and γ-herpesviruses with conserved core sequences (colored) and unique N- and C-terminal extensions (gray). Diagram is approximately to scale with a ∼190 amino acid portion of HSV-1 ICP6 omitted to fit the figure. Scale bar is 100 amino acids.

Techniques Used: Sequencing, Construct

( A ) Co-immunoprecipitation of transfected HSV-1 FLAG-ICP6 with the indicated A3-HA constructs in 293T cells. Cells were lysed 48 hours post-transfection for anti-FLAG pulldown and resulting proteins were analyzed by immunoblot. EBV FLAG-BORF2 transfected with A3B and A3G were used as positive and negative co-IP controls, respectively. ( B ) Representative images of U2OS cells transfected with either A3-mCherry or FLAG-RNR constructs. Cells were fixed 48 hours post-transfection, permeabilized, and stained with anti-FLAG antibody and Hoechst. Co-transfection with A3B-mCherry and EBV FLAG-BORF2 was used as positive controls for relocalization from nuclear to cytoplasmic aggregates. A3 localization was compared in the presence and absence of HSV-1 FLAG-ICP6 co-transfection.
Figure Legend Snippet: ( A ) Co-immunoprecipitation of transfected HSV-1 FLAG-ICP6 with the indicated A3-HA constructs in 293T cells. Cells were lysed 48 hours post-transfection for anti-FLAG pulldown and resulting proteins were analyzed by immunoblot. EBV FLAG-BORF2 transfected with A3B and A3G were used as positive and negative co-IP controls, respectively. ( B ) Representative images of U2OS cells transfected with either A3-mCherry or FLAG-RNR constructs. Cells were fixed 48 hours post-transfection, permeabilized, and stained with anti-FLAG antibody and Hoechst. Co-transfection with A3B-mCherry and EBV FLAG-BORF2 was used as positive controls for relocalization from nuclear to cytoplasmic aggregates. A3 localization was compared in the presence and absence of HSV-1 FLAG-ICP6 co-transfection.

Techniques Used: Immunoprecipitation, Transfection, Construct, Western Blot, Co-Immunoprecipitation Assay, Staining, Cotransfection

Representative images of U2OS cells transfected with A3-mCherry constructs, followed by mock or HSV-1 K26GFP infection 48 hours post-transfection. Cells were fixed 8 hpi and stained with Hoechst, then imaged directly. The viral capsid protein VP26 is tagged with GFP which marks infected cells.
Figure Legend Snippet: Representative images of U2OS cells transfected with A3-mCherry constructs, followed by mock or HSV-1 K26GFP infection 48 hours post-transfection. Cells were fixed 8 hpi and stained with Hoechst, then imaged directly. The viral capsid protein VP26 is tagged with GFP which marks infected cells.

Techniques Used: Transfection, Construct, Infection, Staining

(A) Representative images of Vero cells transfected with A3-mCherry constructs, followed by mock, wild-type HSV-1 KOS1.1, or HSV-1 KOS1.1ΔICP6 infection 48 hours post-transfection. Cells were fixed 8 hours after HSV-1 infection, permeabilized, and stained with anti-ICP27 antibody to mark infected cells and Hoechst. (B) Representative images from an experiment similar to that described in panel A, except using U2OS cells and the mutant virus HSV-1 KOS1.1ΔICP4.
Figure Legend Snippet: (A) Representative images of Vero cells transfected with A3-mCherry constructs, followed by mock, wild-type HSV-1 KOS1.1, or HSV-1 KOS1.1ΔICP6 infection 48 hours post-transfection. Cells were fixed 8 hours after HSV-1 infection, permeabilized, and stained with anti-ICP27 antibody to mark infected cells and Hoechst. (B) Representative images from an experiment similar to that described in panel A, except using U2OS cells and the mutant virus HSV-1 KOS1.1ΔICP4.

Techniques Used: Transfection, Construct, Infection, Staining, Mutagenesis


Structured Review

Santa Cruz Biotechnology hsv 1 infection
Hsv 1 Infection, supplied by Santa Cruz Biotechnology, 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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Structured Review

Santa Cruz Biotechnology hsv 1 infection
Bortezomib is effective when added prior to 3 h p.i. <t>HSV-1</t> KOS was added to Vero cells (MOI of 0.001). At the indicated times p.i., 200 nM bortezomib was added. At 18 to 24 h p.i., plaques were enumerated. The values representing mock-treated samples were set to 100%. Data presented are representative of results from three experiments. Error bars, SEM.
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1) Product Images from "Early Steps in Herpes Simplex Virus Infection Blocked by a Proteasome Inhibitor"

Article Title: Early Steps in Herpes Simplex Virus Infection Blocked by a Proteasome Inhibitor

Journal: mBio

doi: 10.1128/mBio.00732-19

Bortezomib is effective when added prior to 3 h p.i. HSV-1 KOS was added to Vero cells (MOI of 0.001). At the indicated times p.i., 200 nM bortezomib was added. At 18 to 24 h p.i., plaques were enumerated. The values representing mock-treated samples were set to 100%. Data presented are representative of results from three experiments. Error bars, SEM.
Figure Legend Snippet: Bortezomib is effective when added prior to 3 h p.i. HSV-1 KOS was added to Vero cells (MOI of 0.001). At the indicated times p.i., 200 nM bortezomib was added. At 18 to 24 h p.i., plaques were enumerated. The values representing mock-treated samples were set to 100%. Data presented are representative of results from three experiments. Error bars, SEM.

Techniques Used:

Bortezomib does not exhibit virucidal activity. HSV-1 KOS virions were treated with 100 nM bortezomib at 37°C for 1 h. Bortezomib was diluted to reach noninhibitory concentrations, and titers were determined on Vero cells. Data presented are representative of results from three experiments. Error bars, SEM. ns, not significant (compared to no-drug treatment).
Figure Legend Snippet: Bortezomib does not exhibit virucidal activity. HSV-1 KOS virions were treated with 100 nM bortezomib at 37°C for 1 h. Bortezomib was diluted to reach noninhibitory concentrations, and titers were determined on Vero cells. Data presented are representative of results from three experiments. Error bars, SEM. ns, not significant (compared to no-drug treatment).

Techniques Used: Activity Assay

Bortezomib does not affect HSV attachment to cells but inhibits transport of the entering capsid to the nucleus. (A) HSV-1 KOS was added to Vero cells (40 genome copies/cell) in the presence of DMSO control (No drug), 500 nM bortezomib, or 2 µg/ml heparin control. Samples were subjected to spinoculation at 200 × g at 4°C for 1 h. After three washes, cell-associated HSV levels were determined by qPCR. Data presented represent means of results from three experiments. Error bars, SEM; ns, not significant; *, P value of <0.05 (compared to no drug). (B to D) HSV-1 K26GFP was added to Vero cells on coverslips in the presence of (B) DMSO control or (C) 100 nM bortezomib or (D) 500 nM bortezomib for 2.5 h. Cells were fixed and stained with DAPI nuclear stain and visualized. Data presented are representative of results from at least two experiments.
Figure Legend Snippet: Bortezomib does not affect HSV attachment to cells but inhibits transport of the entering capsid to the nucleus. (A) HSV-1 KOS was added to Vero cells (40 genome copies/cell) in the presence of DMSO control (No drug), 500 nM bortezomib, or 2 µg/ml heparin control. Samples were subjected to spinoculation at 200 × g at 4°C for 1 h. After three washes, cell-associated HSV levels were determined by qPCR. Data presented represent means of results from three experiments. Error bars, SEM; ns, not significant; *, P value of <0.05 (compared to no drug). (B to D) HSV-1 K26GFP was added to Vero cells on coverslips in the presence of (B) DMSO control or (C) 100 nM bortezomib or (D) 500 nM bortezomib for 2.5 h. Cells were fixed and stained with DAPI nuclear stain and visualized. Data presented are representative of results from at least two experiments.

Techniques Used: Staining

Bortezomib prevents virus-induced ND10 disruption. Vero cells were pretreated with (A to D) DMSO control or (E to H) 200 nM bortezomib or (I to L) 500 nM bortezomib for 15 to 18 min at 37°C. HSV-1 KOS was added to Vero cells (MOI of ∼0.8) for 6 h at 37°C in the continued presence of agent. Cells were fixed, permeabilized, and stained for PML and ICP4. Panels D, H, and L represent zoomed-out views to show more of the surrounding cells. The ICP4 staining results were consistent with ICP4 expressed by the infected cell . Data presented are representative of results from three experiments.
Figure Legend Snippet: Bortezomib prevents virus-induced ND10 disruption. Vero cells were pretreated with (A to D) DMSO control or (E to H) 200 nM bortezomib or (I to L) 500 nM bortezomib for 15 to 18 min at 37°C. HSV-1 KOS was added to Vero cells (MOI of ∼0.8) for 6 h at 37°C in the continued presence of agent. Cells were fixed, permeabilized, and stained for PML and ICP4. Panels D, H, and L represent zoomed-out views to show more of the surrounding cells. The ICP4 staining results were consistent with ICP4 expressed by the infected cell . Data presented are representative of results from three experiments.

Techniques Used: Staining, Infection

Bortezomib and acyclovir act synergistically to inhibit HSV infection. HSV-1 KOS was added to Vero cells (MOI of 0.1) in the presence of various combinations of acyclovir and bortezomib. At 24 h p.i., cells were fixed, and titers were determined on Vero cells. (A) 3D graph depicting viral titers at the various drug combinations. (B) Isobologram depicting synergistic profiles of bortezomib and acyclovir. “Fa” (fraction affected) refers to fraction inhibition. Each colored line depicts a certain level of fraction inhibition, with endpoints signifying the amount of each drug alone needed to achieve that amount of inhibition. Colored symbols signify how much of each of the two drugs working together is needed to achieve the same inhibition. Symbols below the respective colored lines indicate synergism, those on or near the respective lines indicate additivity, and those above the respective lines indicate antagonism. Data presented are representative of results from three experiments. (C) Software-determined CI values at the specified fractions affected (Fa). Here, “Fa” refers to inhibition of HSV-1 plaque formation (fraction of control). Data are presented as means of results from three experiments ± SEM.
Figure Legend Snippet: Bortezomib and acyclovir act synergistically to inhibit HSV infection. HSV-1 KOS was added to Vero cells (MOI of 0.1) in the presence of various combinations of acyclovir and bortezomib. At 24 h p.i., cells were fixed, and titers were determined on Vero cells. (A) 3D graph depicting viral titers at the various drug combinations. (B) Isobologram depicting synergistic profiles of bortezomib and acyclovir. “Fa” (fraction affected) refers to fraction inhibition. Each colored line depicts a certain level of fraction inhibition, with endpoints signifying the amount of each drug alone needed to achieve that amount of inhibition. Colored symbols signify how much of each of the two drugs working together is needed to achieve the same inhibition. Symbols below the respective colored lines indicate synergism, those on or near the respective lines indicate additivity, and those above the respective lines indicate antagonism. Data presented are representative of results from three experiments. (C) Software-determined CI values at the specified fractions affected (Fa). Here, “Fa” refers to inhibition of HSV-1 plaque formation (fraction of control). Data are presented as means of results from three experiments ± SEM.

Techniques Used: Infection, Inhibition, Software


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Santa Cruz Biotechnology hsv 1 infection
<t>HSV-1</t> infection increases NEAT1 expression and paraspeckle formation. a HeLa and MEF cells were infected with HSV-1 and harvested at the indicated time points. The expression levels of NEAT1 relative to those of β-actin mRNA were determined with real-time PCR. The data were normalized to the NEAT1 level at 0 h after HSV-1 infection. b HeLa cells were infected with HSV-1 and collected at the indicated time points for western blot to analyze the expression of ICP0, P54nrb, PSPC1, STAT3, pSTAT3 Y705 and β-actin. c HeLa cells were fixed 4 h after HSV-1 infection and incubated with the NEAT1 probe ( red ). Fluorescence images were captured with a confocal microscope. Scale bars 10 μm. The number of NEAT1 puncta per cell was analyzed. * p < 0.01
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Article Title: NEAT1 modulates herpes simplex virus-1 replication by regulating viral gene transcription

Journal: Cellular and Molecular Life Sciences

doi: 10.1007/s00018-016-2398-4

HSV-1 infection increases NEAT1 expression and paraspeckle formation. a HeLa and MEF cells were infected with HSV-1 and harvested at the indicated time points. The expression levels of NEAT1 relative to those of β-actin mRNA were determined with real-time PCR. The data were normalized to the NEAT1 level at 0 h after HSV-1 infection. b HeLa cells were infected with HSV-1 and collected at the indicated time points for western blot to analyze the expression of ICP0, P54nrb, PSPC1, STAT3, pSTAT3 Y705 and β-actin. c HeLa cells were fixed 4 h after HSV-1 infection and incubated with the NEAT1 probe ( red ). Fluorescence images were captured with a confocal microscope. Scale bars 10 μm. The number of NEAT1 puncta per cell was analyzed. * p < 0.01
Figure Legend Snippet: HSV-1 infection increases NEAT1 expression and paraspeckle formation. a HeLa and MEF cells were infected with HSV-1 and harvested at the indicated time points. The expression levels of NEAT1 relative to those of β-actin mRNA were determined with real-time PCR. The data were normalized to the NEAT1 level at 0 h after HSV-1 infection. b HeLa cells were infected with HSV-1 and collected at the indicated time points for western blot to analyze the expression of ICP0, P54nrb, PSPC1, STAT3, pSTAT3 Y705 and β-actin. c HeLa cells were fixed 4 h after HSV-1 infection and incubated with the NEAT1 probe ( red ). Fluorescence images were captured with a confocal microscope. Scale bars 10 μm. The number of NEAT1 puncta per cell was analyzed. * p < 0.01

Techniques Used: Infection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Fluorescence, Microscopy

STAT3 is involved in NEAT1-expression-mediated HSV-1 infection. a HeLa cells were transfected with STAT3 siRNA or the negative control siRNA for 36 h. The levels of STAT3 and pSTAT3 Y705 were determined by western blotting. b HeLa cells transfected with STAT3 or the control siRNA were mock infected or infected with HSV-1 for 4 h. Relative NEAT1(v1 + v2) levels (compared with β-actin mRNA) were analyzed with real-time PCR. The data were normalized to the control level in mock-infected cells. c Schematic representation of the STAT3-binding site in the human NEAT1 gene. The black box shows the potential binding site, and the red characters indicate matching sequences. Two luciferase promoter reporter constructs are shown, designated Fragment 1 and Fragment 2. FP: forward primer; RP reverse primer. d Luciferase activity assay in HeLa cells transfected with STAT3 siRNA and the luciferase reporter containing either Fragment 1 or Fragment 2 of the NEAT1 gene. The data were normalized to the control level in mock-infected cells. e ChIP assays were performed with an anti-pSTAT3 Y705 antibody to determine the fold enrichment of the NEAT1 fragment by pSTAT3 Y705. NEAT1 P1 and NEAT1 P2 refer to the region that was amplified by paired primers (FP1/RP1 and FP2/RP2, respectively). NEAT1 P2 was used as a negative control. * p < 0.01, ** p < 0.05
Figure Legend Snippet: STAT3 is involved in NEAT1-expression-mediated HSV-1 infection. a HeLa cells were transfected with STAT3 siRNA or the negative control siRNA for 36 h. The levels of STAT3 and pSTAT3 Y705 were determined by western blotting. b HeLa cells transfected with STAT3 or the control siRNA were mock infected or infected with HSV-1 for 4 h. Relative NEAT1(v1 + v2) levels (compared with β-actin mRNA) were analyzed with real-time PCR. The data were normalized to the control level in mock-infected cells. c Schematic representation of the STAT3-binding site in the human NEAT1 gene. The black box shows the potential binding site, and the red characters indicate matching sequences. Two luciferase promoter reporter constructs are shown, designated Fragment 1 and Fragment 2. FP: forward primer; RP reverse primer. d Luciferase activity assay in HeLa cells transfected with STAT3 siRNA and the luciferase reporter containing either Fragment 1 or Fragment 2 of the NEAT1 gene. The data were normalized to the control level in mock-infected cells. e ChIP assays were performed with an anti-pSTAT3 Y705 antibody to determine the fold enrichment of the NEAT1 fragment by pSTAT3 Y705. NEAT1 P1 and NEAT1 P2 refer to the region that was amplified by paired primers (FP1/RP1 and FP2/RP2, respectively). NEAT1 P2 was used as a negative control. * p < 0.01, ** p < 0.05

Techniques Used: Expressing, Infection, Transfection, Negative Control, Western Blot, Real-time Polymerase Chain Reaction, Binding Assay, Luciferase, Construct, Activity Assay, Amplification

NEAT1 modulates HSV-1 replication and viral gene expression. a Analysis of NEAT1 expression in HeLa cells transfected with NEAT1 siRNA or the negative control for 36 h. b HeLa cells transfected with NEAT siRNA were infected with HSV-1 and immuno-stained with an anti-HSV-1 glycoprotein antibody ( green ). Images were captured with a confocal microscope. Nuclei were stained with DAPI ( blue ). Scale bars 80 μm. c Plaque-forming assay in HeLa cells transfected with NEAT siRNA and infected with HSV-1 at the indicated time points. d Quantification of viral DNA levels in HeLa cells transfected with NEAT1 siRNA and infected with HSV-1 at indicated time points. e The relative mRNA expression levels of ICP0 and TK relative to that of ACTB were determined in HeLa cells transfected with NEAT1 siRNA and infected with HSV-1 for 4 h. f The samples in e were subjected to western blot to analyze the expression of ICP0, TK and β-actin. g Analysis of NEAT1 expression in MEF cells transfected with NEAT1 siRNA or the negative control for 36 h. h The relative mRNA expression levels of ICP0 and TK were determined in MEF cells transfected with NEAT1 siRNA and infected with HSV-1 for 4 h. i Western blot analysis of ICP0, TK and β-actin in MEF cells transfected with NEAT1 siRNA and infected with HSV-1 for 4 h. * p < 0.01, ** p < 0.05
Figure Legend Snippet: NEAT1 modulates HSV-1 replication and viral gene expression. a Analysis of NEAT1 expression in HeLa cells transfected with NEAT1 siRNA or the negative control for 36 h. b HeLa cells transfected with NEAT siRNA were infected with HSV-1 and immuno-stained with an anti-HSV-1 glycoprotein antibody ( green ). Images were captured with a confocal microscope. Nuclei were stained with DAPI ( blue ). Scale bars 80 μm. c Plaque-forming assay in HeLa cells transfected with NEAT siRNA and infected with HSV-1 at the indicated time points. d Quantification of viral DNA levels in HeLa cells transfected with NEAT1 siRNA and infected with HSV-1 at indicated time points. e The relative mRNA expression levels of ICP0 and TK relative to that of ACTB were determined in HeLa cells transfected with NEAT1 siRNA and infected with HSV-1 for 4 h. f The samples in e were subjected to western blot to analyze the expression of ICP0, TK and β-actin. g Analysis of NEAT1 expression in MEF cells transfected with NEAT1 siRNA or the negative control for 36 h. h The relative mRNA expression levels of ICP0 and TK were determined in MEF cells transfected with NEAT1 siRNA and infected with HSV-1 for 4 h. i Western blot analysis of ICP0, TK and β-actin in MEF cells transfected with NEAT1 siRNA and infected with HSV-1 for 4 h. * p < 0.01, ** p < 0.05

Techniques Used: Expressing, Transfection, Negative Control, Infection, Staining, Microscopy, Western Blot

NEAT1, P54nrb, and PSPC1 co-localize with HSV-1 genomic DNA. The biotin-labeled HSV-1 genomic DNA probe (green) incubated with denatured ( a ) or undenatured ( b ) HeLa cells nuclei infected with HSV-1( blue ) and then incubated with the NEAT1 probe ( red ), the anti-P54nrb antibody ( red ) or anti-PSPC1 antibodies ( red ). The images were captured with a confocal microscope. The intensity plots for the red and green channels were analyzed with ImageJ software. DAPI ( blue ) was used to stain the nuclei. Scale bars 10 μm
Figure Legend Snippet: NEAT1, P54nrb, and PSPC1 co-localize with HSV-1 genomic DNA. The biotin-labeled HSV-1 genomic DNA probe (green) incubated with denatured ( a ) or undenatured ( b ) HeLa cells nuclei infected with HSV-1( blue ) and then incubated with the NEAT1 probe ( red ), the anti-P54nrb antibody ( red ) or anti-PSPC1 antibodies ( red ). The images were captured with a confocal microscope. The intensity plots for the red and green channels were analyzed with ImageJ software. DAPI ( blue ) was used to stain the nuclei. Scale bars 10 μm

Techniques Used: Labeling, Incubation, Infection, Microscopy, Software, Staining

P54nrb and PSPC1 modulate HSV-1 replication and gene expression. a HeLa cells transfected with P54nrb, PSPC1 or the control siRNA were infected with HSV-1 for 4 h. The samples were collected for western blot to analyze the expression of P54nrb, PSPC1 and β-actin. b The samples in a. were fixed and immuno-stained with an anti-HSV-1 glycoprotein antibody ( green ). Images were captured with a confocal microscope. Nuclei were stained with DAPI ( blue ). Scale bars 80 μm. c Plaque-forming assay in HeLa cells transfected with P54nrb, PSPC1 or the control siRNA and infected with HSV-1 for the indicated time points. d Western blot analysis of ICP0, TK and β-actin in HeLa cells transfected with P54nrb or PSPC1 siRNA and infected with HSV-1. e HeLa cells transfected with the indicated siRNAs were infected with HSV-1 for 4 h. The relative expression of ICP0 and TK relative to that of ACTB was measured with real-time PCR. f MEF cells transfected with the indicated siRNAs were infected with HSV-1 for 4 h. Samples were collected for western blot to analyze the expression of P54nrb, PSPC1, ICP0, and TK. g The samples in f were collected to analyze the mRNA levels of ICP0 and TK relative to that of ACTB . * p < 0.01
Figure Legend Snippet: P54nrb and PSPC1 modulate HSV-1 replication and gene expression. a HeLa cells transfected with P54nrb, PSPC1 or the control siRNA were infected with HSV-1 for 4 h. The samples were collected for western blot to analyze the expression of P54nrb, PSPC1 and β-actin. b The samples in a. were fixed and immuno-stained with an anti-HSV-1 glycoprotein antibody ( green ). Images were captured with a confocal microscope. Nuclei were stained with DAPI ( blue ). Scale bars 80 μm. c Plaque-forming assay in HeLa cells transfected with P54nrb, PSPC1 or the control siRNA and infected with HSV-1 for the indicated time points. d Western blot analysis of ICP0, TK and β-actin in HeLa cells transfected with P54nrb or PSPC1 siRNA and infected with HSV-1. e HeLa cells transfected with the indicated siRNAs were infected with HSV-1 for 4 h. The relative expression of ICP0 and TK relative to that of ACTB was measured with real-time PCR. f MEF cells transfected with the indicated siRNAs were infected with HSV-1 for 4 h. Samples were collected for western blot to analyze the expression of P54nrb, PSPC1, ICP0, and TK. g The samples in f were collected to analyze the mRNA levels of ICP0 and TK relative to that of ACTB . * p < 0.01

Techniques Used: Expressing, Transfection, Infection, Western Blot, Staining, Microscopy, Real-time Polymerase Chain Reaction

NEAT1 and paraspeckle protein components regulate viral gene transcription. Luciferase activity assay in HeLa ( a ) or MEF cells ( b ) co-transfected with NEAT siRNA and the luciferase reporter plasmid containing either the ICP0 promoter or the TK promoter. NEAT1 Fragment 2 reporter was used as the control. Luciferase activity assay in HeLa ( c ) or MEF cells ( d ) co-transfected with P54nrb or PSPC1 siRNA and a luciferase reporter plasmid containing either the ICP0 promoter or the TK promoter. The NEAT1 Fragment 2 reporter was used as the control. HeLa cells transfected with P54nrb siRNA ( e ) or PSPC1 siRNA ( f ) were infected with HSV-1 for 4 h. A ChIP assay was performed with anti-P54nrb or anti-PSPC1 antibody, and the fold enrichment of the ICP0 or TK promoter by P54nrb or PSPC1 relative to the input level was determined with real-time PCR. NEAT1 P2 refers to the region of NEAT1 Fragment 2 that was amplified by paired primers (FP2 and RP2, in Fig. b) and was used as a negative control. g HeLa cells transfected with NEAT1 siRNA were infected with HSV-1 for 4 h. ChIP assays were performed with anti-P54nrb or anti-PSPC1 antibody, and the fold enrichment of the ICP0 and TK promoters by P54nrb or PSPC1 relative to the input level was examined with real-time PCR. NEAT1 P2 refers to the region of NEAT1 Fragment 2 that was amplified by paired primers (FP2 and RP2, in Fig. b) and was used as a negative control. * p < 0.01
Figure Legend Snippet: NEAT1 and paraspeckle protein components regulate viral gene transcription. Luciferase activity assay in HeLa ( a ) or MEF cells ( b ) co-transfected with NEAT siRNA and the luciferase reporter plasmid containing either the ICP0 promoter or the TK promoter. NEAT1 Fragment 2 reporter was used as the control. Luciferase activity assay in HeLa ( c ) or MEF cells ( d ) co-transfected with P54nrb or PSPC1 siRNA and a luciferase reporter plasmid containing either the ICP0 promoter or the TK promoter. The NEAT1 Fragment 2 reporter was used as the control. HeLa cells transfected with P54nrb siRNA ( e ) or PSPC1 siRNA ( f ) were infected with HSV-1 for 4 h. A ChIP assay was performed with anti-P54nrb or anti-PSPC1 antibody, and the fold enrichment of the ICP0 or TK promoter by P54nrb or PSPC1 relative to the input level was determined with real-time PCR. NEAT1 P2 refers to the region of NEAT1 Fragment 2 that was amplified by paired primers (FP2 and RP2, in Fig. b) and was used as a negative control. g HeLa cells transfected with NEAT1 siRNA were infected with HSV-1 for 4 h. ChIP assays were performed with anti-P54nrb or anti-PSPC1 antibody, and the fold enrichment of the ICP0 and TK promoters by P54nrb or PSPC1 relative to the input level was examined with real-time PCR. NEAT1 P2 refers to the region of NEAT1 Fragment 2 that was amplified by paired primers (FP2 and RP2, in Fig. b) and was used as a negative control. * p < 0.01

Techniques Used: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction, Amplification, Negative Control

NEAT1 and paraspeckle protein components regulate STAT3-mediated viral gene expression. a HeLa cells co-transfected with FLAG-STAT3 and NEAT siRNA were infected with HSV-1. The levels of ICP0 and TK were measured with real-time PCR and western blotting. b HeLa cells co-transfected with FLAG–STAT3 and the indicated siRNAs were subjected to HSV-1 infection. The levels of ICP0 and TK were analyzed with real-time PCR and western blotting. HeLa cells were co-transfected with FLAG–STAT3 and NEAT1 siRNA ( c ) or FLAG–STAT3 and P54nrb, PSPC1 siRNAs ( d ) and luciferase reporter plasmids. Cells were infected with HSV-1 for 4 h and collected for the luciferase activity assay. The NEAT1 Fragment 2 reporter was used as the control. HeLa cells infected with NEAT1 siRNA ( e ) or PSPC1 and P54nrb siRNAs ( f ) were subjected to HSV-1 infection. Samples were collected for ChIP assays to analyze the fold enrichment of the ICP0 and TK promoters by pSTAT3 Y705 relative to the input level. NEAT1 P2 refers to the region of NEAT1 Fragment 2 that was amplified by paired primers (FP2 and RP2, in Fig. b) and was used as a negative control. g HeLa cells transfected with FLAG-STAT3 or the empty vector were infected with HSV-1 for 4 h. The cell lysates were collected and immuno-precipitated with an anti-FLAG antibody. The immuno-precipitates were subjected to western blotting analysis of FLAG, pSTAT3 Y705, PSPC1 and β-actin. h HeLa cells infected with HSV-1 were immuno-stained with PSPC1 ( green ) and pSTAT3 Y705 ( red ) and subjected to confocal analysis. The intensity plots for the red and green channels were analyzed with ImageJ software. Scale bars 10 μm. * p < 0.01
Figure Legend Snippet: NEAT1 and paraspeckle protein components regulate STAT3-mediated viral gene expression. a HeLa cells co-transfected with FLAG-STAT3 and NEAT siRNA were infected with HSV-1. The levels of ICP0 and TK were measured with real-time PCR and western blotting. b HeLa cells co-transfected with FLAG–STAT3 and the indicated siRNAs were subjected to HSV-1 infection. The levels of ICP0 and TK were analyzed with real-time PCR and western blotting. HeLa cells were co-transfected with FLAG–STAT3 and NEAT1 siRNA ( c ) or FLAG–STAT3 and P54nrb, PSPC1 siRNAs ( d ) and luciferase reporter plasmids. Cells were infected with HSV-1 for 4 h and collected for the luciferase activity assay. The NEAT1 Fragment 2 reporter was used as the control. HeLa cells infected with NEAT1 siRNA ( e ) or PSPC1 and P54nrb siRNAs ( f ) were subjected to HSV-1 infection. Samples were collected for ChIP assays to analyze the fold enrichment of the ICP0 and TK promoters by pSTAT3 Y705 relative to the input level. NEAT1 P2 refers to the region of NEAT1 Fragment 2 that was amplified by paired primers (FP2 and RP2, in Fig. b) and was used as a negative control. g HeLa cells transfected with FLAG-STAT3 or the empty vector were infected with HSV-1 for 4 h. The cell lysates were collected and immuno-precipitated with an anti-FLAG antibody. The immuno-precipitates were subjected to western blotting analysis of FLAG, pSTAT3 Y705, PSPC1 and β-actin. h HeLa cells infected with HSV-1 were immuno-stained with PSPC1 ( green ) and pSTAT3 Y705 ( red ) and subjected to confocal analysis. The intensity plots for the red and green channels were analyzed with ImageJ software. Scale bars 10 μm. * p < 0.01

Techniques Used: Expressing, Transfection, Infection, Real-time Polymerase Chain Reaction, Western Blot, Luciferase, Activity Assay, Amplification, Negative Control, Plasmid Preparation, Staining, Software

Depletion of STAT3 reduces the development of zosteriform lesions. a MEF cells transfected with STAT3 siRNA or negative control siRNA were infected with HSV-1, and the expression levels of STAT3, pSTAT3 Y705, ICP0, and TK were analyzed with western blot. b Quantification of the mRNA levels of ICP0 and TK in MEF cells transfected with STAT3 siRNA and infected with HSV-1 with real-time PCR. c Thermosensitive gel (100 μL) containing M siSTAT3-2, M siNEAT1v2, negative control, or mock was placed on the skin of C57BL/6 mice. Two days later, the skin was cut off, and the cell lysates were harvested. The relative expression of STAT3 or NEAT1 was determined with real-time PCR. d The mice that developed zosteriform lesions were treated with thermosensitive gel containing M siSTAT3-2, M siNEAT1v2 or the negative control siRNA on their skin. The zosteriform lesions were observed on days 0, 1, 3, and 6 after incubation with the gel. The relative sizes of the zosteriform lesions were quantified by measuring the widths of the zosteriform lesions at indicated time points ( e ). ** p < 0.05. f . Schematic model of the roles of NEAT1, P54nrb and PSPC1 in viral gene expression
Figure Legend Snippet: Depletion of STAT3 reduces the development of zosteriform lesions. a MEF cells transfected with STAT3 siRNA or negative control siRNA were infected with HSV-1, and the expression levels of STAT3, pSTAT3 Y705, ICP0, and TK were analyzed with western blot. b Quantification of the mRNA levels of ICP0 and TK in MEF cells transfected with STAT3 siRNA and infected with HSV-1 with real-time PCR. c Thermosensitive gel (100 μL) containing M siSTAT3-2, M siNEAT1v2, negative control, or mock was placed on the skin of C57BL/6 mice. Two days later, the skin was cut off, and the cell lysates were harvested. The relative expression of STAT3 or NEAT1 was determined with real-time PCR. d The mice that developed zosteriform lesions were treated with thermosensitive gel containing M siSTAT3-2, M siNEAT1v2 or the negative control siRNA on their skin. The zosteriform lesions were observed on days 0, 1, 3, and 6 after incubation with the gel. The relative sizes of the zosteriform lesions were quantified by measuring the widths of the zosteriform lesions at indicated time points ( e ). ** p < 0.05. f . Schematic model of the roles of NEAT1, P54nrb and PSPC1 in viral gene expression

Techniques Used: Transfection, Negative Control, Infection, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Incubation


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Santa Cruz Biotechnology hsv 1 infection
Hsv 1 Infection, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology hsv 1 infection
Hsv 1 Infection, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology hsv 1 infection
Hsv 1 Infection, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/hsv 1 infection/product/Santa Cruz Biotechnology
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    Santa Cruz Biotechnology hsv 1 infection
    A (upper panel). Polarized tonsil epithelial cells were treated with active or inactive recombinant HIV tat and gp120 in combination for 5 days, and TER was then measured. A (lower panel). The same cells were used to evaluate paracellular permeability after 5 days of treatment, as determined by leakage of IgG (Fab’) 2 from the apical chamber to the basolateral chamber. OD, optical density. B. The same cells were immunostained for ZO-1 (green). Cell nuclei are stained in blue. C. <t>HSV-1</t> at an MOI of 10 PFU per cell was added to the upper chamber of polarized cells, and culture medium was collected from the basolateral chamber after 1, 2, or 4 h. HSV-1 paracellular spread was confirmed by detection of ICP4 protein in Vero cells (green) 4 h after infection. Cell nuclei were stained with propidium iodide (red). Yellow indicates colocalization of ICP4 with the nuclear marker. D. HSV-1 paracellular spread was quantified by counting of HSV-1-infected Vero cells in 10 random microscopic fields and determining the percentage of cells positive for ICP4. A, D: Error bars indicate SEM (n = 3).
    Hsv 1 Infection, supplied by Santa Cruz Biotechnology, 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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    Santa Cruz Biotechnology hsv
    A (upper panel). Polarized tonsil epithelial cells were treated with active or inactive recombinant HIV tat and gp120 in combination for 5 days, and TER was then measured. A (lower panel). The same cells were used to evaluate paracellular permeability after 5 days of treatment, as determined by leakage of IgG (Fab’) 2 from the apical chamber to the basolateral chamber. OD, optical density. B. The same cells were immunostained for ZO-1 (green). Cell nuclei are stained in blue. C. <t>HSV-1</t> at an MOI of 10 PFU per cell was added to the upper chamber of polarized cells, and culture medium was collected from the basolateral chamber after 1, 2, or 4 h. HSV-1 paracellular spread was confirmed by detection of ICP4 protein in Vero cells (green) 4 h after infection. Cell nuclei were stained with propidium iodide (red). Yellow indicates colocalization of ICP4 with the nuclear marker. D. HSV-1 paracellular spread was quantified by counting of HSV-1-infected Vero cells in 10 random microscopic fields and determining the percentage of cells positive for ICP4. A, D: Error bars indicate SEM (n = 3).
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    A (upper panel). Polarized tonsil epithelial cells were treated with active or inactive recombinant HIV tat and gp120 in combination for 5 days, and TER was then measured. A (lower panel). The same cells were used to evaluate paracellular permeability after 5 days of treatment, as determined by leakage of IgG (Fab’) 2 from the apical chamber to the basolateral chamber. OD, optical density. B. The same cells were immunostained for ZO-1 (green). Cell nuclei are stained in blue. C. HSV-1 at an MOI of 10 PFU per cell was added to the upper chamber of polarized cells, and culture medium was collected from the basolateral chamber after 1, 2, or 4 h. HSV-1 paracellular spread was confirmed by detection of ICP4 protein in Vero cells (green) 4 h after infection. Cell nuclei were stained with propidium iodide (red). Yellow indicates colocalization of ICP4 with the nuclear marker. D. HSV-1 paracellular spread was quantified by counting of HSV-1-infected Vero cells in 10 random microscopic fields and determining the percentage of cells positive for ICP4. A, D: Error bars indicate SEM (n = 3).

    Journal: PLoS ONE

    Article Title: HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

    doi: 10.1371/journal.pone.0088803

    Figure Lengend Snippet: A (upper panel). Polarized tonsil epithelial cells were treated with active or inactive recombinant HIV tat and gp120 in combination for 5 days, and TER was then measured. A (lower panel). The same cells were used to evaluate paracellular permeability after 5 days of treatment, as determined by leakage of IgG (Fab’) 2 from the apical chamber to the basolateral chamber. OD, optical density. B. The same cells were immunostained for ZO-1 (green). Cell nuclei are stained in blue. C. HSV-1 at an MOI of 10 PFU per cell was added to the upper chamber of polarized cells, and culture medium was collected from the basolateral chamber after 1, 2, or 4 h. HSV-1 paracellular spread was confirmed by detection of ICP4 protein in Vero cells (green) 4 h after infection. Cell nuclei were stained with propidium iodide (red). Yellow indicates colocalization of ICP4 with the nuclear marker. D. HSV-1 paracellular spread was quantified by counting of HSV-1-infected Vero cells in 10 random microscopic fields and determining the percentage of cells positive for ICP4. A, D: Error bars indicate SEM (n = 3).

    Article Snippet: To determine HSV-1 infection, we immunostained infected cells with mouse antibodies against HSV-1 ICP4 and gB (both from Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit anti-gD (HSV-1) (MyBioSource, San Diego, CA), and goat anti-HSV-1/2 antiserum (AbD Serotec, Raleigh, NC).

    Techniques: Recombinant, Permeability, Staining, Infection, Marker

    A. Polarized tonsil epithelial cells were incubated with dual X4- and R5-tropic HIV-1 SF33 for 5 days. One set of cells was exposed to UV-inactivated virions. Culture medium was changed daily to add fresh virus, and TER was measured. B. HSV-1 was added to the apical surface of polarized cells upon complete disruption of TJs at 5 days. HSV paracellular spread at 1, 2, and 4 h after incubation was examined in Vero cells grown in the basolateral chamber of filter inserts by immunostaining of ICP4 protein. HSV-1 paracellular spread was quantified by counting HSV-1-infected Vero cells, and the percentage of cells positive for ICP4 was determined. A, B: Error bars indicate SEM (n = 3).

    Journal: PLoS ONE

    Article Title: HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

    doi: 10.1371/journal.pone.0088803

    Figure Lengend Snippet: A. Polarized tonsil epithelial cells were incubated with dual X4- and R5-tropic HIV-1 SF33 for 5 days. One set of cells was exposed to UV-inactivated virions. Culture medium was changed daily to add fresh virus, and TER was measured. B. HSV-1 was added to the apical surface of polarized cells upon complete disruption of TJs at 5 days. HSV paracellular spread at 1, 2, and 4 h after incubation was examined in Vero cells grown in the basolateral chamber of filter inserts by immunostaining of ICP4 protein. HSV-1 paracellular spread was quantified by counting HSV-1-infected Vero cells, and the percentage of cells positive for ICP4 was determined. A, B: Error bars indicate SEM (n = 3).

    Article Snippet: To determine HSV-1 infection, we immunostained infected cells with mouse antibodies against HSV-1 ICP4 and gB (both from Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit anti-gD (HSV-1) (MyBioSource, San Diego, CA), and goat anti-HSV-1/2 antiserum (AbD Serotec, Raleigh, NC).

    Techniques: Incubation, Immunostaining, Infection

    A. Polarized tonsil epithelial cells were coimmunostained for nectin-1 and E-cadherin. Yellow in the merged panel indicates colocalization of nectin-1 and E-cadherin. B. Polarized tonsil cells were treated with active or inactive tat and gp120 in combination for 5 days. In parallel experiments, cells were exposed to HIV-1 SF33 for 5 days. Cells were then immunostained for E-cadherin and nectin-1. C. Polarized cells were treated with active or inactive tat and gp120 in combination or with cell-free HIV-1 SF33 for 5 days. Cells were then extracted, and E-cadherin and nectin-1 were detected by Western blot assay. D. Apical or basolateral membranes of polarized epithelial cells were treated with inactive or active HIV tat and labeled with sulfo-NHS-LC-biotin. Nectin-1 was detected in the avidin-precipitated total membrane proteins by Western blot assay. E. HSV-1 gD(306t) at 20 µg/ml was added to apical or basolateral membranes of polarized epithelial cells treated with inactive or active HIV tat/gp120. After 30 min the cell surface was labeled with sulfo-NHS-LC-biotin. Proteins biotinylated at the cell surface were precipitated with streptavidin–agarose beads, and gD was detected by Western blot assay. AP, apical; BL, basolateral.

    Journal: PLoS ONE

    Article Title: HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

    doi: 10.1371/journal.pone.0088803

    Figure Lengend Snippet: A. Polarized tonsil epithelial cells were coimmunostained for nectin-1 and E-cadherin. Yellow in the merged panel indicates colocalization of nectin-1 and E-cadherin. B. Polarized tonsil cells were treated with active or inactive tat and gp120 in combination for 5 days. In parallel experiments, cells were exposed to HIV-1 SF33 for 5 days. Cells were then immunostained for E-cadherin and nectin-1. C. Polarized cells were treated with active or inactive tat and gp120 in combination or with cell-free HIV-1 SF33 for 5 days. Cells were then extracted, and E-cadherin and nectin-1 were detected by Western blot assay. D. Apical or basolateral membranes of polarized epithelial cells were treated with inactive or active HIV tat and labeled with sulfo-NHS-LC-biotin. Nectin-1 was detected in the avidin-precipitated total membrane proteins by Western blot assay. E. HSV-1 gD(306t) at 20 µg/ml was added to apical or basolateral membranes of polarized epithelial cells treated with inactive or active HIV tat/gp120. After 30 min the cell surface was labeled with sulfo-NHS-LC-biotin. Proteins biotinylated at the cell surface were precipitated with streptavidin–agarose beads, and gD was detected by Western blot assay. AP, apical; BL, basolateral.

    Article Snippet: To determine HSV-1 infection, we immunostained infected cells with mouse antibodies against HSV-1 ICP4 and gB (both from Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit anti-gD (HSV-1) (MyBioSource, San Diego, CA), and goat anti-HSV-1/2 antiserum (AbD Serotec, Raleigh, NC).

    Techniques: Western Blot, Labeling, Avidin-Biotin Assay

    A. Polarized tonsil cells were treated with HIV tat/gp120 or HIV virions for 5 days and infected with HSV-1. After 24 h, cells were fixed and immunostained using anti-HSV-1 gB antibodies (red). Cell nuclei are stained in blue. B. HSV-1 infection was quantitatively evaluated, and the percentage of cells positive for gB was determined. Error bars indicate SEM. C. Cells were incubated with antibodies against nectin-1 for 1 h and then infected with HSV-1. Cells were fixed after 24 h, HSV-1 infection was confirmed by detection of goat anti-HSV-1 immune serum, and the number of infected cells was counted. ab, cells incubated with antibodies. c, control cells without antibodies. Error bars indicate SEM. *P<0.01, **P<0.001, all compared with the control group.

    Journal: PLoS ONE

    Article Title: HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

    doi: 10.1371/journal.pone.0088803

    Figure Lengend Snippet: A. Polarized tonsil cells were treated with HIV tat/gp120 or HIV virions for 5 days and infected with HSV-1. After 24 h, cells were fixed and immunostained using anti-HSV-1 gB antibodies (red). Cell nuclei are stained in blue. B. HSV-1 infection was quantitatively evaluated, and the percentage of cells positive for gB was determined. Error bars indicate SEM. C. Cells were incubated with antibodies against nectin-1 for 1 h and then infected with HSV-1. Cells were fixed after 24 h, HSV-1 infection was confirmed by detection of goat anti-HSV-1 immune serum, and the number of infected cells was counted. ab, cells incubated with antibodies. c, control cells without antibodies. Error bars indicate SEM. *P<0.01, **P<0.001, all compared with the control group.

    Article Snippet: To determine HSV-1 infection, we immunostained infected cells with mouse antibodies against HSV-1 ICP4 and gB (both from Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit anti-gD (HSV-1) (MyBioSource, San Diego, CA), and goat anti-HSV-1/2 antiserum (AbD Serotec, Raleigh, NC).

    Techniques: Infection, Staining, Incubation

    A. Polarized tonsil cells were treated with active or inactive tat/gp120 and HIV virions for 5 days. Disrupted cells were infected with HSV-1 at 0.01 PFU per cell from basolateral membranes of polarized cells. After 3 days, cells were fixed and immunostained using goat anti-HSV immune serum (green). Cell nuclei are stained in red. Yellow represents colocalization of HSV proteins and nuclei. B. (upper panel) Plaque numbers were counted from 3 independent filter inserts and data are presented as the average number of HSV-infected plaques per insert. (lower panel) Cell-to-cell spread of HSV-1 was quantitatively evaluated by counting HSV-infected cells in the plaques. Results are presented as the average number of HSV-infected cells per plaque. Error bars indicate SEM. C. Polarized cells were infected with HSV-1. After 4 h, antibodies to nectin-1 and gD were added separately and in combination. Cell medium was changed daily to add fresh antibodies. Cells were fixed and immunostained for HSV-1, and the plaque numbers (upper panel) and the number of HSV-1-positive cells in plaques were counted (lower panel). Error bars indicate SEM. *P<0.05, *P<0.01, **P<0.001, all compared with the control group.

    Journal: PLoS ONE

    Article Title: HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

    doi: 10.1371/journal.pone.0088803

    Figure Lengend Snippet: A. Polarized tonsil cells were treated with active or inactive tat/gp120 and HIV virions for 5 days. Disrupted cells were infected with HSV-1 at 0.01 PFU per cell from basolateral membranes of polarized cells. After 3 days, cells were fixed and immunostained using goat anti-HSV immune serum (green). Cell nuclei are stained in red. Yellow represents colocalization of HSV proteins and nuclei. B. (upper panel) Plaque numbers were counted from 3 independent filter inserts and data are presented as the average number of HSV-infected plaques per insert. (lower panel) Cell-to-cell spread of HSV-1 was quantitatively evaluated by counting HSV-infected cells in the plaques. Results are presented as the average number of HSV-infected cells per plaque. Error bars indicate SEM. C. Polarized cells were infected with HSV-1. After 4 h, antibodies to nectin-1 and gD were added separately and in combination. Cell medium was changed daily to add fresh antibodies. Cells were fixed and immunostained for HSV-1, and the plaque numbers (upper panel) and the number of HSV-1-positive cells in plaques were counted (lower panel). Error bars indicate SEM. *P<0.05, *P<0.01, **P<0.001, all compared with the control group.

    Article Snippet: To determine HSV-1 infection, we immunostained infected cells with mouse antibodies against HSV-1 ICP4 and gB (both from Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit anti-gD (HSV-1) (MyBioSource, San Diego, CA), and goat anti-HSV-1/2 antiserum (AbD Serotec, Raleigh, NC).

    Techniques: Infection, Staining

    The oral mucosal epithelium consists of stratified squamous epithelial cells. Each layer of epithelial cells forms lateral intercellular junctional complexes, including AJs and TJs. HSV-1 gD receptor nectin-1 is sequestered within the intact AJs area of lateral membranes of epithelial cells (left panel). HIV-induced disruption of AJs exposes nectin-1 from its sequestered areas (right panel), which binds to HSV gD and thereby promotes HSV infection and cell-to-cell spread within the oral epithelium. HIV-induced disruption of TJs leads to paracellular spread of HSV virions, which may facilitate penetration of virus from the apical to the basolateral direction into the deeper part of the epithelium, and from the basolateral to the apical direction leading to release of virus into saliva. Thus, HIV-induced disruption of epithelial junctions may facilitate the spread of HSV-1 infection within the mucosal epithelium, leading to the rapid progression of HSV-mediated mucosal lesions and ulcers.

    Journal: PLoS ONE

    Article Title: HIV-Associated Disruption of Tight and Adherens Junctions of Oral Epithelial Cells Facilitates HSV-1 Infection and Spread

    doi: 10.1371/journal.pone.0088803

    Figure Lengend Snippet: The oral mucosal epithelium consists of stratified squamous epithelial cells. Each layer of epithelial cells forms lateral intercellular junctional complexes, including AJs and TJs. HSV-1 gD receptor nectin-1 is sequestered within the intact AJs area of lateral membranes of epithelial cells (left panel). HIV-induced disruption of AJs exposes nectin-1 from its sequestered areas (right panel), which binds to HSV gD and thereby promotes HSV infection and cell-to-cell spread within the oral epithelium. HIV-induced disruption of TJs leads to paracellular spread of HSV virions, which may facilitate penetration of virus from the apical to the basolateral direction into the deeper part of the epithelium, and from the basolateral to the apical direction leading to release of virus into saliva. Thus, HIV-induced disruption of epithelial junctions may facilitate the spread of HSV-1 infection within the mucosal epithelium, leading to the rapid progression of HSV-mediated mucosal lesions and ulcers.

    Article Snippet: To determine HSV-1 infection, we immunostained infected cells with mouse antibodies against HSV-1 ICP4 and gB (both from Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit anti-gD (HSV-1) (MyBioSource, San Diego, CA), and goat anti-HSV-1/2 antiserum (AbD Serotec, Raleigh, NC).

    Techniques: Infection