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  • 96
    Thermo Fisher hiv 1
    Evidence supporting a direct effect of hA3G on relative infectivity of <t>HIV-1</t> virions produced from Th1 and Th2 cells. In vitro cytokine-derived Th2 cells were transduced with a control lentiviral vector (Th2-Empty) or a vector expressing hA3G (Th2-A3G). Protein levels of hA3G packaged into virions produced from Th1, Th2, and hA3G-transduced Th2 cells were determined by Western Blotting on the LICOR Odyssey system (Relative Light Units (RLU) of hA3G as normalized by HIV-1 p24 antigen) Shown is a representative blot of three experiments with similar trends. (A). The infectivity of vif -deleted HIV-1(NL4-3) virions produced from those cells from 6 donors was determined using the TZM-bl indicator cell line. Error bars represent median and interquartile range (*p = .03). (B) In vitro cytokine-derived Th1 cells were activated and incubated with either an isotype control or neutralizing IFN-γ antibodies for 48 hours. Protein concentrations were determined by Western blot (C,open bars). The infectivity of vif -deleted HIV-1(NL4-3) virions produced from those cells was determined using the TZM-bl indicator cell line (C, closed bars).
    Hiv 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hiv 1 - by Bioz Stars, 2022-08
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    86
    Merck KGaA recombinant hiv 1 rt enzyme
    Evidence supporting a direct effect of hA3G on relative infectivity of <t>HIV-1</t> virions produced from Th1 and Th2 cells. In vitro cytokine-derived Th2 cells were transduced with a control lentiviral vector (Th2-Empty) or a vector expressing hA3G (Th2-A3G). Protein levels of hA3G packaged into virions produced from Th1, Th2, and hA3G-transduced Th2 cells were determined by Western Blotting on the LICOR Odyssey system (Relative Light Units (RLU) of hA3G as normalized by HIV-1 p24 antigen) Shown is a representative blot of three experiments with similar trends. (A). The infectivity of vif -deleted HIV-1(NL4-3) virions produced from those cells from 6 donors was determined using the TZM-bl indicator cell line. Error bars represent median and interquartile range (*p = .03). (B) In vitro cytokine-derived Th1 cells were activated and incubated with either an isotype control or neutralizing IFN-γ antibodies for 48 hours. Protein concentrations were determined by Western blot (C,open bars). The infectivity of vif -deleted HIV-1(NL4-3) virions produced from those cells was determined using the TZM-bl indicator cell line (C, closed bars).
    Recombinant Hiv 1 Rt Enzyme, supplied by Merck KGaA, 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/recombinant hiv 1 rt enzyme/product/Merck KGaA
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    recombinant hiv 1 rt enzyme - by Bioz Stars, 2022-08
    86/100 stars
      Buy from Supplier

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    Evidence supporting a direct effect of hA3G on relative infectivity of HIV-1 virions produced from Th1 and Th2 cells. In vitro cytokine-derived Th2 cells were transduced with a control lentiviral vector (Th2-Empty) or a vector expressing hA3G (Th2-A3G). Protein levels of hA3G packaged into virions produced from Th1, Th2, and hA3G-transduced Th2 cells were determined by Western Blotting on the LICOR Odyssey system (Relative Light Units (RLU) of hA3G as normalized by HIV-1 p24 antigen) Shown is a representative blot of three experiments with similar trends. (A). The infectivity of vif -deleted HIV-1(NL4-3) virions produced from those cells from 6 donors was determined using the TZM-bl indicator cell line. Error bars represent median and interquartile range (*p = .03). (B) In vitro cytokine-derived Th1 cells were activated and incubated with either an isotype control or neutralizing IFN-γ antibodies for 48 hours. Protein concentrations were determined by Western blot (C,open bars). The infectivity of vif -deleted HIV-1(NL4-3) virions produced from those cells was determined using the TZM-bl indicator cell line (C, closed bars).

    Journal: PLoS Pathogens

    Article Title: Differences in APOBEC3G Expression in CD4+ T Helper Lymphocyte Subtypes Modulate HIV-1 Infectivity

    doi: 10.1371/journal.ppat.1000292

    Figure Lengend Snippet: Evidence supporting a direct effect of hA3G on relative infectivity of HIV-1 virions produced from Th1 and Th2 cells. In vitro cytokine-derived Th2 cells were transduced with a control lentiviral vector (Th2-Empty) or a vector expressing hA3G (Th2-A3G). Protein levels of hA3G packaged into virions produced from Th1, Th2, and hA3G-transduced Th2 cells were determined by Western Blotting on the LICOR Odyssey system (Relative Light Units (RLU) of hA3G as normalized by HIV-1 p24 antigen) Shown is a representative blot of three experiments with similar trends. (A). The infectivity of vif -deleted HIV-1(NL4-3) virions produced from those cells from 6 donors was determined using the TZM-bl indicator cell line. Error bars represent median and interquartile range (*p = .03). (B) In vitro cytokine-derived Th1 cells were activated and incubated with either an isotype control or neutralizing IFN-γ antibodies for 48 hours. Protein concentrations were determined by Western blot (C,open bars). The infectivity of vif -deleted HIV-1(NL4-3) virions produced from those cells was determined using the TZM-bl indicator cell line (C, closed bars).

    Article Snippet: After determination of the concentration of viral particles by HIV-1 CA p24 ELISA, 300 ng of p24-equivalents of HIV-1 were spinoculated (300×g, 30 min) on 1×106 Th1, Th2, or TH2-A3G cells that had been activated by anti-CD3/CD28 coated beads (Invitrogen Dynal, Carlsbad, CA) for 60 hours .

    Techniques: Infection, Produced, In Vitro, Derivative Assay, Transduction, Plasmid Preparation, Expressing, Western Blot, Incubation

    Increased Infectivity of HIV-1 produced from Th2 cells compared to Th1 cells. vif -competent (+) or vif -deleted (−) HIV-1(NL4-3) was used to infect cultures of Th1 and Th2 cells as described in Materials and Methods . Infectivity of the virions produced was determined by infection of the TZM-bl indicator cell line and determination of luciferase activity. An example from an individual donor is shown (A). The experiment was repeated on a total of seven donors with both the vif -competent (B) and vif -deleted virus(C). Error bars represent median and interquartile range of difference in infectivity of virions produced from Th1 cells versus Th2 cells for vif-positive virus (B; *p = .031) and vif-negative virus (C; **p = .016). To test for correlation, individual donor cells' levels of hA3G protein expression were plotted against infectivity of the vif -competent and vif -deleted virions produced from that individual's Th1 cells (D) and Th2 cells (E).

    Journal: PLoS Pathogens

    Article Title: Differences in APOBEC3G Expression in CD4+ T Helper Lymphocyte Subtypes Modulate HIV-1 Infectivity

    doi: 10.1371/journal.ppat.1000292

    Figure Lengend Snippet: Increased Infectivity of HIV-1 produced from Th2 cells compared to Th1 cells. vif -competent (+) or vif -deleted (−) HIV-1(NL4-3) was used to infect cultures of Th1 and Th2 cells as described in Materials and Methods . Infectivity of the virions produced was determined by infection of the TZM-bl indicator cell line and determination of luciferase activity. An example from an individual donor is shown (A). The experiment was repeated on a total of seven donors with both the vif -competent (B) and vif -deleted virus(C). Error bars represent median and interquartile range of difference in infectivity of virions produced from Th1 cells versus Th2 cells for vif-positive virus (B; *p = .031) and vif-negative virus (C; **p = .016). To test for correlation, individual donor cells' levels of hA3G protein expression were plotted against infectivity of the vif -competent and vif -deleted virions produced from that individual's Th1 cells (D) and Th2 cells (E).

    Article Snippet: After determination of the concentration of viral particles by HIV-1 CA p24 ELISA, 300 ng of p24-equivalents of HIV-1 were spinoculated (300×g, 30 min) on 1×106 Th1, Th2, or TH2-A3G cells that had been activated by anti-CD3/CD28 coated beads (Invitrogen Dynal, Carlsbad, CA) for 60 hours .

    Techniques: Infection, Produced, Luciferase, Activity Assay, Expressing

    The m 6 A writers and erasers affect HIV-1 Gag expression in virus-producing cells. ( A and B ) Individual or combined knockdown of endogenous METTL3 and METTL14 inhibits HIV-1 Gag protein expression. HEK293T cells were transfected with indicated siRNA, and then with an HIV-1 proviral DNA plasmid (pNL4-3). Cells and supernatants were collected for analyses at 36 hr post-transfection. ( A ) Expression of METTL3, METTL14 and HIV-1 Gag proteins in the transfected HEK293T cells was detected by immunoblotting. ( C and D ) Knockdown of endogenous AlkBH5, FTO, or both promotes HIV-1 Gag protein expression. HEK293T cells were transfected with indicated siRNA, and then with pNL4-3. Cells and supernatants were collected at 36 hr post-transfection. ( C ) Expression of AlkBH5, FTO and HIV-1 Gag proteins in the cells was detected by immunoblotting. ( A and C ) GAPDH was used as a loading control. Relative levels of Gag expression were normalized to GAPDH levels. ( B and D ) HIV-1 capsid p24 levels in supernatants were measured by ELISA. The relative levels (%) are also shown. *p

    Journal: eLife

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    doi: 10.7554/eLife.15528

    Figure Lengend Snippet: The m 6 A writers and erasers affect HIV-1 Gag expression in virus-producing cells. ( A and B ) Individual or combined knockdown of endogenous METTL3 and METTL14 inhibits HIV-1 Gag protein expression. HEK293T cells were transfected with indicated siRNA, and then with an HIV-1 proviral DNA plasmid (pNL4-3). Cells and supernatants were collected for analyses at 36 hr post-transfection. ( A ) Expression of METTL3, METTL14 and HIV-1 Gag proteins in the transfected HEK293T cells was detected by immunoblotting. ( C and D ) Knockdown of endogenous AlkBH5, FTO, or both promotes HIV-1 Gag protein expression. HEK293T cells were transfected with indicated siRNA, and then with pNL4-3. Cells and supernatants were collected at 36 hr post-transfection. ( C ) Expression of AlkBH5, FTO and HIV-1 Gag proteins in the cells was detected by immunoblotting. ( A and C ) GAPDH was used as a loading control. Relative levels of Gag expression were normalized to GAPDH levels. ( B and D ) HIV-1 capsid p24 levels in supernatants were measured by ELISA. The relative levels (%) are also shown. *p

    Article Snippet: All HIV-1 stocks used for PCR assays were treated with DNaseI (40 U/ml; Ambion, Waltham, MA) prior to infections to avoid plasmid DNA contamination.

    Techniques: Expressing, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    YTHDF1–3 proteins negatively regulate post-entry HIV-1 infection in HeLa cells. ( A – B ) Overexpression of YTHDF1–3 proteins in HeLa cells significantly inhibits HIV-1 infection compared to vector control cells. ( A ) Overexpression of YTHDF1–3 proteins in HeLa cells was confirmed by immunoblotting. ( B ) HeLa cells overexpressing YTHDF1–3 proteins were infected with HIV-1 Luc/VSV-G at an MOI of 0.5 and viral infection was measured by luciferase activity at 24 hpi. ( C ) Overexpression of YTHDF1–3 proteins inhibits HIV-1 Gag protein synthesis in infected cells. HeLa cells overexpressing individual YTHDF1–3 proteins or the vector control cells were infected by HIV-1-Luc/VSV-G at an MOI of 0.5. At 24 hpi, the expression of HIV-1 Gag and YTHDF1–3 proteins (FLAG-tagged) was determined using immunoblotting. GAPDH was used as a loading control and mock-infected vector control cells were used as a negative control. ( D and E ) Individual knockdown of endogenous YTHDF1–3 proteins in HeLa cells significantly increases HIV-1 infection compared to vector control cells. HIV-1 infection assays were performed as described for panel B . *p

    Journal: eLife

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    doi: 10.7554/eLife.15528

    Figure Lengend Snippet: YTHDF1–3 proteins negatively regulate post-entry HIV-1 infection in HeLa cells. ( A – B ) Overexpression of YTHDF1–3 proteins in HeLa cells significantly inhibits HIV-1 infection compared to vector control cells. ( A ) Overexpression of YTHDF1–3 proteins in HeLa cells was confirmed by immunoblotting. ( B ) HeLa cells overexpressing YTHDF1–3 proteins were infected with HIV-1 Luc/VSV-G at an MOI of 0.5 and viral infection was measured by luciferase activity at 24 hpi. ( C ) Overexpression of YTHDF1–3 proteins inhibits HIV-1 Gag protein synthesis in infected cells. HeLa cells overexpressing individual YTHDF1–3 proteins or the vector control cells were infected by HIV-1-Luc/VSV-G at an MOI of 0.5. At 24 hpi, the expression of HIV-1 Gag and YTHDF1–3 proteins (FLAG-tagged) was determined using immunoblotting. GAPDH was used as a loading control and mock-infected vector control cells were used as a negative control. ( D and E ) Individual knockdown of endogenous YTHDF1–3 proteins in HeLa cells significantly increases HIV-1 infection compared to vector control cells. HIV-1 infection assays were performed as described for panel B . *p

    Article Snippet: All HIV-1 stocks used for PCR assays were treated with DNaseI (40 U/ml; Ambion, Waltham, MA) prior to infections to avoid plasmid DNA contamination.

    Techniques: Infection, Over Expression, Plasmid Preparation, Luciferase, Activity Assay, Expressing, Negative Control

    Distribution of m 6 A in cellular RNAs and the frequency of m 6 A motifs in HIV-1-infected cells. ( A – B ) Pie charts show the distribution of m 6 A peaks in the 5′ UTR, coding DNA sequence (CDS), 3′ UTR, and noncoding regions of transcripts from uninfected and HIV-1-infected Jurkat T-cells ( A ) or primary CD4 + T-cells ( B ). The m 6 A peak distribution in HIV-1-specific RNAs is also shown. ( C – D ) Frequency of the RRACH motif ( C ) and the GGACU motif ( D ) within the m 6 A peaks in cellular RNAs from the uninfected control and HIV-1-infected cells. Data presented are the average results of duplicated samples (n=2). DOI: http://dx.doi.org/10.7554/eLife.15528.006

    Journal: eLife

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    doi: 10.7554/eLife.15528

    Figure Lengend Snippet: Distribution of m 6 A in cellular RNAs and the frequency of m 6 A motifs in HIV-1-infected cells. ( A – B ) Pie charts show the distribution of m 6 A peaks in the 5′ UTR, coding DNA sequence (CDS), 3′ UTR, and noncoding regions of transcripts from uninfected and HIV-1-infected Jurkat T-cells ( A ) or primary CD4 + T-cells ( B ). The m 6 A peak distribution in HIV-1-specific RNAs is also shown. ( C – D ) Frequency of the RRACH motif ( C ) and the GGACU motif ( D ) within the m 6 A peaks in cellular RNAs from the uninfected control and HIV-1-infected cells. Data presented are the average results of duplicated samples (n=2). DOI: http://dx.doi.org/10.7554/eLife.15528.006

    Article Snippet: All HIV-1 stocks used for PCR assays were treated with DNaseI (40 U/ml; Ambion, Waltham, MA) prior to infections to avoid plasmid DNA contamination.

    Techniques: Infection, Sequencing

    YTHDF1–3 proteins negatively regulate post-entry HIV-1 infection in CD4 + T-cells. ( A ) Individual knockdown of endogenous YTHDF1–3 proteins in Jurkat CD4 + T cells was confirmed by immunoblotting. ( B ) Knockdown of YTHDF1–3 proteins does not affect proliferation of Jurkat cells. Jurkat cells (2 × 10 4 ) were seeded and cultured for 3 days. At the times indicated, cell proliferation was measured using the MTS assay. ( C ) Knockdown of YTHDF1–3 proteins significantly increases HIV-1 infection compared to vector control cells. ( D ) Individual knockdown of YTHDF1–3 proteins in activated primary CD4+ T-cells from a healthy donor. ( E ) Knockdown of YTHDF1–3 proteins significantly increases HIV-1 infection compared to vector control cells. ( A and D ) GAPDH was used as a loading control. ( C and E ) The vector controls without AZT were set as 100%. The reverse transcriptase inhibitor AZT treated cells were used as positive control for productive HIV-1 infection. *p

    Journal: eLife

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    doi: 10.7554/eLife.15528

    Figure Lengend Snippet: YTHDF1–3 proteins negatively regulate post-entry HIV-1 infection in CD4 + T-cells. ( A ) Individual knockdown of endogenous YTHDF1–3 proteins in Jurkat CD4 + T cells was confirmed by immunoblotting. ( B ) Knockdown of YTHDF1–3 proteins does not affect proliferation of Jurkat cells. Jurkat cells (2 × 10 4 ) were seeded and cultured for 3 days. At the times indicated, cell proliferation was measured using the MTS assay. ( C ) Knockdown of YTHDF1–3 proteins significantly increases HIV-1 infection compared to vector control cells. ( D ) Individual knockdown of YTHDF1–3 proteins in activated primary CD4+ T-cells from a healthy donor. ( E ) Knockdown of YTHDF1–3 proteins significantly increases HIV-1 infection compared to vector control cells. ( A and D ) GAPDH was used as a loading control. ( C and E ) The vector controls without AZT were set as 100%. The reverse transcriptase inhibitor AZT treated cells were used as positive control for productive HIV-1 infection. *p

    Article Snippet: All HIV-1 stocks used for PCR assays were treated with DNaseI (40 U/ml; Ambion, Waltham, MA) prior to infections to avoid plasmid DNA contamination.

    Techniques: Infection, Cell Culture, MTS Assay, Plasmid Preparation, Positive Control

    YTHDF1–3 proteins bind to HIV-1 gRNA in infected cells. ( A ) Immunoblotting of YTHDF1–3 proteins in the input and immunoprecipitation (IP) samples from HIV-1-Luc/VSV-G infected HeLa cells. FLAG antibodies were used to immunoprecipitate FLAG-tagged YTHDF1–3 proteins overexpressed in HeLa cells after HIV-1 infection. A short and long exposure of the immunoblot is shown. ( B ) HIV-1 gRNA is bound by YTHDF1–3 proteins expressed in HeLa cells. HeLa cells stably overexpressing FLAG-tagged YTHDF1–3 proteins or empty vector control cells (Ctrl) were infected with HIV-1-Luc/VSV-G at an MOI of 5 for 3 hr. Cell lysates were immunoprecipitated with anti-FLAG, RNA was extracted and HIV-1 gag RNA levels were measured. **p

    Journal: eLife

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    doi: 10.7554/eLife.15528

    Figure Lengend Snippet: YTHDF1–3 proteins bind to HIV-1 gRNA in infected cells. ( A ) Immunoblotting of YTHDF1–3 proteins in the input and immunoprecipitation (IP) samples from HIV-1-Luc/VSV-G infected HeLa cells. FLAG antibodies were used to immunoprecipitate FLAG-tagged YTHDF1–3 proteins overexpressed in HeLa cells after HIV-1 infection. A short and long exposure of the immunoblot is shown. ( B ) HIV-1 gRNA is bound by YTHDF1–3 proteins expressed in HeLa cells. HeLa cells stably overexpressing FLAG-tagged YTHDF1–3 proteins or empty vector control cells (Ctrl) were infected with HIV-1-Luc/VSV-G at an MOI of 5 for 3 hr. Cell lysates were immunoprecipitated with anti-FLAG, RNA was extracted and HIV-1 gag RNA levels were measured. **p

    Article Snippet: All HIV-1 stocks used for PCR assays were treated with DNaseI (40 U/ml; Ambion, Waltham, MA) prior to infections to avoid plasmid DNA contamination.

    Techniques: Infection, Immunoprecipitation, Stable Transfection, Plasmid Preparation

    Proposed mechanisms and dynamics of m 6 A modification of HIV-1 RNA in regulating viral infection in cells. In the nucleus, the m 6 A writers (METTL3 and METTL14) add the m 6 A marker to HIV-1 genomic RNA (gRNA) or mRNA, and the m 6 A erasers (FTO and AlkBH5) remove the m 6 A modifications of HIV-1 RNA. The m 6 A modification of HIV-1 RNA can promote viral protein translation in cells. In contrast, cytoplasmic m 6 A readers (YTHDF1–3) bind to m 6 A-modified HIV-1 gRNA, which can result in inhibition of HIV-1 reverse transcription (RT), viral mRNA expression, and thereby HIV-1 infection in cells. DOI: http://dx.doi.org/10.7554/eLife.15528.014

    Journal: eLife

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    doi: 10.7554/eLife.15528

    Figure Lengend Snippet: Proposed mechanisms and dynamics of m 6 A modification of HIV-1 RNA in regulating viral infection in cells. In the nucleus, the m 6 A writers (METTL3 and METTL14) add the m 6 A marker to HIV-1 genomic RNA (gRNA) or mRNA, and the m 6 A erasers (FTO and AlkBH5) remove the m 6 A modifications of HIV-1 RNA. The m 6 A modification of HIV-1 RNA can promote viral protein translation in cells. In contrast, cytoplasmic m 6 A readers (YTHDF1–3) bind to m 6 A-modified HIV-1 gRNA, which can result in inhibition of HIV-1 reverse transcription (RT), viral mRNA expression, and thereby HIV-1 infection in cells. DOI: http://dx.doi.org/10.7554/eLife.15528.014

    Article Snippet: All HIV-1 stocks used for PCR assays were treated with DNaseI (40 U/ml; Ambion, Waltham, MA) prior to infections to avoid plasmid DNA contamination.

    Techniques: Modification, Infection, Marker, Inhibition, Expressing

    YTHDF1–3 proteins negatively regulate HIV-1 gag mRNA expression. Specific shRNAs or scrambled shRNA vector-treated cells were infected with HIV-1 Luc/VSV-G at an MOI of 0.5. Total RNA was isolated from the cells 24 hr post-infection and HIV-1 gag mRNA levels were quantified using qRT-PCR. ( A and B ) HIV-1 gag mRNA levels in the infected HeLa cells with overexpression ( A ) or knockdown ( B , shRNA) of YTHDF1–3 proteins. ( C ) HIV-1 gag mRNA levels in the HIV-1 infected Jurkat cells after knockdown of YTHDF1–3 proteins. AZT treated vector control cells were used as a negative control of HIV-1 infection ( A – C ). The vector controls without AZT were set as 100%. *p

    Journal: eLife

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    doi: 10.7554/eLife.15528

    Figure Lengend Snippet: YTHDF1–3 proteins negatively regulate HIV-1 gag mRNA expression. Specific shRNAs or scrambled shRNA vector-treated cells were infected with HIV-1 Luc/VSV-G at an MOI of 0.5. Total RNA was isolated from the cells 24 hr post-infection and HIV-1 gag mRNA levels were quantified using qRT-PCR. ( A and B ) HIV-1 gag mRNA levels in the infected HeLa cells with overexpression ( A ) or knockdown ( B , shRNA) of YTHDF1–3 proteins. ( C ) HIV-1 gag mRNA levels in the HIV-1 infected Jurkat cells after knockdown of YTHDF1–3 proteins. AZT treated vector control cells were used as a negative control of HIV-1 infection ( A – C ). The vector controls without AZT were set as 100%. *p

    Article Snippet: All HIV-1 stocks used for PCR assays were treated with DNaseI (40 U/ml; Ambion, Waltham, MA) prior to infections to avoid plasmid DNA contamination.

    Techniques: Expressing, shRNA, Plasmid Preparation, Infection, Isolation, Quantitative RT-PCR, Over Expression, Negative Control

    HIV-1 RNA contains m 6 A modifications. HEK293 T cells were transfected with a proviral DNA-containing plasmid (pNL4-3). Total RNA was extracted at 48 hr post-transfection and immunoprecipitated with an m 6 A-specific antibody. Enriched RNA was subjected to next generation sequencing. Peaks show the relative abundance of m 6 A sites on the HIV-1 genome. The distribution of m 6 A reads from m 6 A-seq mapped to HIV-1 genome (red line). Baseline signal from the RNA-seq of input samples is shown as a black line. A schematic diagram of HIV-1 NL4-3 genome features is shown above. TAR, transacting response element; RRE, Rev response element. The data presented are representative of two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.15528.004

    Journal: eLife

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    doi: 10.7554/eLife.15528

    Figure Lengend Snippet: HIV-1 RNA contains m 6 A modifications. HEK293 T cells were transfected with a proviral DNA-containing plasmid (pNL4-3). Total RNA was extracted at 48 hr post-transfection and immunoprecipitated with an m 6 A-specific antibody. Enriched RNA was subjected to next generation sequencing. Peaks show the relative abundance of m 6 A sites on the HIV-1 genome. The distribution of m 6 A reads from m 6 A-seq mapped to HIV-1 genome (red line). Baseline signal from the RNA-seq of input samples is shown as a black line. A schematic diagram of HIV-1 NL4-3 genome features is shown above. TAR, transacting response element; RRE, Rev response element. The data presented are representative of two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.15528.004

    Article Snippet: All HIV-1 stocks used for PCR assays were treated with DNaseI (40 U/ml; Ambion, Waltham, MA) prior to infections to avoid plasmid DNA contamination.

    Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Next-Generation Sequencing, RNA Sequencing Assay

    Gene ontology (GO) analysis of m 6 A-modified cellular genes in HIV-1 infected cells. ( A and B ) GO terms specific to virus related pathways and corresponding p values, clustered from methylated genes detected in Jurkat cells ( A ) or primary CD4 + T cells ( B ) infected with HIV-1. ( C and D ) GO graphs showing functional clusters from genes with unique m 6 A peaks identified in HIV-1-infected Jurkat cells ( C ) or primary CD4 + T-cells ( D ) when compared to uninfected cells. Data presented are the average results of duplicated samples (n=2). DOI: http://dx.doi.org/10.7554/eLife.15528.007

    Journal: eLife

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    doi: 10.7554/eLife.15528

    Figure Lengend Snippet: Gene ontology (GO) analysis of m 6 A-modified cellular genes in HIV-1 infected cells. ( A and B ) GO terms specific to virus related pathways and corresponding p values, clustered from methylated genes detected in Jurkat cells ( A ) or primary CD4 + T cells ( B ) infected with HIV-1. ( C and D ) GO graphs showing functional clusters from genes with unique m 6 A peaks identified in HIV-1-infected Jurkat cells ( C ) or primary CD4 + T-cells ( D ) when compared to uninfected cells. Data presented are the average results of duplicated samples (n=2). DOI: http://dx.doi.org/10.7554/eLife.15528.007

    Article Snippet: All HIV-1 stocks used for PCR assays were treated with DNaseI (40 U/ml; Ambion, Waltham, MA) prior to infections to avoid plasmid DNA contamination.

    Techniques: Modification, Infection, Methylation, Functional Assay

    Quantification of HIV-1 RNA m 6 A level using liquid chromatography-mass spectrometry. HIV-1 RNA (250 ng) was isolated from highly purified HIV-1 MN virions (total 600 μg of p24 capsid) and subjected to quantitative analysis of the m 6 A level using LC-MS/MS (n=3 of each sample). The results are presented are from representative of two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.15528.005

    Journal: eLife

    Article Title: N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    doi: 10.7554/eLife.15528

    Figure Lengend Snippet: Quantification of HIV-1 RNA m 6 A level using liquid chromatography-mass spectrometry. HIV-1 RNA (250 ng) was isolated from highly purified HIV-1 MN virions (total 600 μg of p24 capsid) and subjected to quantitative analysis of the m 6 A level using LC-MS/MS (n=3 of each sample). The results are presented are from representative of two independent experiments. DOI: http://dx.doi.org/10.7554/eLife.15528.005

    Article Snippet: All HIV-1 stocks used for PCR assays were treated with DNaseI (40 U/ml; Ambion, Waltham, MA) prior to infections to avoid plasmid DNA contamination.

    Techniques: Liquid Chromatography, Mass Spectrometry, Isolation, Purification, Liquid Chromatography with Mass Spectroscopy