Journal: Journal of Virology

Article Title: Single-base m 6 A epitranscriptomics reveals novel HIV-1 host interaction targets in primary CD4 + T cells

doi: 10.1128/jvi.01536-25

Figure Lengend Snippet: m 6 A modification of PLIN3 mRNA is increased by HIV-1 infection in primary CD4 + T cells. Activated primary CD4 + T cells isolated from PBMCs of three donors were mock-infected or infected with HIV-1 NL4–3 at an MOI of 1 for 96 h. Total cellular RNA was subjected to meRIP, and the level of m 6 A-modified transcripts in the meRIP was determined relative to ( A ) input or ( B ) mock-infected controls by RT-qPCR. Data are shown as mean ± SD. A two-tailed unpaired t -test was used for statistical analysis ( P values are shown on the figures).

Article Snippet: Antibodies used for immunoblotting were as follows: HIV-1 p24 (clone #24-2, the AIDS Research and Reference Reagent Program, NIH), GAPDH (AHP1628, Bio-Rad), METTL3 (15073, Proteintech), METTL14 (CL4252, Abcam), PLIN3 (10694-1-AP, Proteintech), and HIV-Ig (3957, the AIDS Research and Reference Reagent Program, NIH).

Techniques: Modification, Infection, Isolation, Quantitative RT-PCR, Two Tailed Test

Journal: Journal of Virology

Article Title: Single-base m 6 A epitranscriptomics reveals novel HIV-1 host interaction targets in primary CD4 + T cells

doi: 10.1128/jvi.01536-25

Figure Lengend Snippet: HIV-1 infection increases PLIN3 mRNA levels but decreases PLIN3 protein levels in primary CD4 + T cells. ( A–D ) Primary CD4 + T cells were mock-infected or infected with HIV-1 NL4–3 at an MOI of 1 for 96 h. The HIV-1 reverse transcription inhibitor NVP was used to block viral replication (HIV-1 + NVP). GAPDH was used as a loading control. ( A ) PLIN3 and HIV-1 protein expression were measured by IB. A representative IB is shown. ( B ) Relative levels of PLIN3 protein expression normalized with GAPDH, as shown in ( A ), from three individual donors. ( C ) PLIN3 mRNA levels were measured by RT-qPCR. N = 6 (Mock, HIV-1) or N = 3 (HIV-1 + NVP). ( D ) Cells were treated with actinomycin D at 96 hpi. Samples were collected at the indicated time points, and PLIN3 mRNA levels were detected by RT-qPCR. Data are shown as means ± SD of results from three donors’ cells (raw data in ). Ordinary one-way ANOVA with Dunnett correction (B, and C) and multiple unpaired t -test ( D ) were used for statistical analysis ( P values are shown on figures). ns, not significant. * P < 0.05.

Article Snippet: Antibodies used for immunoblotting were as follows: HIV-1 p24 (clone #24-2, the AIDS Research and Reference Reagent Program, NIH), GAPDH (AHP1628, Bio-Rad), METTL3 (15073, Proteintech), METTL14 (CL4252, Abcam), PLIN3 (10694-1-AP, Proteintech), and HIV-Ig (3957, the AIDS Research and Reference Reagent Program, NIH).

Techniques: Infection, Reverse Transcription, Blocking Assay, Control, Expressing, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Single-base m 6 A epitranscriptomics reveals novel HIV-1 host interaction targets in primary CD4 + T cells

doi: 10.1128/jvi.01536-25

Figure Lengend Snippet: HIV-1 infection increases the levels of PLIN3 mRNA in the nucleus of primary CD4 + T cells. ( A–D ) Activated primary CD4 + T cells from three healthy donors were mock-infected or infected with HIV-1 NL4–3 at an MOI of 1 for 96 h. Each dot represents the result from one donor’s cells. Data are shown as means ± SD. ( A ) PLIN3 mRNA levels in total cell lysates were measured by RT-qPCR and normalized with GAPDH. ( B ) MALAT1 lncRNA and HPRT mRNA levels from each fraction were measured by RT-qPCR to confirm successful separation of the nucleus and cytoplasm, respectively. ( C and D ) Cellular RNA was separated into nuclear and cytoplasmic fractions prior to RT-qPCR analysis. PLIN3 mRNA levels from the nuclear and cytoplasmic fractions are shown relative to MALAT1 and HPRT , respectively. A two-tailed unpaired t -test was used for statistical analysis. P value is shown on the figure. ns, not significant.

Article Snippet: Antibodies used for immunoblotting were as follows: HIV-1 p24 (clone #24-2, the AIDS Research and Reference Reagent Program, NIH), GAPDH (AHP1628, Bio-Rad), METTL3 (15073, Proteintech), METTL14 (CL4252, Abcam), PLIN3 (10694-1-AP, Proteintech), and HIV-Ig (3957, the AIDS Research and Reference Reagent Program, NIH).

Techniques: Infection, Quantitative RT-PCR, Two Tailed Test

Journal: Journal of Virology

Article Title: Single-base m 6 A epitranscriptomics reveals novel HIV-1 host interaction targets in primary CD4 + T cells

doi: 10.1128/jvi.01536-25

Figure Lengend Snippet: Polysome profile analysis of HIV-1-infected primary CD4 + T cells from three donors. ( A–E ) Activated primary CD4 + T cells from three healthy donors were mock-infected or infected with HIV-1 NL4–3 at an MOI of 1 for 96 h. ( A ) Polysome profile analysis (fractions 1-10, from top to bottom of the sucrose gradient). ( B–D ) Relative mRNA levels of ( B ) HIV-1 gag, ( C ) HPRT, and ( D ) PLIN3 from each fraction were measured by RT-qPCR. ( E ) Relative abundance of PLIN3 and HPRT in combined fractions 9 and 10 was measured by RT-qPCR. A two-tailed unpaired t -test was used for statistical analysis. P value is shown on the figure. ns, not significant. ( B–E ) The total detected quantity of a transcript in all fractions is set to 100% and the proportion of transcript found in each fraction ( B–D ) or combined two fractions ( E ) is represented as a percentage.

Article Snippet: Antibodies used for immunoblotting were as follows: HIV-1 p24 (clone #24-2, the AIDS Research and Reference Reagent Program, NIH), GAPDH (AHP1628, Bio-Rad), METTL3 (15073, Proteintech), METTL14 (CL4252, Abcam), PLIN3 (10694-1-AP, Proteintech), and HIV-Ig (3957, the AIDS Research and Reference Reagent Program, NIH).

Techniques: Infection, Quantitative RT-PCR, Two Tailed Test

Journal: Journal of Virology

Article Title: Single-base m 6 A epitranscriptomics reveals novel HIV-1 host interaction targets in primary CD4 + T cells

doi: 10.1128/jvi.01536-25

Figure Lengend Snippet: Knockdown of PLIN3 in primary CD4 + T cells decreases HIV-1 production but increases viral infectivity. ( A–D ) Primary CD4 + T cells were transduced with lentiviral vectors expressing non-targeting (Ctrl) or PLIN3 small guide (sg) RNA to achieve partial stable knockdown of PLIN3. Cells were then infected with HIV-1 NL4–3 at an MOI of 1 for 96 h. ( A ) Relative levels of PLIN3 expression and HIV-1 infection were measured by IB in cells from three independent donors. ( B ) Relative quantification of PLIN3 protein expression is shown in ( A ). ( C ) Relative levels of HIV-1 protein expression are shown in ( A ). ( D ) Cell supernatant p24 levels from HIV-1-infected cells were quantified by ELISA. ( E ) TZM-bl cells were infected with HIV-1 collected from sgCtrl or sgPLIN3 cell culture supernatants. Luciferase activity was measured at 48 hpi. ( F–H ) Primary activated CD4 + T cells from one additional donor were nucleofected with control crRNA or one of three different crRNAs specifically targeting PLIN3 . ( F ) Western blot analysis of PLIN3 knockdown efficiency. GAPDH was used as a loading control. Relative PLIN3 levels were normalized to GAPDH. ( G ) The cells used in ( F ) were subsequently infected with HIV-1 NL4-3 at an MOI of 1 for 96 h, and p24 levels in the supernatant were measured by ELISA. ( H ) TZM-bl cells were infected with HIV-1 collected from Ctrl or crPLIN3 cell culture supernatants. Luciferase activity was measured at 48 hpi and normalized to protein amounts. Data are shown as means ± SD from three individual donors or 2–4 technical replicates. A two-tailed unpaired t -test ( B ), multiple unpaired t -test ( C–E ), and one-way ANOVA ( G and H ) were used for statistical analysis ( P values are shown on figures). ns, not significant.

Article Snippet: Antibodies used for immunoblotting were as follows: HIV-1 p24 (clone #24-2, the AIDS Research and Reference Reagent Program, NIH), GAPDH (AHP1628, Bio-Rad), METTL3 (15073, Proteintech), METTL14 (CL4252, Abcam), PLIN3 (10694-1-AP, Proteintech), and HIV-Ig (3957, the AIDS Research and Reference Reagent Program, NIH).

Techniques: Knockdown, Infection, Transduction, Expressing, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay, Cell Culture, Luciferase, Activity Assay, Control, Western Blot, Two Tailed Test

Journal: Journal of Virology

Article Title: Single-base m 6 A epitranscriptomics reveals novel HIV-1 host interaction targets in primary CD4 + T cells

doi: 10.1128/jvi.01536-25

Figure Lengend Snippet: Summary and proposed model. In primary CD4 + T cells, HIV-1 infection promotes the interaction between METTL3 and METTL14. HIV-1 infection increases m 6 A level and nuclear accumulation of PLIN3 mRNA but reduces PLIN3 protein expression and translation efficiency. Knockdown of PLIN3 in primary CD4 + T cells decreases HIV-1 release but increases viral infectivity in TZM-bl cells. Ctrl, control; KD, knockdown.

Article Snippet: Antibodies used for immunoblotting were as follows: HIV-1 p24 (clone #24-2, the AIDS Research and Reference Reagent Program, NIH), GAPDH (AHP1628, Bio-Rad), METTL3 (15073, Proteintech), METTL14 (CL4252, Abcam), PLIN3 (10694-1-AP, Proteintech), and HIV-Ig (3957, the AIDS Research and Reference Reagent Program, NIH).

Techniques: Infection, Expressing, Knockdown, Control