Fig 2: A3A suppresses HIV-1 production via TTTC motifs. (A and B) 293T cells were transfected with WT, single-, or double-TTTC motif mutant NL4- (1 µg) and A3A-HA (0.5, 1 µg) plasmids. Total transfected plasmid quantity was normalized with pcDNA3.1 empty plasmid. (A) Total RNA was recovered 24 h after transfection and analyzed for HIV-1 Gag and 18s mRNA expression by qRT-PCR; 18s mRNA was used as an internal control. (B) The whole-cell lysate was recovered 48 h after transfection and HIV-1 p24 protein expression was measured by ELISA. For A and B, data are means ± SE (n = 3). Sample means were compared to control and assessed by ANOVA with Tukey–Kramer’s test. ***P < 0.001, **P < 0.01, *P < 0.05; n.s., not significant. (C and D) Recombinant A3A protein was pulled down using biotinylated HIV-1 LTR encoding ssDNA oligos with indicated spacer lengths between the two A3A-targeted TTTC motifs. The amount of bound A3A was measured by Western blot (C). A3A binding intensity for oligos with indicated spacer lengths was normalized to the WT LTR oligo (9 nt) signal (D).

Fig 3: A3A binds to TTTC/GAAA motifs in the ISG15 promoter and suppresses IFN-a–induced pSTAT1 recruitment. (A) 293T cells were transfected with WT A3A-HA or pcDNA3.1 and treated with IFN-a (1,000 IU/mL) for 3 h. A3A-HA and p-STAT-1 binding to the ISRE region of the ISG15 promoter was analyzed using ChIP-qPCR. Data are means ± SE (n = 3). For ***P < 0.001, **P < 0.01, the sample means were compared to nontreated control. For †††P < 0.001, the indicated sample mean was compared to IFN-a–treated control. All comparisons were assessed by ANOVA with Tukey–Kramer’s test. n.s., not significant. (B and C) WT dsDNA, WT sense-, WT antisense-ssDNA, and indicated TTTC mutant (TTT to AAA) antisense-ssDNA oligos were used to pull down recombinant A3A protein (B). Five percent of input and DNA bound A3A protein was analyzed by Western blot (C).

Fig 4: A3A knockout enhanced IFN-a–induced ISG expression. (A) CRISPR-control or -A3A J-Lat10.6 cells were treated with IFN-a (1,000 IU/mL) for 3 h. Nuclear STAT1, p-STAT1, A3A, HDAC1 protein expression was measured by Western blot. HDAC1 expression served as an internal loading control. (B) ISG15 and 18s mRNA were measured in CRISPR-control or -A3A J-Lat10.6 cells treated with IFN-a (1,000 IU/mL) for 18 h. Data are means ± SE (n = 3). Sample means were compared to CRISPR-control and assessed by ANOVA with Tukey–Kramer’s test. ***P < 0.001; n.s., not significant. (C) CRISPR-control and -A3A J-Lat10.6 cells were treated with IFN-a (1,000 IU/mL) for 3 h and p-STAT-1 and RNA Pol II binding to the ISRE region in the ISG15 promoter was analyzed by ChIP-qPCR. Data are means ± SE (n = 3). Sample means were compared to IFN-a–treated CRISPR-control and assessed by ANOVA with Tukey–Kramer’s test. ***P < 0.001. (D) CRISPR-control or CRISPR-A3A J-Lat10.6 cells were treated with IFN-a (1,000 IU/mL) for 18 h and gene expression was measured by RNA-seq (n = 3). Volcano plot (59) depicts the difference in IFN-a–responsive gene induction in CRISPR-A3A vs. -control J-Lat10.6 cells. Genes with significant differential expression (Benjamini–Hochberg false-discovery rate < 0.1) (58) are highlighted in red. (E) ClusterProfiler (51) GO enrichment analysis of genes significantly up-regulated (log2 fold-change = 0.1, Benjamini–Hochberg P-adjusted = 0.1) in CRISPR-A3A cells. (F) ISRE-like motif identified by HOMER2 (48) enrichment analysis. (G) TTTC motif locations and sequences (red) within the IFIT1 promoter are shown. (H) Boxplots denote the distribution of TTTC motif counts per promoter sequence of common and DE-ISGs. *P = 0.05, Welch’s two-sample t test. (I) HOMER2 enrichment analyses were performed using a custom library encompassing the indicated TTTC motifs. Enrichment ratios are reported from the HOMER2 ‘known’ output.

Fig 5: A3A binds TTTC motifs located in the HIV-1 LTR. (A) The sequence encoding the U3/R region of the HXB2 HIV-1 LTR contains TTTC motifs (highlighted in blue) predicted to be bound by A3A. (B) 5'-biotinylated or Alexa 488-conjugated HIV-1 LTR oligos were used for DNA pull-down and deamination assays, respectively. These oligos encompassed WT sense-stranded ssDNA, WT dsDNA, and dsDNA containing 3- to 15-nt-long mismatches at the A3A-targeted TTTC motifs. Recombinant A3A protein was pulled down using biotinylated-oligo–linked dynabeads. Ten percent of input and bound A3A protein was analyzed by Western blot (B, Upper). Deamination activity of A3A-transfected 293T cell lysate was analyzed using Alexa 488-conjugated oligos (B, Lower). As shown in the Right, nondeaminated oligos migrate as a 50 mer-DNA band. Cytidine deamination within TTTC motifs at positions 31 and 17 results in the appearance of 30 mer- and 16 mer-DNA bands, respectively. (C) The relative fluorescent intensity of deamination products (shown in B) was compared to WT dsDNA. (D) 293T cells were transfected with WT, ??B, ?Sp1 HIV-1 LTR luciferase constructs and cotransfected with or without A3A. Twenty-four hours after transfection, cells were treated with 10 nM PMA for 48 h. Then, luciferase activity was measured for each LTR and normalized to spontaneous reporter signal in the absence of A3A. Data are means ± SE (n = 3). Sample means were compared to ??B signal and assessed by ANOVA with Tukey–Kramer’s test. ***P < 0.001, **P < 0.01, *P < 0.05; n.s., not significant. (E) Recombinant A3A protein was pulled down using ssDNA oligos with indicated TTTC motif mutations. Five percent of input and DNA-bound A3A protein was analyzed by Western blot.