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. Author manuscript; available in PMC: 2024 Dec 11.
Published in final edited form as: Nat Microbiol. 2020 Jul 20;5(10):1247–1261. doi: 10.1038/s41564-020-0753-6

Extended Data Fig. 9. Effect of Vpu and RAD52 or BLM on unintegrated viral cDNA, integration and superinfection in primary CD4+ T cells.

Extended Data Fig. 9

Effect of Vpu and RAD52 or BLM on unintegrated viral cDNA, integration and superinfection in primary CD4+ T cells. (a-c) RT-PCR analysis of 1-LTR, 2-LTR circles and integration in CD4+ T cells infected with wt or vpu* pHIV-1-NL4-3 in the presence or absence of a RAD52 inhibitor (5 μM 6-Hydroxy-DL-DOPA) 48 h post infection. Bars represent means ± SEM, n=3 biologically independent experiments in triplicates, (a) *p=0.0156, **p=0.0039, (b) **p=0.0078, (c) *p=0.0273, **p=0.0039. (d) Propidium iodide staining of acetone/ethanol fixed cells and p24 stain of primary CD4+ T cells treated as in Fig. 7 (a-c). n=3, bars represent means ± SEM of 6 (apoptosis) or 4 measurements, **p=0.022. (e) Western blot analysis of p24 protein expression in CD4+ T cells infected with wt or vpu* pHIV-1-NL4-3. Experiment conducted two times with similar outcome. (f-k) RT-PCR analysis of 1-LTR, 2-LTR circles and integration in CD4+ T cells transduced with wt, vpu* or vpr* defective pHIV-1-NL4-3-env* constructs (f-h) or infected with replication-competent pHIV-1-NL4-3 In the presence of the protease inhibitor saquinavir (i-k). Bars represent means ± SEM from 6 (f,g), 5 (h), 3(i), 2(j, k) donors are shown. Analysis done 48 h post-infection/transduction. (l) Western blot demonstrating BLM knockdown efficiency in Jurkat cells. Experiment conducted 2 times with similar outcome. (m-o) RT-PCR analysis of 1-LTR and 2-LTR circles and integration in BLM silenced Jurkat cells infected with wt or vpu* VSV-G pseudotyped pHIV-1-NL4-3. Bars represent means ± SEM, n=3 biologically independent experiments in triplicates, (m) *p=0.0156, **p=0.0039; (n) *p=0.0313, **p=0.0039, (o) **p=0.0039. Analysis done 48 h post-infection. (p) Supernatants from m-o were taken to analyze infectious yields determined by β-galactosidase assay in TZM-bl cells. Bars represent means ± SEM, n=2 biologically independent experiments in triplicates, *p=0.0313. Two sided Wilcoxon matched-pairs test in a-d, m-p.