DNA damage induced by topoisomerase inhibitors activates SAMHD1 and blocks HIV-1 infection of macrophages (2017)
Figure 1

Figure 1

Etoposide/Camptothecin induced DNA damage inhibits HIV-1 infection

Monocyte derived macrophages (MDM) were treated with 5μM etoposide (ETO) for 18h. Cells were stained for γH2AX and 53BP1 nuclear foci, acquired and analysed using the automated cell‐imaging system Hermes WiScan and ImageJ. On average 104 cells were acquired and analysed. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; paired t‐test). Scale bars, 10μm.

MDM were treated with increasing concentrations of etoposide (ETO) and camptothecin (CTH) 18h before infection. Cells were infected with VSV-G pseudotyped HIV-1 GFP in the presence of ETO/CTH and 104 cells in each experiment were recorded and analysed for GFP expression 48h post-infection using an automated cell‐imaging system Hermes WiScan and ImageJ. (n = 3, mean ± s.e.m).

MDM were treated with increasing concentrations of ETO and CTH for 66h. Cells were stained to distinguish between live and dead cells using LIVE/DEAD fixable Dead cell stain protocol. Percentages of live/dead cells were determined using automated cell‐imaging system Hermes WiScan and ImageJ. Addition of 20% ethanol for 10min into cells treated with ETO was used as positive control. (n = 3, mean ± s.e.m).

MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP viruses wild type (wt) HIV-1 capsid mutants (N74D or P90A), HIV-2, SIV sooty mangabey (SIVsm E543), replication competent Adenovirus type 5 AdV (AdV), Semliki Forest Virus (SFV) and HSV-1. The percentages of infected cells were determined using an automated cell‐imaging system Hermes WiScan and ImageJ and normalized to untreated control ~100%. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; ***P‐value ≤ 0.001; (ns) non‐significant, paired t‐test). Cells from a representative donor were used for immunoblotting.

MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with VSV-G pseudotyped HIV-1 GFP. 104 cells were recorded and analysed for GFP expression 48h post-infection using an automated cell‐imaging system Hermes WiScan and ImageJ. (n = 4, mean ± s.e.m; ***P‐value ≤ 0.001, paired t‐test).

MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with full-length replication competent macrophage tropic HIV-1 virus BaL. Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post‐infection by FACS (n = 3, mean ± s.e.m; ***P‐value ≤ 0.001, paired t‐test).

G-I MDM were treated with 5μM ETO for 18h and infected in the presence of ETO with HIV-1 BaL and DNA isolated 18 h post‐infection for qPCR.

Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).

2LTR circles. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05, paired t‐test).

Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; ***P‐value ≤ 0.001, paired t‐test).

MDM were treated with 5μM ETO for 18h and infected with HIV-1 BaL (3, 6, 12 ffu/cell). Cells were stained for intracellular p24 and the percentage of infected cells was quantified 48 h post‐infection by FACS (n = 3, mean ± s.e.m; ** P‐value ≤ 0.01; ***P‐value ≤ 0.001, paired t‐test).

MDM were treated with 5μM ETO for 18h, infected with HIV-1 BaL (3, 6, 12 ffu/cell) and DNA isolated 18 h post‐infection for qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).

MDM were treated with 5μM ETO for 18h, infected with HIV-1 BaL and DNA isolated 4, 6, and 18 h post‐infection for qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).