Figure 3

Vpx relieves SAMHD1-mediated restriction of MLV RT but does not allow for productive infection of non-dividing cells. (A) MDM were preincubated with Vpx-containing (+Vpx) or control VLP (−Vpx). The cells were then incubated with control supernatant (mock) or infected with VSV-G pseudotyped HIV-GFP, N-tropic MLV-GFP (N-MLV), or B-tropic MLV (B-MLV) at a MOI = 1 or (B) with increasing amounts of NB-tropic MLV-GFP (NB-MLV). (C) THP-1 cells that express shSAMHD1 (shSAMHD1) or control shRNA (shControl) were differentiated with PMA, preincubated with VLP and then infected with HIV-GFP or MLV-GFP at MOI = 1. (D) MDM were incubated with Vpx-containing (+Vpx) or control VLP (−Vpx). After 2 h, the cells were infected with control supernatant (mock) or with NB-tropic MLV reporter virus. Cellular DNA was isolated 24 and 48 h postinfection and used as a template in qPCR to amplify late reverse transcriptase products and 2-LTR circles. AZT was added to one well treated with Vpx-containing VLP prior to infection to control for contaminating plasmid. The data are presented as the average of triplicates with error bars indicating the standard deviation. Reporter gene expression in the infected cells was analyzed three days postinfection by flow cytometry. Similar results were obtained in three independent experiments with different donors.