FIGURE 4.

UNG2 is required for HIV-1 NLAD8 (R5) but not HIV-1 NL4. 3 (X4) infection and viral cDNA synthesis. PBMCs from pooled donors (A) or freshly isolated monocytes (B) were either mock nucleofected, nucleofected with a non-silencing control siRNA (siRandom), or nucleofected with a UNG2-specific siRNA (siUNG2). Monocytes were then cultured on plastic and differentiated to macrophages for 5 days. siRNA-treated cells were then used as target cells for infection with either 293T cell-derived NL43, NLAD8, or NLAD8 virus pseudotyped with the VSV envelope glycoprotein. Cells were lysed either 24 h (A) or 48 h (B) post infection, and HIV-1 cDNA synthesis was quantified by qPCR and normalized to cell number. Virus production from siRNA-treated PBMCs from pooled donors infected with VSV-G-pseudotyped NL4.3 and NLAD8 was measured was measured 4 days post infection by quantification of HIV-1 p24 (CA) protein in cell culture supernatants (C). Infectivity of NL4.3 and NLAD8 virus produced from siRNA-treated PBMCs was examined in freshly isolated PHA-stimulated PBMCs (NL43 and NLAD8) or MDMs (NLAD8) (D). Equivalent amounts of virus, as determined by HIV-1 p24 CA, were used for all infections. Cells were lysed either 24 h (PBMC) or 48 h (MDM) post infection and HIV-1 cDNA was quantified by qPCR and normalized to cell number. Data shown are means (±S.E.) from independent blood donors.