Lentiviral Vpx Accessory Factor Targets VprBP/DCAF1 Substrate Adaptor for Cullin 4 E3 Ubiquitin Ligase to Enable Macrophage Infection
Figure 6
VprBP is important for the ability of SIVmac 239 to transduce macrophages.
(A) Depletion of VprBP levels in macrophages by RNAi. Detergent extracts prepared from macrophages (lane 1) transfected with a control non-targeting siRNA (lane 2, scr) or siRNA pool targeting human VprBP (lane 3, VBP) four days after initiation of RNAi were analyzed by immunoblotting with rabbit anti-VprBP IgG, or with an antibody to the α-subunit of the AP-2 clathrin adaptor complex (α-ad), to control for equal loading. (B) and (C) VprBP-depleted macrophages resist SIVmac 239 infection. Macrophages transfected with the indicated amounts of VprBP-targeting (panels 3 and 5) or non-targeting (panels 4 and 6) siRNA pools and nontransfected macrophages (panel 2) were infected with VSV-G pseudotyped SIVmac 239(GFP) virus two days after initiation of RNAi. Flow cytometric analysis of GFP expression (B) and real time PCR quantification of gag DNA (C) in the transduced populations were performed 3 days later.