Figure 8. DDB1 is required for the ability of Vpx to counteract the restriction to macrophage infection by HIV-1.

(A) SIV Vpx (or GFP as a control) was packaged into HIV-1_ΔVpr virions as described in Figure 5. Infectivity of those viruses for DDB1-depleted macrophages (DDB1 siRNA) or control macrophages (ScrΙ siRNA) were evaluated from levels of viral cDNA 24 h later (+, p>0.2; *, p<0.002). (B) DDB1 packaging partially substitutes for Vpx. A Vpx/Vpr deletion mutant of SIV (SIV_ΔXR) was co-transfected with vectors expressing SIV Vpr, SIV Vpx, DDB1 or a Vpr-DDB1 fusion. Infectivity of the resulting viruses for macrophages was evaluated from levels of SIV cDNA at 24 and 48 h post infection (*, p<0.005). (C) Impact of DDB1 silencing on Vpx ubiquitylation. 293T cells were cotransfected with DDB1 or scrambled siRNAs and with HIS-ubiquitin and HA-Vpx or IRES-GFP expression plasmids as outlined in Figure 3. Ubiquitin-conjugated proteins were nickel purified and immunoblotted for Vpx (HA). Cell lysates were directly blotted for Vpx and DDB1 proteins (lower two panels).