Figure 4

Neddylation is important for Vpr mediated depletion of UNG2 through CRL4. HEK293T cells were either mock treated or treated with 250 nM MLN4924 for four hours and infected with VSV-G-pseudotyped-HIV-1 or -HIV-1 with a frame shift mutation in Vpr. Twenty-four hours after infection, cells were harvested and the lysates immunoblotted for endogenous UNG2, HIV-1 p24 and tubulin (A endog.). HEK293T cells were transfected with an expression vector for UNG2 with two HA epitope tags (UNG2–2HA). Forty-eight hours later, cultures were mock treated or treated with 250 nM MLN4924 for four hours and then infected with VSV-G-pseudotyped-HIV-1 or -HIV-1 with a frame shift mutation in Vpr. Twenty-four hours later, cells were lysed and immunoblotted for the HA epitope, HIV-1 p24, and tubulin (A exog.). HEK293T cells were treated for 2 hours with concentrations of MLN4924 as indicated and then either mock infected or infected, in parallel, with a reduced MOI (approximately 2) of VSV-G-pseudotyped HIV-1. The cells were harvested for immunoblotting 24 and 48 hrs after infection. Blots were probed for endogenous UNG2, CUL4A, tubulin or HIV-1 p24 (B). Twenty-four hours after transfection with UNG2–2HA expression vector, HEK293T cells were treated and infected as described for panel B. Forty-eight hours after infection, cultures were harvested for western blot analysis and probed for UNG2–2HA (HA), CUL4A, HIV-1 p24, or tubulin (C). HEK293T cells were transfected with UNG2–2HA expression vector and either an expression vector (pcDNA3.1(−)) or ones for DN UBC12 or DN CUL4A. Twenty-four hours later cultures were mock infected or infected with VSV-G-pseudotyped-HIV-1. Twenty-four hours after infection cultures were lysed for immunoblotting and probed for UNG2–2HA (HA), endogenous UNG2, CUL4A, DN CUL4A (HA), DN UBC12, HIV-1 p24, or tubulin (D).