Fig 2: Exo1 and HLTF epistasis studies. (A) CEM.SS T cells were subjected to NT, Exo1-targeting, and/or HLTF-targeting RNAi, as indicated. PRCA with a 1:1 mixture of HIV-1.mRFP.vpr.wt and HIV-1.RFP.vpr.Q8* was initiated 3 d later, and cell-associated DNA for each of the competing viruses was quantified at 7 dpi. Fold-change (Left) and relative percentages (Right) of cell-associated HIV-1 DNA copies per cell for the competing viruses are shown at 7 dpi. A representative of three experiments is shown. Only P values for HIV-1.RFP.vpr.Q8* lower than 0.05 are shown: *P < 0.05; **P < 0.01. (B) Exo1 and HLTF levels in extracts from cells subjected to RNAi assessed at the time of HIV-1 infection were revealed by immunoblotting. Lamin B1 provided a loading control.

Fig 3: HIV-1 Vpr antagonizes HLTF-mediated restriction of HIV-1 replication in T cells. (A) HLTF restricts HIV-1 replication. CEM.SS T cells harboring doxycycline-inducible codon-optimized HLTF transgene (CEM.SS_iHLTFo) were subjected to NT or targeting endogenous HLTF mRNA (HLTF) RNAi in the absence or presence of the indicated concentrations of doxycycline (ng/mL). (Left) Three days after initiation of RNAi, PRCA was performed with a 1:1 mixture of HIV-1.mRFP.vpr.wt and HIV-1.RFP.vpr.Q8*. (Right) As a control, PRCA was also performed in parental CEM.SS T cells cultured in the absence or presence of doxycycline. (Upper) Cell-associated DNA of the competing viruses was quantified at 7 dpi. (Middle) Fold-change in HIV-1 DNA copies in cells subjected to HLTF RNAi, normalized to those in cells subjected to NT RNAi. (Lower) Percentages of cell-associated HIV-1 DNA for the competing viruses. (B) HLTF levels in CEM.SS_iHLTFo and parental CEM.SS T cell populations used in experiments shown in A were revealed by immunoblotting. Lamin B1 provided a loading control. (C) HLTF depletion partially restores the HIV-1 replication fitness advantage provided by Vpr. CEM.SS T cells were subjected to NT or HLTF-targeting (HLTF) RNAi and infected with a 1:1 mixture of HIV-1.mRFP.vpr.wt and HIV-1.RFP.vpr.Q8*. Percentages of cell-associated DNA for the competing viruses at 7 dpi (Left) and fold-change in HIV-1 DNA copies per cell in cells subjected to HLTF-targeting RNAi normalized to those in cells subjected to NT RNAi (Right) are shown. The data shown are from five biological replicate experiments. ***P < 0.001.

Fig 4: Pulsed SILAC Method to Identify Direct Targets for Vpr-Mediated Degradation(A) Graphical summary of the pulsed SILAC experiment.(B) Scatterplots showing the changes to protein stability of proteins after 6 or 24 h of exposure to a Vpr-bearing lentivirus compared with a control lentivirus, with previously defined groups of Vpr-depleted (blue) and -increased (red) proteins highlighted.(C) Expanded view of proteins degraded within 6 h of Vpr exposure. Significantly degraded (significance B [Sig.B] < 0.01) proteins are highlighted in gold. The previously described Vpr targets HLTF, MUS81, and ZGPAT are shown in purple.

Fig 5: HIV-1 Vpr depletes Exo1 levels in CD4+ T cells. (A) HIV-1 infection depletes Exo1 in primary CD4+ T cells in a Vpr-dependent manner. Human peripheral blood CD4+ T cells were activated with a-CD3/a-CD28 beads and 2 days later challenged with HIV-1 NL4-3.GFP.R+ (or R–) single-cycle viruses expressing GFP marker protein. Exo1, HLTF, MUS81, and UNG2/UNG1 levels in extracts from productively infected cells, isolated by cell sorting for GFP fluorescence, were characterized by immunoblotting. Twofold serial dilutions of control CD4+ T cell extracts provided quantification standards. Levels of HIV-1 Gag were also characterized to confirm similar infections with the R– and R+ viruses. TFIID provided a loading control. (B) Exo1 is rapidly depleted in cells expressing HIV-1 Vpr. U2OS-iH1vpr (iH1vpr) and control U2OS (mock) cells were cultured in the presence of doxycycline (100 ng/ml). Extracts prepared from the cells at the indicated times postaddition of doxycycline were analyzed by immunoblotting for Exo1 and other postreplication DNA repair proteins known to be antagonized by HIV-1 Vpr. The arrow indicates the position of the Vpr band, and the asterisk indicates a nonspecifically cross-reacting background band. (C) Exo1 is a conserved target of main group HIV-1 and SIVcpz Vpr proteins. Levels of Exo1, Vpr, and a tubulin loading control in extracts from U2OS cells induced with doxycycline (100 ng/ml) to express the HIV-1 M, N, or O main group consensus Vpr proteins or SIVcpz P. troglodytes troglodytes (Ptt) or SIVcpz P. troglodytes schweinfurthii (Pts) consensus Vpr proteins were revealed by immunoblotting. The cells were harvested 24 h postaddition of doxycycline. U2OS cells not expressing Vpr (–) and U2OS-iH1vpr cells expressing the HIV-1 NL4-3 vpr allele (NL) provided negative and positive controls, respectively. Tubulin (Tub) provided loading controls.