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. 2005 Sep;79(17):10978-87.
doi: 10.1128/JVI.79.17.10978-10987.2005.

Human immunodeficiency virus type 1 Vpr induces the degradation of the UNG and SMUG uracil-DNA glycosylases

Bärbel Schröfelbauer  1 , Qin Yu, Samantha G Zeitlin, Nathaniel R Landau
Affiliations

Human immunodeficiency virus type 1 Vpr induces the degradation of the UNG and SMUG uracil-DNA glycosylases

Bärbel Schröfelbauer et al. J Virol. 2005 Sep.

Abstract

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr has previously been shown to bind to the cellular uracil DNA glycosylase UNG. We show here that the binding of Vpr to UNG and to the related enzyme SMUG induces their proteasomal degradation. UNG and SMUG were found to be encapsidated in Deltavpr HIV-1 virions but were significantly less abundant in vpr(+) virions. Deltavpr virions contained readily detectable uracil-DNA glycosylase enzymatic activity, while the activity was reduced to undetectable levels in vpr(+) virions. Consistent with proteasomal degradation, complexes that contained Vpr and the E3 ubiquitin ligase components Cul1 and Cul4 were detected in cell lysates. We hypothesized that the interaction of Vpr might be a means for the virus to reduce the frequency of abasic sites in viral reverse transcripts at uracil residues caused by APOBEC3-catalyzed deamination of cytosine residues. Although APOBEC3 is largely neutralized by the Vif accessory protein, residual enzyme could remain in virions that would generate uracils. In support of this, Deltavif vpr(+) HIV-1 produced in the presence of limited amounts of APOBEC3G was significantly more infectious than Deltavif Deltavpr virus. In Addition, vpr(+) HIV-1 replicated more efficiently than vpr(-) virus in cells that expressed limited amounts of APOBEC3G. The findings highlight the importance of cytidine deamination in the virus replication cycle and present a novel function for Vpr.

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Figures

FIG. 1.
FIG. 1.
Encapsidation of UNG and SMUG is reduced by Vpr. (A) Vpr reduces UNG and SMUG encapsidation. Wild-type and Δvpr virions were produced in 293T cells cotransfected with HA-tagged UNG or SMUG expression vector. The virions (50 ng p24) were analyzed on an immunoblot probed with anti-HA MAb to detect UNG and SMUG (top right), anti-Vpr MAb (middle left and right), and anti-HIV-1 serum (bottom right). The cell lysates were analyzed in parallel (top left). Controls lacking viral DNA are labeled “no virus.” (B) Vpr reduces intracellular UNG and SMUG. 293T cells were cotransfected with UNG expression vector and decreasing amounts of pcHA.Vpr (left). The proteins were detected in the cell lysates by immunoblot analysis with anti-HA MAb. 293T cells were cotransfected with HA-tagged SMUG or TDG expression vector, with or without 0.5 μg pcVpr (right), and detected with anti-HA MAb. The total amount of plasmid DNA in each transfection was kept constant. (C) Vpr reduces UNG more efficiently in the absence of p6. 293T cells were transfected with UNG.HA expression vector and the indicated proviral DNA. Cell lysates and virions were analyzed on immunoblots probed with anti-HA MAb (top left and right) and anti-tubulin (bottom left) or anti-HIV-1 serum (bottom right). (D) Vpr reduces the UDG enzymatic activity of HIV-1 virions. The uracil glycosylase activities in wild-type and Δvpr sucrose gradient-purified virions were measured using a 5′-32P-labeled deoxyoligonucleotide containing a single uracil. Controls included recombinant UDG and mock virions (no virus) in which viral DNA was omitted from the transfection. (E) 5.25 cells were infected with WT or Δvpr NL4-3, harvested 6 days postinfection, and purified through a sucrose gradient. The uracil glycosylase activities of the virions were measured as for panel D.
FIG. 2.
FIG. 2.
Immunofluorescence detection of Vpr and endogenous UNG. HeLa cells were transfected with HA-Vpr expression vector. The cells were fixed and stained with anti-HA MAb (green) and anti-UNG serum (red). Upper left, UNG staining; upper right, Vpr staining; lower left, DAPI nuclear staining; lower right, merged UNG, Vpr, and DAPI. The cells shown are representative of those contained in several fields. Secondary-antibody controls yielded black fields.
FIG. 3.
FIG. 3.
Vpr induces ubiquitination and proteasomal degradation of UNG and SMUG. (A) Proteasome inhibitor blocks the effect of Vpr on UNG. 293T cells were cotransfected with UNG expression vector with (+) or without (−) pcHA.Vpr and then cultured for 16 h with DMSO, MG132, or clasto-lactacystin. Cell lysates were analyzed on immunoblots probed with anti-HA MAb, anti-Vpr MAb, and anti-tubulin MAb. (B) Vpr induces polyubiquitination of UNG and SMUG. 293T cells were transfected with UNG (left) or SMUG (right) expression vector, Vpr, and Ub-His6-myc or UbK48R-His6-myc expression vector. The cell lysates were incubated with Ni-nitrilotriacetic acid beads, and coprecipitated UNG and SMUG were detected on an immunoblot probed with anti-HA MAb. (C) Vpr reduces the half-life of SMUG. The stability of SMUG in the presence and absence of Vpr was determined by pulse-chase metabolic labeling. Bands were quantified on a phosphorimager, and cpm at time zero was normalized to 100.
FIG. 4.
FIG. 4.
Vpr interacts with UNG, SMUG, and Cullins. (A) Vpr interacts with UNG and SMUG. 293T cells were cotransfected with (+) UNG (left) or SMUG (right) expression vector with or without (−) Vpr expression plasmid and were then cultured for 16 h with MG132. Cell lysates were immunoprecipitated with anti-HA MAb, and the coimmunoprecipitated proteins were detected on an immunoblot probed with anti-Vpr MAb or anti-HA MAb. Vpr in the cell lysates was quantitated by immunoblot analysis with anti-Vpr MAb (bottom). (B) Vpr interacts with Cul1 and Cul4a. Vpr-Cullin complexes were detected by coimmunoprecipitation from 293T cells cotransfected with the indicated Cullin-myc and HA.Vpr expression vectors. Cell lysates were immunoprecipitated with anti-myc MAb, and the coimmunoprecipitated proteins were detected on an immunoblot probed with anti-HA MAb. The membrane was stripped and probed with anti-myc MAb to confirm Cullin expression. Vpr in the cell lysates was detected by probing with anti-HA MAb. (C) Vpr W54R does not decrease levels of UNG. 293T cells were cotransfected with UNG or SMUG expression vector and HA.Vpr, HA.VprW54R plasmid, or empty vector. Cell lysates were analyzed on an immunoblot probed with anti-HA MAb.
FIG. 5.
FIG. 5.
Vpr partially rescues Δvif HIV-1 infectivity. (A) Wild-type, Δvif, Δvpr, and Δvif Δvpr luciferase reporter viruses were produced in 293T cells cotransfected with the indicated amounts of human APOBEC3G expression vector. Luciferase activities generated by infection of HOS cells with 1 ng of each virus are shown. The error bars indicate standard deviations. (B) Same as panel A, but infectivity was measured in PBMCs. (C) Reporter viruses were generated in cells cotransfected with wild-type or W54R Vpr in trans with or without human APOBEC3G, and their infectivities were determined by luciferase assay. The data shown are representative of three independent repetitions of the experiment. (D) HOS.CD4.X4 (left) or HOS.CD4.X4 APOBEC3G.HA (right) cells were infected with the indicated viruses at a multiplicity of infection of 0.1. Virus production was determined at the indicated time points by p24 ELISA.
FIG. 6.
FIG. 6.
Vpr increases the copy number of late reverse transcription (RT) products. The relative copy number of reverse transcripts in cells newly infected with wild-type, Δvif, Δvpr, or Δvif Δvpr that had been generated in 293T cells cotransfected with or without APOBEC3G expression vector was determined by fluorescence-detected quantitative PCR. DNA was isolated from the infected cells at the indicated times postinfection, and the relative copy number of the reverse transcript was determined with primers specific for early or late products. The experiments shown are representative of at least two repetitions.
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