The HIV1 Protein Vpr Acts to Enhance Constitutive DCAF1-Dependent UNG2 Turnover

Abstract

BackgroundThe HIV1 protein Vpr assembles with and acts through an ubiquitin ligase complex that includes DDB1 and cullin 4 (CRL4) to cause G2 cell cycle arrest and to promote degradation of both uracil DNA glycosylase 2 (UNG2) and single-strand selective mono-functional uracil DNA glycosylase 1 (SMUG1). DCAF1, an adaptor protein, is required for Vpr-mediated G2 arrest through the ubiquitin ligase complex. In work described here, we used UNG2 as a model substrate to study how Vpr acts through the ubiquitin ligase complex. We examined whether DCAF1 is essential for Vpr-mediated degradation of UNG2 and SMUG1. We further investigated whether Vpr is required for recruiting substrates to the ubiquitin ligase or acts to enhance its function and whether this parallels Vpr-mediated G2 arrest.Methodology/Principal FindingsWe found that DCAF1 plays an important role in Vpr-independent UNG2 and SMUG1 depletion. UNG2 assembled with the ubiquitin ligase complex in the absence of Vpr, but Vpr enhanced this interaction. Further, Vpr-mediated enhancement of UNG2 degradation correlated with low Vpr expression levels. Vpr concentrations exceeding a threshold blocked UNG2 depletion and enhanced its accumulation in the cell nucleus. A similar dose-dependent trend was seen for Vpr-mediated cell cycle arrest.Conclusions/SignificanceThis work identifies UNG2 and SMUG1 as novel targets for CRL4DCAF1-mediated degradation. It further shows that Vpr enhances rather than enables the interaction between UNG2 and the ubiquitin ligase. Vpr augments CRL4DCAF1-mediated UNG2 degradation at low concentrations but antagonizes it at high concentrations, allowing nuclear accumulation of UNG2. Further, the protein that is targeted to cause G2 arrest behaves much like UNG2. Our findings provide the basis for determining whether the CRL4DCAF1 complex is alone responsible for cell cycle-dependent UNG2 turnover and will also aid in establishing conditions necessary for the identification of additional targets of Vpr-enhanced degradation.

Base Excision Repair, a Pathway Regulated by Posttranslational Modifications

“…Similarly to UNG2, SMUG1 can be ubiquitylated in vivo by Cul1-and Cul4-based ubiquitin ligases, with the Cul4 complex likewise dependent on the presence of DDB1 (20,21). The Vpr accessory protein of HIV-1 has been shown to interact with Cul4A within the DDB1-Cul4A complex and induces the degradation of both UNG2 and SMUG1, presumably via the ubiquitin proteasome pathway (UPP) (20)(21)(22). As mentioned above for UNG2, the relevance of Cul1/Cul4-based ubiquitin ligases in modulating SMUG1 protein levels required for cellular BER has not yet been studied.…”

Vpr Enhances Tumor Necrosis Factor Production by HIV-1-Infected T Cells

“…Besides the SLX4com, Vpr interacts with numerous cellular proteins, but the relevance of these interactions is not always clear (66). For instance, Vpr degrades UNG2, a cellular protein involved in the removal of misincorporated uracil in DNA and for which a role during the HIV-1 life cycle remains a subject of debate (67)(68)(69). Vpr also binds to TAK1, a kinase involved in NF-B signaling (70,71).…”

Cullin4A and Cullin4B Are Interchangeable for HIV Vpr and Vpx Action through the CRL4 Ubiquitin Ligase Complex

“…2609) was obtained from Lee Ratner through the NIH AIDS Reagent Program (73). For plasmids, the LaminC-eGFP (enhanced green fluorescent protein) construct was a gift from Ronald Goldman, and the pcDNA3.1(Ϫ)HIV-2FLAG-huVpr and UNG2-2HA expression constructs were generated as described previously (31,38). The neddylation inhibitor MLN4924 was a gift from Millennium Pharmaceuticals.…”

“…Whether HIV-2/SIV Vpr acts similarly remains to be determined. HIV-1 Vpr also boosts the turnover of uracil-N-glycosylase 2 (UNG2) and single-strand-selective monofunctional uracil DNA glycosylase (SMUG1) through the CRL4 complex (38)(39)(40). The impact of G 2 cell cycle arrest or UNG2 and SMUG1 degradation on HIV replication and pathogenesis remains unclear; however, G 2 arrest has been linked to immune evasion (37) and increased viral production (41) by HIV-1.…”

“…Reports showing that SAMHD1 depletion is triggered in the nucleus (67-69) led us to posit that CUL4B is required for this phenotype as well. UNG2 can be found in both the cytoplasm and the nucleus (38), suggesting that HIV-1 Vpr-directed UNG2 degradation could be set off through CUL4A or CUL4B. In this work, we investigated the need for CUL4A and/or CUL4B for the Vpr and Vpx phenotypes that require the CRL4 ubiquitin ligase and show that the two CUL4 types are exchangeable for G 2 cell cycle arrest, UNG2 turnover, and SAMHD1 depletion.…”