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

The 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.We 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.This work identifies UNG2 and SMUG1 as novel targets for CRL4(DCAF1)-mediated degradation. It further shows that Vpr enhances rather than enables the interaction between UNG2 and the ubiquitin ligase. Vpr augments CRL4(DCAF1)-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 CRL4(DCAF1) 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

Model for HIV1 Vpr-mediated degradation.

<p>In the absence of HIV1 Vpr expression, UNG2 engages DCAF1, is ubiquitinated and thus marked for proteasomal degradation (A). In the presence of HIV1 Vpr, CRL4^DCAF1-mediated ubiquitination of UNG2 is increased because Vpr enhances the interaction between UNG2 and DCAF1 (B). In the presence of high HIV1 Vpr levels, UNG2 degradation is no longer increased and instead, UNG2 accumulates in the cell nucleus (C).</p

HIV1 Vpr enhances the interaction between UNG2 and the CRL4^DCAF1 ubiquitin ligase complex.

<p>293T HEK cells were transfected with either UNG2–myc (5 µg, lanes 1 and 4), 5 µg of UNG2–2HA expression vector (lanes 2,3 5 and 6), together with empty vector or expression vector for HIV1 FLAG–Vpr (1.25 µg, lanes 3 and 6). At 48 hours post-transfection, cells were lysed and the lysates were incubated with HA-specific antibody linked to agarose beads. The bound proteins were eluted with HA peptide. The eluted proteins and pre-immunoprecipitation samples were characterized by immunoblotting with antibodies specific for DCAF1, DDB1, the HA epitope tag or the FLAG epitope tag.</p

HIV1 Vpr-mediated degradation and subcellular redistribution of UNG2 are dose-dependent.

<p>293T HEK cell cultures were transfected with empty vector alone and together with increasing quantities of HIV1 FLAG–Vpr expression vector as indicated. 48 hours after transfection nuclear and cytoplasmic fractions were prepared and analyzed by immunoblotting with antibodies specific for UNG2, FLAG epitope tag (FLAG–Vpr), α tubulin (cytoplasmic fraction control) and Histone H3 (nuclear fraction control) (A). 293T cultures were transfected with 1 µg of UNG2–HA expression vector alone, together with increasing quantities of HIV1 FLAG–Vpr expression vector as indicated. 48 hours after transfection nuclear and cytoplasmic fractions were prepared. Immunoblotting with anti-HA (UNG2–2HA), anti-FLAG (FLAG–Vpr), anti-α tubulin (cytoplasmic fraction control) and anti-Histone H3 (nuclear fraction control) antibodies was used to determine relative quantities of the respective protein that were present in the fractions (B). HEK 293T cells, either untransfected (left) or transfected with 1 µg of UNG2–2HA expression vector and 3 µg of empty vector (C, right) were mock-infected (none), infected with <i>vpr(–), env(–)</i>, VSV-G-pseudotyped virus (<i>vpr(–)</i>) or with wild-type, <i>env(–)</i>, VSV-G-pseudotyped virus (wild-type).</p

The pattern of Vpr-induced cell cycle arrest mirrors that of Vpr-mediated UNG2 depletion.

<p>293T HEK cells were transfected with empty vector or increasing amounts of HIV1 FLAG–Vpr expression vector as indicated. Total plasmid DNA in each transfection was kept constant by addition of empty vector. The cells in each sample were co-transfected with 175 ng of laminC−GFP to allow identification of nuclei from transfected cells by flow cytometry. The cell nuclei were isolated 48 hours after transfection, treated with RNaseA and stained with propidium iodide. The DNA content was determined by flow cytometry (A). Panel B shows the (G2+M)/G1 ratios for comparison.</p

HEK 293T cells were transfected with 1 µg of UNG2–2HA expression vector together with the indicated expression vectors.

<p>Forty-eight hours after transfection nuclear and cytoplasmic fractions were prepared. The composition of these fractions was characterized by immunoblotting with HA-, DDB1-,α tubulin- or anti-Histone H3-specific antibodies (A). 293T cells were transfected with UNG2–2HA expression vector, together with empty vector, or expression vector for wild type HIV1 FLAG–Vpr (3 µg). MG132 (12.5 µM) or DMSO (vehicle control) were added 24 hours after transfection. 16 hours later nuclear and cytoplasmic fractions were prepared and characterized by immunoblotting with anti-HA, anti-FLAG, anti-α tubulin (cytoplasmic fraction control) and anti-Histone H3 (nuclear fraction control) antibodies (B).</p

HIV1 Vpr-mediated UNG2 degradation is dose-dependent.

<p>293T HEK cells were transfected with empty vector or increasing amounts of HIV1 FLAG–Vpr expression vector as indicated. 24 hours later the cells were lysed and the expression levels of UNG2, UNG1, HIV1 FLAG–Vpr and β-actin were determined by immunoblotting (A). Quantitation of relative UNG1/2 degradation for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030939#pone-0030939-g003" target="_blank">Figure 3A</a> was plotted (B). 293T HEK cells were transfected with 1 µg of UNG2–2HA expression vector, together with empty vector or increasing amounts of HIV1 FLAG–Vpr expression vector as indicated. Twenty-four hours later cell lysates were prepared and subjected to immunoblotting with anti-HA, anti-FLAG and anti-β-actin antibodies (C). Quantitation of relative UNG2 degradation for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030939#pone-0030939-g003" target="_blank">Figure 3C</a> is shown in D. 293T HEK cells were transfected with 1 µg of UNG2–2HA expression vector, together with empty vector or increasing amounts of HIV1 FLAG–VprR90K expression vector as indicated. Forty-eight hours later cell lysates were prepared and subjected to immunoblotting with anti-HA, anti-FLAG and anti-β-actin antibodies (E). Quantitation of relative UNG2 degradation for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030939#pone-0030939-g003" target="_blank">Figure 3E</a> is shown (F) 293T HEK cells were transfected with 1 µg of UNG2–2HA expression vector, together with empty vector or increasing amounts of HIV1 FLAG–Vpr expression vector as indicated. Forty-eight hours later cell lysates were prepared and subjected to immunoblotting with anti-HA, anti-FLAG and anti-β-actin antibodies (G). Quantitation of relative UNG2 degradation for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030939#pone-0030939-g003" target="_blank">Figure 3G</a> is shown (H).</p

CRL4^DCAF1 is involved in constitutive turnover of UNG2 and SMUG1.

<p>Cultures of 293T HEK cells were transfected with 1 µg of UNG2–2HA expression vector, together with 2 µg of empty vector or 2 µg of an expression vector for DCAF1-directed shRNA and additional empty vector (1 µg) or HIV1 FLAG–Vpr expression vector (1 µg). 48 hours after transfection cell lysates were harvested and tested for expression of UNG2–2HA, HIV1 FLAG–Vpr, endogenous DCAF1 and β-actin by immunoblotting (A). 293T HEK cells were transfected with empty vector (4 µg), or expression vector for DDB1 (4 µg) or DDB1-directed shRNA (4 µg) as indicated. Fourty-eight hours after transfection cell lysates were prepared and tested for expression of endogenous UNG2, DDB1, and α-tubulin by immunoblotting (B). Cultures of 293T HEK cells were transfected with SMUG1-3HA expression vector (1 µg), together with empty vector (2 µg) or an expression vector for DCAF1-directed shRNA (2 µg) and additional empty vector (1 µg) or HIV1 FLAG–Vpr expression vector (1 µg). 48 hours after transfection cell lysates were harvested and tested for expression of SMUG1–3HA, HIV1 FLAG–Vpr, DCAF1 and β-actin by immunoblotting (C). 293T HEK cells were transfected with empty vector (1 µg) or SMUG1–3HA expression vector (1 µg) together with additional empty vector (3 µg) expression vectors for DDB1 (3 µg) or DDB1-directed shRNA (3 µg) as indicated. 48 hours after transfection cell lysates were prepared and tested for expression of SMUG1-3HA, DDB1, and β-actin by immunoblotting (D). 293T HEK cells were transfected with UNG2–2HA expression vector, together with empty vector or an expression vector for untagged DCAF1 or transfected with FLAG–HA–DCAF1 expression vector, together with empty vector or an expression vector for UNG2–2HA. After 48 hours, the cells were lysed and the cleared lysates were incubated with anti-FLAG agarose beads. The bound proteins were eluted with FLAG peptide. The eluted proteins and pre-immunoprecipitation samples were characterized by immunoblotting with HA-specific antibody (E).</p