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

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

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-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

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

The SFN-mediated HIV infection block is reversible.

<p>(A), Study design: PMA-differentiated THP1 cells were pretreated with media supplemented with vehicle only (DMSO) or with 10 μM, 20 μM or 30 μM SFN. After twenty-four hours of treatment, the culture media was replaced with fresh SFN-free media. Cultures were infected 1, 2, 3, 4, or 5 days after treatment with freshly thawed aliquots of VSV-G pseudotyped HIV-1 encoding firefly luciferase in place of <i>nef</i>. Twenty-four hours after the start of each time-point infection, the cells were harvested and (B), luciferase activity was measured by photon emission. The bar graph represents the data for replicate experiments (n = 3).</p

SFN blocks infection after entry and but before 2-LTR circle formation.

<p>Replicate cultures of hMDMs were pretreated with vehicle (DMSO)-containing media, with 5 μM AZT or with 10 μM SFN. Twenty four hours after treatment, the samples were infected with VSV-G-pseudotyped HIV-1 encoding GFP in place of <i>nef</i>. Cultures treated with heat-inactivated virus served as controls for plasmid carry over and for impaired viral entry. Cells were harvested and DNA was isolated 24 hours after infection. Viral DNA products were detected by real-time PCR using primer sets specific for the indicated stage of reverse transcription. (A), Relative quantities of late reverse transcription products, (B), 2-LTR circles, and (C), integrated proviruses. The bar graph represents the data for replicate experiments (n = 3).</p

SFN does not trigger expression of interferon-stimulated anti-viral factors SAMHD1 or MX2.

<p>PMA-differentiated THP1 cells were mock-treated or treated with media supplemented with vehicle only (DMSO) or with 10 μM SFN or with 500 U/mL of IFNα. (A), Proteins from whole cell lysates were resolved by SDS-PAGE and identified by western blotting using antibodies with the indicated specificities. (B), Densitometric analysis was performed on the Nrf2, SAMHD1, MX2, NQO1 and GCLM (NQO1 and GCLM are both indicators of Nrf2 function) bands and normalized to the values of the corresponding tubulin bands. The relative normalized intensities of the bands were then graphed.</p