Lentiviral Vpx accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection - PubMed (original) (raw)

Lentiviral Vpx accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection

Smita Srivastava et al. PLoS Pathog. 2008.

Abstract

Vpx is a small virion-associated adaptor protein encoded by viruses of the HIV-2/SIVsm lineage of primate lentiviruses that enables these viruses to transduce monocyte-derived cells. This probably reflects the ability of Vpx to overcome an as yet uncharacterized block to an early event in the virus life cycle in these cells, but the underlying mechanism has remained elusive. Using biochemical and proteomic approaches, we have found that Vpx protein of the pathogenic SIVmac 239 strain associates with a ternary protein complex comprising DDB1 and VprBP subunits of Cullin 4-based E3 ubiquitin ligase, and DDA1, which has been implicated in the regulation of E3 catalytic activity, and that Vpx participates in the Cullin 4 E3 complex comprising VprBP. We further demonstrate that the ability of SIVmac as well as HIV-2 Vpx to interact with VprBP and its associated Cullin 4 complex is required for efficient reverse transcription of SIVmac RNA genome in primary macrophages. Strikingly, macrophages in which VprBP levels are depleted by RNA interference resist SIVmac infection. Thus, our observations reveal that Vpx interacts with both catalytic and regulatory components of the ubiquitin proteasome system and demonstrate that these interactions are critical for Vpx ability to enable efficient SIVmac replication in primary macrophages. Furthermore, they identify VprBP/DCAF1 substrate receptor for Cullin 4 E3 ubiquitin ligase and its associated protein complex as immediate downstream effector of Vpx for this function. Together, our findings suggest a model in which Vpx usurps VprBP-associated Cullin 4 ubiquitin ligase to enable efficient reverse transcription and thereby overcome a block to lentivirus replication in monocyte-derived cells, and thus provide novel insights into the underlying molecular mechanism.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Conserved amino acid residues in Vpx C-terminal region mediate the association with DDA1-DDB1-VprBP complex.

(A) hfa-tagged wild type (lane 2) or mutant (lanes 3–6) Vpx proteins were transiently expressed in HEK 293T cells and precipitated from detergent extracts with FLAG-M2 affinity resin. DDB1, VprBP, DDA1 and hfa-Vpx were detected in immune complexes (left panel) and cell extracts (right panel) by immunoblotting and visualized by enhanced chemiluminescence. (B) Amino acid sequences of the C-terminal regions of SIVmac 239 Vpx, SIVmac 239 Vpr, and HIV-1 NL43 Vpr are aligned and amino acid substitutions for the conserved residues in Vpx are indicated. Numbers indicate the positions of the first amino acid residue shown in each protein sequence. Dots identify amino acid identities and letters specify amino acids in the single-letter code.

Figure 2. VprBP links Vpx to Cullin 4.

myc-tagged Cullin 4A (m-Cul4) was coexpressed with FLAG- (f-) or myc- (m-) tagged Vpx, VprBP, DDB2 in HEK 293T cells, as indicated. Protein complexes were immunoprecipitated via their FLAG-tagged f-Vpx, f-VprBP, or f-DDB2 subunit, and resolved by SDS-PAGE. f- and m-tagged polypeptides were detected in detergent extracts (extr) and immune precipitates (IP) by immunoblotting with anti-FLAG or anti-myc epitope antibodies, respectively. Asterisk (*) indicated a background band that corresponds to the heavy chain of the anti-FLAG IgG.

Figure 3. Q76A and F80A substitutions in Vpx disrupt ability of SIVmac 239 to transduce macrophages.

(A) Q76A and F80A substituted Vpx are efficiently incorporated into SIVmac virions. Reference VSV-G pseudotyped single cycle SIVmac 239(GFP) virions containing decreasing amounts of wild type Vpx were produced from HEK 293T cells transiently transfected with the SIVmac 239(GFP) proviral clone containing wild type vpx gene alone (lane 1), or co-transfected with isogenic proviral clones containing wild type or inactivated vpx genes, and mixed at 1∶3 (0.25, lane 2), 1∶7 (0.12, lane 3) or 1∶15 (0.06, lane 4) ratios. Partially purified virions were immunoblotted for Vpx (Vpx) and p27 Capsid (CA p27). (B) Human monocyte-derived adherent macrophages (MF, panels 1–8), primary CD4+ T lymphocytes activated with PHA and IL-2 (CD4+, panels 9–16), and Jurkat T cells (JK, panels 17–24) were infected with normalized amounts of VSV-G pseudotyped single cycle SIVmac 239(GFP) virions containing various amounts of wild type (panels 2–6, 10–14, 18–22), or Q76A (panels 7, 15, 23) and F80A (panels 8, 16, 24) substituted Vpx proteins, or mock infected (panels 1, 9 and 17). Cells were harvested 4 days (MF), or 2 days (CD4+ and JK), following infection and GFP expression in the transduced populations was analyzed by flow cytometry. Bivariant plots of GFP expression (abscissa) versus forward scatter (ordinate) are shown. Percent fractions of GFP-positive cells (boxed) are indicated. Of note, SIVmac 239(GFP) containing wild type Vpx transduced between 5% and 60% of macrophages and Vpx enhanced transduction between 20-fold and 100-fold in 6 independent experiments, which probably reflects the donor-dependent variability of macrophage populations used.

Figure 4. HIV-2 Rod Vpx facilitates macrophage transduction through its interaction with VprBP.

(A) HIV-2 Rod Vpx binds VprBP/DCAF1. hfa-tagged wild type (lanes 1 and 4) or Q76A substituted (lane 2 and 5) HIV-2 Rod Vpx proteins were transiently expressed in HEK 293T cells and precipitated from detergent extracts with FLAG-M2 affinity resin. VprBP and hfa-Vpx were detected in immune complexes (left panel) and cell extracts (right panel) by immunoblotting and visualized by enhanced chemiluminescence. (B) Wild type and Q76A substituted HIV-2 Rod Vpx proteins are incorporated into SIVmac virions. VSV-G pseudotyped single cycle SIVmac 239(GFP) virions were produced from HEK 293T cells transiently coexpressing wild type (lane 2) or Q76A mutated (lane 3) Rod Vpx, SIVmac 239(GFP) proviral clone with disrupted vpx and env genes, and VSV-G. Virions were partially purified and analyzed for Vpx and p27 Capsid by Western blotting. (C) Human monocyte-derived macrophages (MF, panels 1–4), and Jurkat T cells (JK, panels 5–8) were infected with normalized amounts of viruses characterized in (B) above, or mock infected (panels 1 and 5), and GFP positive cells were determined after 4 days (MF), or 2 days (JK), by flow cytometry. Percent fractions of GFP-positive cells (boxed) are indicated.

Figure 5. Reverse transcription of SIVmac 239(GFP) virions comprising Vpx(Q76A) and Vpx(F80A) in macrophages is compromised.

(A) Location of reverse transcription intermediates used to gauge the progression of reverse transcription of SIVmac 239(GFP) genome. Regions amplified by “early”, “U3, “gag” and “late” sets of oligonucleotide primers are represented by grey boxes. The thin line represents viral RNA and the locations of the R, U5, primer binding site (PBS), packaging signal (Ψ), polypurine tract (PPT) and U3 regions are indicated. The thick arrows represent viral cDNA. (B) Steady state levels of reverse transcription intermediates following infection with VSV-G pseudotyped single cycle SIVmac 239(GFP) virions deficient in Vpx. Macrophages and Jurkat T cells were infected with the reference set of SIVmac 239(GFP) virions containing various amounts of wild type Vpx, characterized in Figure 3. DNA was prepared from the infected cells 18 hour or 72 hours following infection and 50 ng aliquots were analyzed in duplicate by real time PCR with oligonucleotide primers that recognize “early”(E), “U3”(3), “gag”(G) and “late”(L) reverse transcription products, as indicated below the histograms. The amounts of reverse transcription products were calculated by comparison to standard curves generated with serially diluted SIVmac 239 proviral DNA and are shown in red and blue for the 18-hour and 72-hour timepoint, respectively. The variances between duplicate data points were less than 4%. (C) Q76A and F80A substitutions disrupt Vpx function in macrophages. Macrophages were not infected (u) or infected with single cycle SIVmac 239(GFP) virions containing Q76A (76) or F80A (80) substituted, wild type (V+), or no Vpx (V−), characterized in Figure 3A, and reverse transcription was analyzed with “early” and “late” primers as described above.

Figure 6. VprBP is important for the ability of SIVmac 239 to transduce macrophages.

(A) Depletion of VprBP levels in macrophages by RNAi. Detergent extracts prepared from macrophages (lane 1) transfected with a control non-targeting siRNA (lane 2, scr) or siRNA pool targeting human VprBP (lane 3, VBP) four days after initiation of RNAi were analyzed by immunoblotting with rabbit anti-VprBP IgG, or with an antibody to the α-subunit of the AP-2 clathrin adaptor complex (α-ad), to control for equal loading. (B) and (C) VprBP-depleted macrophages resist SIVmac 239 infection. Macrophages transfected with the indicated amounts of VprBP-targeting (panels 3 and 5) or non-targeting (panels 4 and 6) siRNA pools and nontransfected macrophages (panel 2) were infected with VSV-G pseudotyped SIVmac 239(GFP) virus two days after initiation of RNAi. Flow cytometric analysis of GFP expression (B) and real time PCR quantification of gag DNA (C) in the transduced populations were performed 3 days later.

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Lentiviral Vpx accessory factor targets VprBP/DCAF1 substrate adaptor for cullin 4 E3 ubiquitin ligase to enable macrophage infection - PubMed (original) (raw)