Serine Phosphorylation of HIV-1 Vpu and Its Binding to Tetherin Regulates Interaction with Clathrin Adaptors

HIV-1 Vpu prevents incorporation of tetherin (BST2/ CD317) into budding virions and targets it for ESCRT-dependent endosomal degradation via a clathrin-dependent process. This requires a variant acidic dileucine-sorting motif (ExxxLV) in Vpu. Structural studies demonstrate that recombinant Vpu/tetherin fusions can form a ternary complex with the clathrin adaptor AP-1. However, open questions still exist about Vpu's mechanism of action. Particularly, whether endosomal degradation and the recruitment of the E3 ubiquitin ligase SCFβTRCP1/2 to a conserved phosphorylated binding site, DSGNES, are required for antagonism. Re-evaluation of the phenotype of Vpu phosphorylation mutants and naturally occurring allelic variants reveals that the requirement for the Vpu phosphoserine motif in tetherin antagonism is dissociable from SCFβTRCP1/2 and ESCRT-dependent tetherin degradation. Vpu phospho-mutants phenocopy ExxxLV mutants, and can be rescued by direct clathrin interaction in the absence of SCFβTRCP1/2 recruitment. Moreover, we demonstrate physical interaction between Vpu and AP-1 or AP-2 in cells. This requires Vpu/tetherin transmembrane domain interactions as well as the ExxxLV motif. Importantly, it also requires the Vpu phosphoserine motif and adjacent acidic residues. Taken together these data explain the discordance between the role of SCFβTRCP1/2 and Vpu phosphorylation in tetherin antagonism, and indicate that phosphorylation of Vpu in Vpu/tetherin complexes regulates promiscuous recruitment of adaptors, implicating clathrin-dependent sorting as an essential first step in tetherin antagonism

Identification of Interferon-Stimulated Genes with Antiretroviral Activity

SummaryInterferons (IFNs) exert their anti-viral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). The activity of known ISGs is insufficient to account for the antiretroviral effects of IFN, suggesting that ISGs with antiretroviral activity are yet to be described. We constructed an arrayed library of ISGs from rhesus macaques and tested the ability of hundreds of individual macaque and human ISGs to inhibit early and late replication steps for 11 members of the retroviridae from various host species. These screens uncovered numerous ISGs with antiretroviral activity at both the early and late stages of virus replication. Detailed analyses of two antiretroviral ISGs indicate that indoleamine 2,3-dioxygenase 1 (IDO1) can inhibit retroviral replication by metabolite depletion while tripartite motif-56 (TRIM56) accentuates ISG induction by IFNα and inhibits the expression of late HIV-1 genes. Overall, these studies reveal numerous host proteins that mediate the antiretroviral activity of IFNs

Residual activity of Vpu ELV requires an intact recycling signal in tetherin.

<p>(<b>A</b>) 293T/tetherin or 293T/tetherin Y6,8A mutant were infected with VSV-G-pseudotyped HIV-1 wt, HIV-1 delVpu or HIV Vpu ELV at an MOI of 0.5. Supernatants and cell lysates were harvested 48 h later and analyzed for infectious virus release on HeLa-TZMbl as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002609#ppat-1002609-g001" target="_blank">Figure 1C</a>. (<b>B</b>) Corresponding Western blots of cell lysates and virions from <b>A</b>. (<b>C</b>) 293T/tetherin Y6,8A cells treated with control or UBAP1 specific siRNAs were infected with VSV-G-pseudotyped HIV-1 wt, HIV Vpu ELV, HIV-1 Vpu A14L/W22A or HIV-1 delVpu at an MOI of 2. 48 h post infection, cells were lysed and immunoprecipitated with an anti-tetherin antibody. Lysates and immunoprecipitates were subjected to SDS-PAGE and analyzed by Western Blotting for tetherin, UBAP1 and Vpu, and analyzed by LiCor quantitative imager. Ratios of Vpu/tetherin band intensities in the coIP are plotted on the histogram below.</p

A Cytoplasmic Tail Determinant in HIV-1 Vpu Mediates Targeting of Tetherin for Endosomal Degradation and Counteracts Interferon-Induced Restriction

<div><p>The HIV-1 accessory protein Vpu counteracts tetherin (BST-2/CD317) by preventing its incorporation into virions, reducing its surface expression, and ultimately promoting its degradation. Here we characterize a putative trafficking motif, EXXXLV, in the second alpha helix of the subtype-B Vpu cytoplasmic tail as being required for efficient tetherin antagonism. Mutation of this motif prevents ESCRT-dependent degradation of tetherin/Vpu complexes, tetherin cell surface downregulation, but not its physical interaction with Vpu. Importantly, this motif is required for efficient cell-free virion release from CD4+ T cells, particularly after their exposure to type-1 interferon, indicating that the ability to reduce surface tetherin levels and promote its degradation is important to counteract restriction under conditions that the virus likely encounters <em>in vivo</em>. Vpu EXXXLV mutants accumulate with tetherin at the cell surface and in endosomal compartments, but retain the ability to bind both β-TrCP2 and HRS, indicating that this motif is required for a post-binding trafficking event that commits tetherin for ESCRT-dependent degradation and prevents its transit to the plasma membrane and viral budding zones. We further found that while Vpu function is dependent on clathrin, and the entire second alpha helix of the Vpu tail can be functionally complemented by a clathrin adaptor binding peptide derived from HIV-1 Nef, none of the canonical clathrin adaptors nor retromer are required for this process. Finally we show that residual activity of Vpu EXXXLV mutants requires an intact endocytic motif in tetherin, suggesting that physical association of Vpu with tetherin during its recycling may be sufficient to compromise tetherin activity to some degree.</p> </div

Vpu ELV mutants are defective for tetherin degradation and cell-surface downregulation.

<p>(<b>A</b>) HT1080 cells stably expressing tetherin-HA were infected with VSV-G-pseudotyped HIV-1 wt, HIV-1 delVpu or HIV-1 Vpu ELV at an MOI of 2. 48 h post infection, cells were harvested and subjected to SDS-PAGE and analyzed by Western blotting for tetherin-HA, Vpu and Hsp90, and analyzed by LiCor quantitative imager. Relative tetherin-HA levels are indicated below each lane. The blot shown is a representative example of 3 independent experiments. (<b>B</b>) 293T cells were transfected with pCR3.1 Vpu-HA, Vpu 2/6A-HA, or Vpu ELV-HA in combination with pCR3.1 myc-β-TrCP2. 48 h post transfection, cells were lysed and immunoprecipitated with anti-HA antibody. Lysates and precipitates were subjected to SDS-PAGE and analyzed by Western blotting for Vpu and myc-β-TrCP2, and analyzed by ImageQuant. The star represents an unspecific band. (<b>C</b>) Similarly, 293T cells were transfected with pCR3.1 HA-HRS in combination with Vpu-GFP or Vpu ELV-GFP expression constructs. Cell lysates were precipitated with an anti-HA antibody and analyzed as in <b>C</b>. (<b>D</b>) HeLa cells were co-transfected with pCR3.1 Vpu-HA or indicated Vpu mutant in combination with a GFP expression construct. Cell surface staining for endogenous tetherin was analyzed by flow cytometry 48 h post transfection. GFP positive cells were gated and tetherin levels (solid lines) were compared to those of untransfected HeLa cells (dotted lines). Numbers indicate median fluorescence intensities of surface tetherin on transfected cells. The solid peak in the upper middle histogram represents the binding of the isotype control. (<b>E</b>) Jurkat cells were infected with VSV-G-pseudotyped HIV-1 wt, HIV-1 delVpu, HIV-1 Vpu ELV or HIV-1 Vpu 2/6A at an MOI of 1. 48 h post infection, cells were stained for cell surface tetherin and intracellular p24CA, and analyzed by flow cytometry. Productively infected cells were identified by comparing with culture infected with the same MOI in the presence of 50 µM AZT to control for p24CA uptake of the inoculum (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002609#ppat.1002609.s001" target="_blank">Figure S1C</a>).</p

Model for the role of the EXXXLV motif in tetherin antagonism.

<p>Tetherin is expressed at the plasma membrane where it can become incorporated into viral particles or recycles constitutively via early/sorting endosomal compartments and the TGN. Vpu interacts with tetherin in the TGN (and perhaps earlier) through TM-domain-mediated interactions. In the presence of a functional EXXXLV motif, tetherin/Vpu complexes are prevented from trafficking to the PM and routed for ESCRT-dependent endosomal degradation via a clathrin-dependent mechanism. In the absence of an EXXXLV motif, tetherin/Vpu complexes recycle via the PM dependent on the YXYXXV sorting sequence in the tetherin cytoplasmic tail, which interacts with AP-2 and AP-1. During the recycling process, physical interaction of Vpu and/or modification by ubiquitin ligases, such as SCF-β-TrCP2, may further interfere with tetherin function to a variable degree in the absence of cell-surface downregulation.</p

The EXXXLV motif is essential to counteract tetherin-mediated restriction of cell-free HIV-1 particle release from CD4+ T cells treated with type-1 interferon.

<p>Jurkat cells were infected with the indicated HIV-1 mutant at an MOI of 1. 16 h later the cells were treated or not with 5000 U/ml universal type-I interferon. Cell lysates and viral supernatants were harvested a further 24 h later and analyzed for infectivity on HeLa-TZM (<b>A</b>) or physical particle yield and cellular viral and tetherin expression by quantitative Western blotting (<b>B</b>). (<b>C</b>) A representative example of primary human CD4+ T cells treated as in (<b>B</b>) and the MFI of surface tetherin levels on these cells with or without 24 h type-I interferon treatment as analyzed by flow cytometry (<b>D</b>). (<b>E</b>) Human CD4+ T cells were infected with the indicated virus. 48 h later cells were stained for surface tetherin and intracellular p24CA and analyzed by flow cytometry.</p

E59, L63 and V64 in the second alpha helix of the Vpu cytoplasmic tail are required to efficiently counteract tetherin.

<p>(<b>A</b>) Schematic representation of alanine scan mutagenesis in the second alpha helix of the codon-optimized HIV-1 NL4.3 Vpu protein. (<b>B</b>) 293T cells were transfected with NL4.3 wt or NL4.3 delVpu proviruses in combination with tetherin and the indicated pCR3.1 Vpu-HA expression vectors. 48 h post transfection, cell lysates and pelleted supernatant virions were harvested and subjected to SDS-PAGE and analyzed by Western blotting for HIV-1 p24CA, Vpu-HA and Hsp90 serving as loading control, and analyzed by LiCor quantitative imager. (<b>C</b>) Viral supernatants from <b>B</b> were assayed for infectivity using HeLa-TZMbl reporter cells. Infectious virus release is plotted on a log scale as β-galactosidase activity in relative light units (RLU). Error bars represent standard deviations of the means of three independent experiments. (<b>D</b>) E59A, L63A and V64A mutations were inserted into the <i>vpu</i> gene of the NL4.3 provirus referred to as NL4.3 Vpu ELV. 293T cells were transfected with NL4.3 wt, NL4.3 delVpu or NL4.3 Vpu ELV proviral plasmids together with increasing doses of tetherin expression vector. The resulting infectivity was determined as in <b>C</b>, error bars represent standard deviations of the means of three independent experiments. (<b>E</b>) Cell lysates and pelleted viral supernatants from 0 ng and 100 ng tetherin input from <b>D</b> were subjected to SDS-PAGE and analyzed by Western blotting for HIV-1 p24CA and Hsp90, and analyzed by LiCor quantitative imager.</p

AP180c inhibits Vpu-mediated tetherin antagonism but AP-1, AP-2, AP-3 and retromer are dispensable.

<p>(<b>A</b>) 293T/tetherin were transfected with NL4.3 wt or NL4.3 delVpu proviral plasmids in combination with either YFP or increasing doses of an AP180c expression vector. 48 h post transfection, cell lysates and pelleted supernatant virions were harvested and subjected to SDS-PAGE and analyzed by Western blotting for HIV-1 p24CA, Vpu and Hsp90 serving as loading control, and analyzed by LiCor quantitative imager. (<b>B</b>) 293T or 293T/tetherin transfected with pCR3.1 Vpu-YFP with or without AP180c co-expression were fixed and imaged after 48 h (<b>C</b>) 293T cells stably expressing tetherin were transfected twice with pooled control or AP-2μ1 siRNAs and co-transfected with NL4.3 wt, NL4.3 delVpu or GFP expression vectors. Cell lysates and supernatants were analyzed by Western blotting 48 h later as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002609#ppat-1002609-g001" target="_blank">Figure 1B</a>. (<b>D</b>) 293T cells stably expressing tetherin were transfected twice with pooled control or siRNA pools against AP-3δ1 and AP-3μ1. 4 h post the second transfection the cells were infected with VSV-G-pseudotyped HIV-1 wt or HIV-1 delVpu virus stock at an MOI of 1. Cell lysates and supernatants were analyzed by Western blotting 48 h later as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002609#ppat-1002609-g001" target="_blank">Figure 1B</a>. (<b>E</b>) HeLa cells expressing a doxycycline-inducible shRNA hairpin against AP-1γ1were transfected twice with pooled control or siRNA pools against AP-1γ1. The cells were infected and analyzed as in <b>C</b>. (<b>F</b>) 293T cells stably expressing tetherin were transfected twice with pooled control or siRNA pools against the retromer subunit Vps26. Cells were infected and analyzed as in <b>C</b>. In all siRNA knockdown experiments, the % knockdown of the indicated protein as determined by the relative band intensity in the western is indicated below the blot panel.</p