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

Measurement of the charge asymmetry in top-antitop quark production with the CDF II experiment

The Fermi National Laboratory (Fermilab) operates the Tevatron proton-antiproton collider at a center-of-mass energy of {radical}s = 1.96 TeV, the is therefore the only collider which is today able to produce the heaviest known particle, the top quark. The top quark was discovered at the Tevatron by the CDF and D0 collaborations in 1995. At the Tevatron, most top quarks are produced via the strong interaction, whereby quark-antiquark annihilation dominates with 85%, and gluon fusion contributes with 15%. Considering next-to-leading order (NLO) contributions in the cross section of top-antitop quark production, leads to a slight positive asymmetry in the differential distribution of the production angle {alpha} of the top quarks. This asymmetry is due to the interference of certain NLO contributions. The charge asymmetry A in the cosine of {alpha} is predicted [14] to amount to 4-6%. Information about the partonic rest frame, necessary for a measurement of A in the observable cos {alpha}, is not accessible in the experiment. Thus, they use the rapidity difference of the top and the antitop quark as sensitive variable. This quantity offers the advantage of Lorentz invariance and is uniquely correlated with the cosine of {alpha}, justifying the choice of the rapidity difference to describe the behavior of cos {alpha}. In preparation for a measurement of the charge asymmetry, they conduct several Monte Carlo based studies concerning the effect of different event selection criteria on the asymmetry in the selected event samples. They observe a strong dependence of the measured asymmetry on the number of required jets in the particular event sample. This motivates further studies to understand the influence of additional gluon radiation, which leads to more than four observed jets in an event, on the rapidity distribution of the produced top quarks. They find, that events containing hard gluon radiation are correlated with a strong negative shift of the rapidity distribution of the top quarks. This leads to large negative values of the charge asymmetry in event samples that contain only events with exactly five, six or more jets. This finding requires a modification of the original analysis strategy, since an asymmetry measured in an inclusive sample will be a composition of the asymmetry in the four-jets and five-jets sub-samples. Therefore, they perform for the first time a measurement of the asymmetry separately in the exclusive four- and five-jets sub-samples to separate the contribution of hard gluon radiation to the asymmetry. They analyze a data sample, collected by the CDF II detector in the years 2002-2006, that corresponds to an integrated luminosity of about 955 pb{sup -1}

Differential sensitivities of tetherin isoforms to counteraction by primate lentiviruses

The mammalian antiviral membrane protein tetherin (BST2/CD317) can be expressed as two isoforms derived from differential translational initiation. The shorter isoform of the human protein (S-tetherin) lacks the first 12 amino acids of the longer (L-tetherin) cytoplasmic tail, which includes a tyrosine motif that acts as both an endocytic recycling signal and a determinant of virus-induced NF-κB activation. S-tetherin is also reported to be less sensitive to the prototypic viral antagonist human immunodeficiency virus type 1 (HIV-1) Vpu. Here we analyzed the relative sensitivities of L- and S-tetherins to primate lentiviral countermeasures. We show that the reduced sensitivity of S-tetherin to HIV-1 Vpu is a feature of all group M proteins, including those of transmitted founder viruses, primarily because it cannot be targeted for endosomal degradation owing to the truncation of its cytoplasmic tail. In contrast, both isoforms of the human and rhesus macaque tetherins display the same sensitivity to nondegradative lentiviral countermeasures of HIV-2 and SIVmac, respectively. Surprisingly, however, the Vpu proteins encoded by simian immunodeficiency viruses (SIVs) of African guenons, as well as that from recently isolated highly pathogenic HIV-1 group N, do not discriminate between tetherin isoforms. Together, these data suggest that the group M HIV-1 Vpu primarily adapted to target L-tetherin upon zoonotic transmission from chimpanzees, and further, we speculate that functions specifically associated with this isoform, such as proinflammatory signaling, play key roles in human tetherin's antiviral function in vivo. IMPORTANCE The ability of HIV-1 and related viruses to counteract a host antiviral protein, tetherin, is strictly maintained. The adaptation of the HIV-1 Vpu protein to counteract human tetherin is thought to have been one of the key events in the establishment of the HIV/AIDS pandemic. Recent evidence shows that tetherin is expressed as two isoforms and that Vpu preferentially targets the longer form. Here we show that unlike other virus-encoded countermeasures, such as those from primate viruses related to HIV-1, the enhanced ability to counteract the long tetherin isoform is conserved among HIV-1 strains that make up the majority of the human pandemic. This correlates with the ability of Vpu to induce long tetherin degradation. We speculate that functions associated with the human version of this isoform, such as an inflammatory signaling capacity, selected for Vpu's enhanced targeting of long tetherin during its adaptation to humans

Phosphorylation-defective Vpu phenocopies trafficking mutants.

<p>(A-B) 293T, 293T tetherin or Y6,8A tetherin cells were infected with VSV-G pseudotyped NL4.3 WT or mutant virus at an MOI of 0.8. (A) 48 hours post infection viral supernatants were assayed for infectivity using HeLa-TZMbl reporter cells as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005141#ppat.1005141.g001" target="_blank">Fig 1</a>. Error bars represent the standard deviation of three independent experiments. (B) Cell lysates and sucrose purified viral supernatants were subjected to SDS-PAGE and analyzed by Western blotting as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005141#ppat.1005141.g001" target="_blank">Fig 1</a>. (C) 293T tetherin cells were infected with NL4.3 HIV-1 WT, ΔVpu, Vpu LILI, Vpu ELV or Vpu 2/6A mutants at an MOI of 2. 48 hours post infection cell lysates were subjected to SDS-PAGE and analyzed by Western blotting for HSP90 and tetherin, and analyzed by LiCor quantitative imager. (D) 293T tetherin expressing cells were transfected with 50 ng of pCR3.1 Vpu-HA or indicated mutants. 16 hours post transfection cells were fixed and stained for HA (green) and the TGN marker TGN46 (red) and examined by widefield fluorescent microscopy. Panels are of representative examples. Bars = 10 μm. (E) Z stacks were taken of all cells (n = 15), images were deconvolved using the AutoQuant X3 software and Pearson’s correlations were calculated for all Z stacks using ImageJ. Results were analyzed by unpaired 2-tailed t-test—*** P = 10–5 or lower. (F) 293T, 293T tetherin or Y6,8A tetherin cells were infected with VSV-G pseudotyped NL4.3 WT at an MOI of 0.8. 6 hours post infection DMSO or 50 μM Tyrphostin was added to the medium. 48 hours post infection supernatants were assayed as in (A). (G) Cell lysates and sucrose purified viral supernatants were processed as in (B). (H) 293T tetherin cells were transfected with 2 μg pCR3.1 Vpu-HA or 2/6 Vpu-HA and treated with DMSO or 50 μM Tyrphostin for 24 h. Cell lysates were electrophoresed as before, or on a 8%, 50 μM Phos-tag gel to separate the phosphorylated species.</p

A proposed model for Vpu engagement of clathrin adaptors during tetherin counteraction.

<p>Vpu and tetherin interactions via TM/TM domain interactions and casein kinase II phosphorylation promote Vpu recruitment of AP-1 or AP-2. This allows the EXXXLV motif to bind to the σ subunit, and potentially through non-canonical interactions between its first alpha helix with the AP-1 or 2 μ subunits. In addition the YCRV motif in tetherin binds to the AP1μ. Thus tetherin/Vpu complexes are sorted into clathrin rich domains of the TGN or PM for subsequent trafficking and ubiquitination.</p

Functional rescue of Vpu phospho- and trafficking mutants by direct interaction with clathrin.

<p>(A) Schematic representation of Vpu CB chimera constructs. (B) 293T tetherin cells were transfected with NL4.3 ΔVpu proviral plasmid in combination with YFP expression vector and pCR3.1 Vpu, pCR3.1 Vpu CB or Vpu CB mut or Vpu mutants thereof. 48 hours post transfection infectivity of viral supernatants was determined on HeLa-TZMbl cells as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005141#ppat.1005141.g001" target="_blank">Fig 1</a>. Error bars represent standard deviation of three independent experiments. (C) Cell lysates and pelleted supernatant virions from (B) were harvested and subjected to SDS-PAGE and analyzed by Western blotting for HIV-1 p24CA, Vpu and HSP90, and analyzed by LiCor quantitative imager. (D) HeLa-TZMbl cells were co-transfected with pCR3.1 Vpu or indicated mutant and a GFP expression vector. Cell-surface tetherin levels were analyzed 48 hours post transfection by flow cytometry in the GFP positive cells. The percentages of tetherin surface expression levels are calculated from median fluorescence intensities.</p

Vpu interacts with clathrin adaptors AP-1 and AP-2 in tetherin-expressing cells.

<p>(A-C) 293T tetherin (A), 293T tetherin Y6,8A (B) or 293 rhesus tetherin (C) cells were transfected with pCR3.1 Vpu-HA, Vpu A14L/W22A-HA, Vpu ELV-HA, Vpu 2/6A-HA, Vpu LILI-HA or Vpu NE-HA mutants. 48 h post transfection, cells were lysed and cross-linked using PFA (0.05% w/v) and immunoprecipitated with anti-HA antibody. Total cell lysates and precipitates were subjected to SDS-PAGE and analyzed by Western blotting for Vpu-HA, tetherin, AP-1γ or AP-2α. Panels are of representative experiments. Histograms represent western blot quantification of the relative AP-1 or AP-2 binding normalized to input control. Error bars represent the standard deviation of three independent experiments.</p

Clathrin box rescue of Vpu mutants is dependent on tetherin’s Y6,8 sorting signal.

<p>(A-B) 293T tetherin STS or 293T tetherin Y6,8A cells were transfected with NL4.3 ΔVpu proviral plasmid in combination with YFP expression vector and increasing concentrations of pCR3.1 Vpu or indicated mutant. 48 hours post transfection infectivity of viral supernatants was determined on HeLa-TZMbl cells as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005141#ppat.1005141.g001" target="_blank">Fig 1</a>. (C) 293T tetherin or 293T tetherin Y6,8A cells were transfected as in (A) with pCR3.1 Vpu or the N54H,E55G (NE) mutant. 48 hours post transfection infectivity of viral supernatants was determined on HeLa-TZMbl cells as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005141#ppat.1005141.g001" target="_blank">Fig 1</a>. Error bars represent standard deviation of three independent experiments. (D) Cell lysates and pelleted supernatant virions from (A) were harvested and subjected to SDS-PAGE and analyzed by Western blotting for HIV-1 p24CA, Vpu and HSP90, and analyzed by LiCor quantitative imager.</p