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Polarity changes in the transmembrane domain core of HIV-1 Vpu inhibits its anti-tetherin activity.

ABSTRACT: Tetherin (BST-2/CD317) is an interferon-inducible antiviral protein that restricts the release of enveloped viruses from infected cells. The HIV-1 accessory protein Vpu can efficiently antagonize this restriction. In this study, we analyzed mutations of the transmembrane (TM) domain of Vpu, including deletions and substitutions, to delineate amino acids important for HIV-1 viral particle release and in interactions with tetherin. The mutants had similar subcellular localization patterns with that of wild-type Vpu and were functional with respect to CD4 downregulation. We showed that the hydrophobic binding surface for tetherin lies in the core of the Vpu TM domain. Three consecutive hydrophobic isoleucine residues in the middle region of the Vpu TM domain, I15, I16 and I17, were important for stabilizing the tetherin binding interface and determining its sensitivity to tetherin. Changing the polarity of the amino acids at these positions resulted in severe impairment of Vpu-induced tetherin targeting and antagonism. Taken together, these data reveal a model of specific hydrophobic interactions between Vpu and tetherin, which can be potentially targeted in the development of novel anti-HIV-1 drugs.

SUBMITTER: Lv M

PROVIDER: S-EPMC3107245 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Polarity changes in the transmembrane domain core of HIV-1 Vpu inhibits its anti-tetherin activity.

Lv Mingyu M Wang Jiawen J Wang Xiaodan X Zuo Tao T Zhu Yingzi Y Kong Wei W Yu Xianghui X

PloS one 20110602 6

Tetherin (BST-2/CD317) is an interferon-inducible antiviral protein that restricts the release of enveloped viruses from infected cells. The HIV-1 accessory protein Vpu can efficiently antagonize this restriction. In this study, we analyzed mutations of the transmembrane (TM) domain of Vpu, including deletions and substitutions, to delineate amino acids important for HIV-1 viral particle release and in interactions with tetherin. The mutants had similar subcellular localization patterns with tha ...[more]

PMID: 21674066

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Project description:BackgroundThe acquisition of effective Vpu-mediated anti-tetherin activity to promote virion release following transmission of SIVcpzPtt from central chimpanzees (Pan troglodytes troglodytes) to humans distinguishes pandemic HIV-1 group M strains from non-pandemic group N, O and P viruses and may have been a prerequisite for their global spread. Some functional motifs in the cytoplasmic region of HIV-1 M Vpus proposed to be important for anti-tetherin activity are more frequently found in the Vpu proteins of SIVcpzPtt than in those of SIVcpzPts infecting eastern chimpanzees (P. t. schweinfurthii), that have not been detected in humans, and SIVgor from gorillas, which is closely related to HIV-1 O and P. Thus, SIVcpzPtt strains may require fewer adaptive changes in Vpu than SIVcpzPts or SIVgor strains to counteract human tetherin.ResultsTo examine whether SIVcpzPtt may only need changes in the transmembrane domain (TMD) of Vpu to acquire anti-tetherin activity, whereas SIVcpzPts and SIVgor may also require changes in the cytoplasmic region, we analyzed chimeras between the TMD of an HIV-1 M Vpu and the cytoplasmic domains of SIVcpzPtt (n?=?2), SIVcpzPts (n?=?2) and SIVgor (n?=?2) Vpu proteins. Unexpectedly, all of these chimeras were capable of counteracting human tetherin to enhance virion release, irrespective of the presence or absence of the putative adaptor protein binding sites and the DSGxxS ?-TrCP binding motif reported to be critical for effective anti-tetherin activity of M Vpus. It was also surprising that in three of the six chimeras the gain of anti-tetherin function was associated with a loss of the CD4 degradation activity since this function was conserved among all parental HIV-1, SIVcpz and SIVgor Vpu proteins.ConclusionsOur results show that changes in the TMD of SIVcpzPtt, SIVcpzPts and SIVgor Vpus are sufficient to render them active against human tetherin. Thus, several previously described domains in the extracellular region of Vpu are not absolutely essential for tetherin antagonism but may be required for other Vpu functions.

Project description:Simian immunodeficiency viruses (SIVs) use their Nef proteins to counteract the restriction factor tetherin. However, a deletion in human tetherin prevents antagonism by the Nef proteins of SIVcpz and SIVgor, which represent the ape precursors of human immunodeficiency virus type 1 (HIV-1). To promote virus release from infected cells, pandemic HIV-1 group M strains evolved Vpu as a tetherin antagonist, while the Nef protein of less widespread HIV-1 group O strains acquired the ability to target a region adjacent to this deletion. In this study, we identified an unusual HIV-1 group O strain (RBF206) that evolved Vpu as an effective antagonist of human tetherin. While both RBF206 Vpu and Nef exert anti-tetherin activity in transient-transfection assays, mainly Vpu promotes RBF206 release in infected CD4+ T cells. Although mutations distinct from the adaptive changes observed in group M Vpus (M-Vpus) were critical for the acquisition of its anti-tetherin activity, RBF206 O-Vpu potently suppresses NF-κB activation and reduces CD4 cell surface expression. Interestingly, RBF206 Vpu counteracts tetherin in a largely species-independent manner, degrading both the long and short isoforms of human tetherin. Downmodulation of CD4, but not counteraction of tetherin, by RBF206 Vpu was dependent on the cellular ubiquitin ligase machinery. Our data present the first example of an HIV-1 group O Vpu that efficiently antagonizes human tetherin and suggest that counteraction by O-Nefs may be suboptimal.IMPORTANCE Previous studies showed that HIV-1 groups M and O evolved two alternative strategies to counteract the human ortholog of the restriction factor tetherin. While HIV-1 group M switched from Nef to Vpu due to a deletion in the cytoplasmic domain of human tetherin, HIV-1 group O, which lacks Vpu-mediated anti-tetherin activity, acquired a Nef protein that is able to target a region adjacent to the deletion. Here we report an unusual exception, identifying a strain of HIV-1 group O (RBF206) whose Vpu protein evolved an effective antagonism of human tetherin. Interestingly, the adaptive changes in RBF206 Vpu are distinct from those found in M-Vpus and mediate efficient counteraction of both the long and short isoforms of this restriction factor. Our results further illustrate the enormous flexibility of HIV-1 in counteracting human defense mechanisms.

Project description:The three-dimensional backbone structure of the transmembrane domain of Vpu from HIV-1 was determined by solid-state NMR spectroscopy in two magnetically-aligned phospholipid bilayer environments (bicelles) that differed in their hydrophobic thickness. Isotopically labeled samples of Vpu(2-30+), a 36-residue polypeptide containing residues 2-30 from the N-terminus of Vpu, were incorporated into large (q = 3.2 or 3.0) phospholipid bicelles composed of long-chain ether-linked lipids (14-O-PC or 16-O-PC) and short-chain lipids (6-O-PC). The protein-containing bicelles are aligned in the static magnetic field of the NMR spectrometer. Wheel-like patterns of resonances characteristic of tilted transmembrane helices were observed in two-dimensional (1)H/(15)N PISEMA spectra of uniformly (15)N-labeled Vpu(2-30+) obtained on bicelle samples with their bilayer normals aligned perpendicular or parallel to the direction of the magnetic field. The NMR experiments were performed at a (1)H resonance frequency of 900 MHz, and this resulted in improved data compared to lower-resonance frequencies. Analysis of the polarity-index slant-angle wheels and dipolar waves demonstrates the presence of a transmembrane alpha-helix spanning residues 8-25 in both 14-O-PC and 16-O-PC bicelles, which is consistent with results obtained previously in micelles by solution NMR and mechanically aligned lipid bilayers by solid-state NMR. The three-dimensional backbone structures were obtained by structural fitting to the orientation-dependent (15)N chemical shift and (1)H-(15)N dipolar coupling frequencies. Tilt angles of 30 degrees and 21 degrees are observed in 14-O-PC and 16-O-PC bicelles, respectively, which are consistent with the values previously determined for the same polypeptide in mechanically-aligned DMPC and DOPC bilayers. The difference in tilt angle in C14 and C16 bilayer environments is also consistent with previous results indicating that the transmembrane helix of Vpu responds to hydrophobic mismatch by changing its tilt angle. The kink found in the middle of the helix in the longer-chain C18 bilayers aligned on glass plates was not found in either of these shorter-chain (C14 or C16) bilayers.

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Polarity changes in the transmembrane domain core of HIV-1 Vpu inhibits its anti-tetherin activity.

Publications

Polarity changes in the transmembrane domain core of HIV-1 Vpu inhibits its anti-tetherin activity.

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