The C-Terminal End of HIV-1 Vpu Has a Clade-Specific Determinant That Antagonizes BST-2 and Facilitates Virion Release.

The cellular protein bone marrow stromal antigen-2 (BST-2)/tetherin acts against a variety of enveloped viruses by restricting their release from the plasma membrane. The HIV-1 accessory protein Vpu counteracts BST-2 by downregulating it from the cell surface and displacing it from virion assembly sites. Previous comparisons of Vpus from transmitted/founder viruses and between viruses isolated during acute and chronic infection led to the identification of a tryptophan at position 76 in Vpu (W76) as a key determinant for the displacement of BST-2 from virion assembly sites. Although present in Vpus from clades B, D, and G, W76 is absent from Vpus from clades A, C, and H. Mutagenesis of the C-terminal region of Vpu from two clade C viruses led to the identification of a conserved LL sequence that is functionally analogous to W76 of clade B. Alanine substitution of these leucines partially impaired virion release. This impairment was even greater when the mutations were combined with mutations of the Vpu ß-TrCP binding site, resulting in Vpu proteins that induced high surface levels of BST-2 and reduced the efficiency of virion release to less than that of virus lacking vpu Microscopy confirmed that these C-terminal leucines in clade C Vpu, like W76 in clade B, contribute to virion release by supporting the displacement of BST-2 from virion assembly sites. These results suggest that although encoded differently, the ability of Vpu to displace BST-2 from sites of virion assembly on the plasma membrane is evolutionarily conserved among clade B and C HIV-1 isolates.IMPORTANCE Although targeted by a variety of restriction mechanisms, HIV-1 establishes chronic infection in most cases, in part due to the counteraction of these host defenses by viral accessory proteins. Using conserved motifs, the accessory proteins exploit the cellular machinery to degrade or mistraffic host restriction factors, thereby counteracting them. The Vpu protein counteracts the virion-tethering factor BST-2 in part by displacing it from virion assembly sites along the plasma membrane, but a previously identified determinant of that activity is clade specific at the level of protein sequence and not found in the clade C viruses that dominate the pandemic. Here, we show that clade C Vpu provides this activity via a leucine-containing sequence rather than the tryptophan-containing sequence found in clade B Vpu. This difference seems likely to reflect the different evolutionary paths taken by clade B and clade C HIV-1 in human populations.

Membrane Anchoring by a C-terminal Tryptophan Enables HIV-1 Vpu to Displace Bone Marrow Stromal Antigen 2 (BST2) from Sites of Viral Assembly.

The restriction factor BST2 (tetherin) prevents the release of enveloped viruses from the host cell and is counteracted by HIV-1 Vpu. Vpu and BST2 interact directly via their transmembrane domains. This interaction enables Vpu to induce the surface down-regulation and the degradation of BST2, but neither of these activities fully accounts for the ability of Vpu to enhance virion release. During a study of naturally occurring Vpu proteins, we found that a tryptophan residue near the Vpu C terminus is particularly important for enhancing virion release. Vpu proteins with a W76G polymorphism degraded and down-regulated BST2 from the cell surface, yet they inefficiently stimulated virion release. Here we explore the mechanism of this anomaly. We find that Trp-76 is critical for the ability of Vpu to displace BST2 from sites of viral assembly in the plane of the plasma membrane. This effect does not appear to involve a general reorganization of the membrane microdomains associated with virion assembly, but rather is a specific effect of Vpu on BST2. Using NMR spectroscopy, we find that the cytoplasmic domain of Vpu and Trp-76 specifically interact with lipids. Moreover, paramagnetic relaxation enhancement studies show that Trp-76 inserts into the lipid. These data are consistent with a model whereby Trp-76 anchors the C terminus of the cytoplasmic tail of Vpu to the plasma membrane, enabling the movement of Vpu-bound BST2 away from viral assembly sites.

Activities of transmitted/founder and chronic clade B HIV-1 Vpu and a C-terminal polymorphism specifically affecting virion release.

UNLABELLED: Acute HIV-1 infection is characterized by a type I interferon response, resulting in the induction of host restriction factors. HIV-1 has evolved to counteract these factors, and one such adaptation, the ability of Vpu to counteract BST2/tetherin, is associated with the evolution of simian immunodeficiency virus (SIVcpz) into pandemic group M human immunodeficiency virus type 1 (HIV-1). During transmission between individuals, very few viruses or even a single virus, the "transmitted/founder" (T/F) virus, gives rise to the new infection, but in the new host the selective pressure of the immune response yields the diverse "quasispecies" of chronic infection. Here we examine the functional characteristics of Vpu proteins encoded by T/F viruses compared to acute and chronic viruses from longitudinally sampled subjects. The studied T/F Vpu proteins showed a trend toward optimized CD4 downregulation compared to chronic Vpu proteins but did not differ substantially in their ability to downregulate BST2 or enhance virion release, although individual clones from each group were impaired in these activities. Analysis of the functionally impaired clones identified a C-terminal residue, W76, as important specifically for Vpu enhancement of virion release. Primary Vpu clones encoding a W76G polymorphism, or site-directed mutants encoding a W76G substitution, were impaired in their ability to enhance virion release, but they were not defective for BST2 surface downregulation. Conversely, the virion release function of impaired primary clones was restored by creating a G76W substitution. The identification of W76 as important for virion release enhancement that is independent of BST2 surface downregulation supports the potential to mechanistically separate these functions of Vpu. IMPORTANCE: To establish infection in a host, HIV-1 must evade the host's immune response, including the production of antiviral factors. HIV-1 encodes proteins that antagonize these defenses, including Vpu. Vpu counteracts the host protein BST2, which blocks the release of progeny viruses from the host cell. To determine the importance of Vpu activity to HIV-1 transmission, this study assessed the functionality of Vpu from viruses isolated soon after transmission ("transmitted/founder" viruses) compared to isolates from chronic infection. Although the anti-BST2 activity of Vpu proteins from the tested transmitted/founder viruses did not differ from the activity of the chronic Vpu proteins, the transmitted/founder Vpu proteins trended toward having superior activity against another host protein, CD4. Further, this study identified an amino acid near the C terminus of Vpu that is specifically important for Vpu's ability to enhance the release of progeny virus from the host cell, supporting the notion of a new mechanism for this function of Vpu.