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

Although Nef is the viral gene product used by most simian immunodeficiency viruses to overcome restriction by tetherin, this activity was acquired by the Vpu protein of HIV-1 group M due to the absence of sequences in human tetherin that confer susceptibility to Nef. Thus, it is widely accepted that HIV-1 group M uses Vpu instead of Nef to counteract tetherin. Challenging this paradigm, we identified Nef alleles of HIV-1 group M isolates with significant activity against human tetherin. These Nef proteins promoted virus release and tetherin downmodulation from the cell surface and, in the context of vpu-deleted HIV-1 recombinants, enhanced virus replication and resistance to antibody-dependent cell-mediated cytotoxicity (ADCC). Further analysis revealed that the Vpu proteins from several of these viruses lack antitetherin activity, suggesting that under certain circumstances, HIV-1 group M Nef may acquire the ability to counteract tetherin to compensate for the loss of this function by Vpu. These observations illustrate the remarkable plasticity of HIV-1 in overcoming restriction by tetherin and challenge the prevailing view that all HIV-1 group M isolates use Vpu to counteract tetherin. IMPORTANCEMost viruses of HIV-1 group M, the main group of HIV-1 responsible for the global AIDS pandemic, use their Vpu proteins to overcome restriction by tetherin (BST-2 or CD317), which is a transmembrane protein that inhibits virus release from infected cells. Here we show that the Nef proteins of certain HIV-1 group M isolates can acquire the ability to counteract tetherin. These results challenge the current paradigm that HIV-1 group M exclusively uses Vpu to counteract tetherin and underscore the importance of tetherin antagonism for efficient viral replication.

Section: Discussionmentioning

confidence: 99%

Tetherin Antagonism by HIV-1 Group M Nef Proteins

Arias

Colomer-Lluch

Bredow

et al. 2016

“…In contrast, HIV-1 group M Vpu (M-Vpu) targets the long and short BST2 isoforms differentially using distinct mechanisms. While MVpu removes long BST2 molecules from the cell surface essentially via intracellular sequestration and degradation mechanisms, the short BST2 isoform is more resistant to M-Vpu-mediated downregulation and antagonism (23,28) and, as such, appears to be counteracted by displacement from sites of virus budding (11,(29)(30)(31). Interestingly, the ability of M-Vpu to displace residual BST2 molecules away from sites of virus budding was found to have a downregulatory role in the pDC IFN-I response since it promoted the engagement of BST2 with the ILT7 inhibitory receptor upon cell contacts between infected cells and pDCs (11).…”

mentioning

confidence: 99%

Differential Control of BST2 Restriction and Plasmacytoid Dendritic Cell Antiviral Response by Antagonists Encoded by HIV-1 Group M and O Strains

Bego

Cong

Mack

et al. 2016

BST2/tetherin is a type I interferon (IFN-I)-stimulated host factor that restricts the release of HIV-1 by entrapping budding virions at the cell surface. This membrane-associated protein can also engage and activate the plasmacytoid dendritic cell (pDC)-specific immunoglobulin-like transcript 7 (ILT7) inhibitory receptor to downregulate the IFN-I response by pDCs. Pandemic HIV-1 group M uses Vpu (M-Vpu) to counteract the two BST2 isoforms (long and short) that are expressed in human cells. MVpu efficiently downregulates surface long BST2, while it displaces short BST2 molecules away from viral assembly sites. We recently found that this attribute is used by M-Vpu to activate the BST2/ILT7-dependent negative-feedback pathway and to suppress pDC IFN-I responses during sensing of infected cells. However, whether this property is conserved in endemic HIV-1 group O, which has evolved Nef (O-Nef) to counteract specifically the long BST2 isoform, remains unknown. In the present study, we validated that O-Nefs have the capacity to downregulate surface BST2 and enhance HIV-1 particle release although less efficiently than M-Vpu. In contrast to M-Vpu, O-Nef did not efficiently enhance viral spread in T cell culture or displace short BST2 from viral assembly sites to prevent its occlusion by tethered HIV-1 particles. Consequently, O-Nef impairs the ability of BST2 to activate negative ILT7 signaling to suppress the IFN-I response by pDC-containing peripheral blood mononuclear cells (PBMCs) during sensing of infected cells. These distinctive features of BST2 counteraction by O-Nefs may in part explain the limited spread of HIV-1 group O in the human population. IMPORTANCEThe geographical distributions and prevalences of different HIV-1 groups show large variations. Understanding drivers of distinctive viral spread may aid in the development of therapeutic strategies for controlling the spread of HIV-1 pandemic strains. The differential spread of HIV-1 groups appears to be linked to their capacities to antagonize the long and short isoforms of the BST2 restriction factor. We found that the endemic HIV-1 group O-encoded BST2 antagonist Nef is unable to counteract the restriction mediated by short BST2, a condition that impairs its ability to activate ILT7 and suppress pDC antiviral responses. This is in contrast to the pandemic HIV-1 group M-specified BST2 countermeasure Vpu, which displays a diverse array of mechanisms to counteract short and long BST2 isoforms, an attribute that allows the effective control of pDC antiviral responses. These findings may help explain the limited spread of HIV-1 group O as well as the continued predominance of HIV-1 group M throughout the world. BST2/tetherin is a type I interferon (IFN-I)-inducible surface protein with an unusual topology. The protein consists of a N-terminal cytosolic tail followed by a transmembrane domain (TMD) and an ectodomain that is membrane associated through a C-terminal glycosylphosphatidylinositol (GPI) anchor (1). BST2 inhibits the release of a broad arr...

“…MLN4924 was from Active Biochem. pVphu (encoding codon-optimized Vpu), pCINL (expressing Nef), pCG-GFP (expressing green fluorescent protein [GFP]), pNL4-3, and related plasmids lacking intact vpu, nef, or vpu and nef genes or encoding vpu mutants have been described previously (33)(34)(35)(36)(37)(38)(39)(40). The plasmid expressing dominant negative (DN) cullin1 (pcDNA3-DN-hCUL1-FLAG; Addgene plasmid 15818) was deposited by Wade Harper (41).…”

Section: Methodsmentioning

confidence: 99%

“…We considered that the minimal effects of NAE inhibition on the Vpu-mediated downregulation of BST2 and consequently on the cell surface levels of Env and on ADCC might be due to relatively low levels of basal BST2 expression. Under such conditions, ubiquitin-mediated degradation of BST2 is potentially dispensable for Vpu's activity; Vpu can mediate surface downregulation of BST2 via endosomal trafficking (19,47), and mutational analyses suggest that Vpu can displace BST2 from sites of virion assembly to stimulate virion release without substantial BST2 downregulation (40,48). To test the hypothesis that NAE inhibition can upregulate BST2 and Env in cells infected with the wild-type virus when BST2 expression is relatively high, we treated CEM.NKR.luc cells with alpha interferon (here IFN), infected the cells with wild-type or vpu-negative virus, and measured the surface levels of BST2 and Env as detected by the 2G12 antibody ( Fig.…”

Section: Mln4924mentioning

confidence: 99%

“…We suspected that even when NAE inhibition together with IFN treatment increases BST2 on the surface of infected cells, Vpu can stimulate virion release via the displacement activity noted above such that cell surface Env remains relatively low (40,48). The ability of Vpu to displace BST2 from sites of virion assembly is independent of serines 52 and 56 and thus the ␤-TrCP containing cullin1-based ubiquitin ligase complex, but it depends on a C-terminal tryptophan (W76) in clade B virus (40). We tested whether this residue was important for the display of Env in infected CEM.NKR cells (Fig.…”

Section: Mln4924mentioning

confidence: 99%

See 1 more Smart Citation

Pharmacologic Inhibition of Nedd8 Activation Enzyme Exposes CD4-Induced Epitopes within Env on Cells Expressing HIV-1

Tokarev

Stoneham

Lewinski

et al. 2016

Self Cite

HIV-1 Vpu decreases the exposure of epitopes within the viral envelope glycoprotein (Env) on the surface of infected cells by downregulating both BST2 and CD4. To test the hypothesis that inhibiting Vpu activity would increase the exposure of these epitopes and sensitize infected cells to antibody-dependent cellular cytotoxicity (ADCC), we treated cells with the Nedd8 activation enzyme (NAE) inhibitor MLN4924, which inhibits the cullin1-based ubiquitin ligase complex coopted by Vpu to degrade cellular targets. Treatment of HeLa cells with MLN4924 or expression of a dominant negative mutant of cullin1 inhibited the Vpu-mediated downregulation of CD4 but not the downregulation of BST2. NAE inhibition also increased the surface exposure of CD4-induced epitopes within Env on HEK293 cells containing an inducible HIV genome, on infected CEM T cells, and on infected primary T cells. In contrast, the Vpu-mediated downregulation of BST2 was substantially inhibited by MLN4924 only when T cells were treated with alpha interferon (IFN-␣) to induce high levels of BST2 expression. As reported previously, the absence of vpu or nef and even more so the combined absence of these two genes sensitized infected cells to ADCC. However, NAE inhibition affected ADCC minimally. Paradoxically, even in infected, IFN-treated cells in which NAE inhibition substantially rescued the surface level of BST2, the surface level of Env detected with an antibody recognizing a CD4-independent epitope (2G12) was minimally increased. Mutation of the C-terminal Vpu residue W76, which supports the ability of Vpu to stimulate virion release by displacing BST2 from assembly sites on the plasma membrane by a cullin1-independent mechanism, increased the exposure of Env detected by 2G12 on infected T cells. Thus, inhibiting the displacement function of Vpu together with its ability to degrade CD4 and BST2 may be required to sensitize infected cells to ADCC. IMPORTANCEPathogenic viruses encode gene products that enable evasion of host immune surveillance mechanisms. One such mechanism is antibody-dependent cellular cytotoxicity (ADCC), whereby host antibodies bind envelope glycoproteins of the virus that are inserted into the cellular membrane and direct the destruction of infected cells. Targeting pharmacologically the activity of HIV-1 Vpu, which contributes to evasion of ADCC, could potentially sensitize infected cells to this immune surveillance mechanism, an outcome that would have therapeutic implications with respect to the goal of curing HIV-1 infection. The Nedd8 activation enzyme inhibitor MLN4924 blocks the activity of the host ubiquitin ligase that Vpu coopts to direct the degradation of CD4 and BST2. We observed that while MLN4924 partially reverses the activity of Vpu and could become part of a therapeutic approach by virtue of CD4-induced epitope exposure, sufficient Vpu activity as an antagonist of BST2 persists despite this drug to allow escape from ADCC.T he accessory proteins of HIV-1 remain undeveloped drug targets whose inhibition...