HIV-1 and HIV-2 differentially regulate NF-κB activity during the late stages of the replication cycle through BST-2/tetherin antagonism

ABSTRACTHIV-2 is the second causative agent of AIDS and is commonly considered as an attenuated form of retroviral infection. Most of HIV-2-infected individuals display a slow-progressing disease, lower viral loads and a stronger immunological control of viral infection as compared with HIV-1-infected patients. The main hypothesis that could explain the difference of disease progression between HIV-1 and HIV-2 implies a more efficient T cell–mediated immunity in the control of HIV-2 infection. Herein, we investigate the effects of the HIV-2 envelope glycoprotein (Env) and its antitetherin function in the NF-κB signaling pathway during single-round infection of CD4+ T cells. First, we report an essential role of the Env cytoplasmic tail (CT) in the activation of this signaling pathway and we also demonstrate that the HIV-2 Env CT activates NF-κB in a TRAF6-dependent but TAK1-independent manner. Further, we show that HIV-2 reference strains and clinical isolates are unable to completely inhibit NF-κB mainly via the Env-mediated BST-2/tetherin antagonism in the late stages of the viral replication cycle in CD4+ T cells, in striking contrast to the HIV-1 Vpu-mediated counteraction of tetherin. We observe that this inability of HIV-2 to suppress NF-κB signaling pathway promotes stimulation of numerous genes involved in the antiviral immune response, such as il-6, il-21 and ifn-β genes. Therefore, HIV-1 and HIV-2 differentially regulate the NF-κB-induced antiviral immune response mainly through the BST-2/tetherin antagonism. These new insights highlight molecular mechanisms determining, at least partly, the distinct immune control and disease outcomes of HIV-1 and HIV-2 infections.IMPORTANCEThis study explores how HIV-1 and HIV-2 diverge in their regulation of the NF-κB signaling pathway. We revealed that HIV-2 fails to completely inhibit NF-κB activity, thereby inducing a stronger antiviral response than HIV-1. We demonstrated that the ability to antagonize the cellular restriction factor BST-2/tetherin largely governs the regulation of the NF-κB pathway: at the late stages of the viral replication cycle, HIV-1 Vpu blocks this pathway whereas HIV-2 Env does not. We also demonstrated that several NF-κB-targeted genes are upregulated in CD4+ T cells infected with HIV-2, but not with HIV-1. This stronger NF-κB-induced antiviral response may explain the better immune control of HIV-2 infection and the differences between HIV-1 and HIV-2 pathogenesis. Moreover, we observed in this study that non-pathogenic isolates of HIV-2 have an impaired NF-κB inhibitory capacity compared to pathogenic ones.

The Nef Protein of the Macrophage Tropic HIV-1 Strain AD8 Counteracts Human BST-2/Tetherin

Bone Marrow Stromal Cell Antigen 2 (BST-2)/tetherin inhibits the release of numerous enveloped viruses by physically tethering nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency virus (HIV), and pandemic main (M) group HIV type 1s (HIV-1s) are thought to rely exclusively on their Vpu proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express Vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 Nef protein can compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous cell surface tetherin levels, physically separating it from the site of viral budding, thus preventing HIV retention. Mechanistically, AD8 Nef enhances internalisation of the long isoform of human tetherin, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, we show that M group HIV-1s can use an accessory protein other than Vpu to antagonise human tetherin.

The Nef protein of the macrophage tropic HIV-1 strain AD8 counteracts human Bst-2/tetherin

Bst-2/tetherin inhibits the release of numerous enveloped viruses by physically attaching nascent particles to infected cells during the process of viral budding from the cell surface. Tetherin also restricts human immunodeficiency viruses (HIV), and pandemic main (M) group HIV-1s are thought to exclusively rely on their Vpu proteins to overcome tetherin-mediated restriction of virus release. However, at least one M group HIV-1 strain, the macrophage-tropic primary AD8 isolate, is unable to express vpu due to a mutation in its translation initiation codon. Here, using primary monocyte-derived macrophages (MDMs), we show that AD8 was able to use its Nef protein to compensate for the absence of Vpu and restore virus release to wild type levels. We demonstrate that HIV-1 AD8 Nef reduces endogenous tetherin levels from the cell surface, physically separating it from the site of viral budding and thus preventing HIV retention. Mechanistically, AD8 Nef enhances l-tetherin internalisation, leading to perinuclear accumulation of the restriction factor. Finally, we show that Nef proteins from other HIV strains also display varying degrees of tetherin antagonism. Overall, this is the first report showing that M group HIV-1s can use an accessory protein other than Vpu to antagonise human tetherin.

Structural basis for tetherin antagonism as a barrier to zoonotic lentiviral transmission

Tetherin is a host defense that physically prevents escape of virions from the plasma membrane. Human tetherin lacks the motif DIWK antagonized by SIV, the antecedent of HIV. Here, we reconstituted the AP-2 clathrin adaptor complex with a simian tetherin and SIV Nef and determined its structure by cryo-EM. Nef refolds the first α-helix of the β2 subunit of AP-2 to a β hairpin, creating a binding site for the DIWK sequence. The tetherin binding site in Nef is distinct from those of MHC-I, CD3, and CD4, but overlaps the site for SERINC5 restricting viral infectivity. The structure explains the dependence of SIVs on the host tetherin DIWK sequence and the consequent barrier to human transmission.

A GXXXA Motif in the Transmembrane Domain of the Ebola Virus Glycoprotein Is Required for Tetherin Antagonism

ABSTRACTThe interferon-induced antiviral host cell protein tetherin can inhibit the release of several enveloped viruses from infected cells. The Ebola virus (EBOV) glycoprotein (GP) antagonizes tetherin, but the domains and amino acids in GP that are required for tetherin antagonism have not been fully defined. A GXXXA motif within the transmembrane domain (TMD) of EBOV-GP was previously shown to be important for GP-mediated cellular detachment. Here, we investigated whether this motif also contributes to tetherin antagonism. Mutation of the GXXXA motif did not impact GP expression or particle incorporation and only modestly reduced EBOV-GP-driven entry. In contrast, the GXXXA motif was required for tetherin antagonism in transfected cells. Moreover, alteration of the GXXXA motif increased tetherin sensitivity of a replication-competent vesicular stomatitis virus (VSV) chimera encoding EBOV-GP. Although these results await confirmation with authentic EBOV, they indicate that a GXXXA motif in the TMD of EBOV-GP is important for tetherin antagonism. Moreover, they provide the first evidence that GP can antagonize tetherin in the context of an infectious EBOV surrogate.IMPORTANCEThe glycoprotein (GP) of Ebola virus (EBOV) inhibits the antiviral host cell protein tetherin and may promote viral spread in tetherin-positive cells. However, tetherin antagonism by GP has so far been demonstrated only with virus-like particles, and it is unknown whether GP can block tetherin in infected cells. Moreover, a mutation in GP that selectively abrogates tetherin antagonism is unknown. Here, we show that a GXXXA motif in the transmembrane domain of EBOV-GP, which was previously reported to be required for GP-mediated cell rounding, is also important for tetherin counteraction. Moreover, analysis of this mutation in the context of vesicular stomatitis virus chimeras encoding EBOV-GP revealed that GP-mediated tetherin counteraction is operative in infected cells. To our knowledge, these findings demonstrate for the first time that GP can antagonize tetherin in infected cells and provide a tool to study the impact of GP-dependent tetherin counteraction on EBOV spread.

The Tetherin Antagonism of the Ebola Virus Glycoprotein Requires an Intact Receptor-Binding Domain and Can Be Blocked by GP1-Specific Antibodies

ABSTRACT The glycoprotein of Ebola virus (EBOV GP), a member of the family Filoviridae , facilitates viral entry into target cells. In addition, EBOV GP antagonizes the antiviral activity of the host cell protein tetherin, which may otherwise restrict EBOV release from infected cells. However, it is unclear how EBOV GP antagonizes tetherin, and it is unknown whether the GP of Lloviu virus (LLOV), a filovirus found in dead bats in Northern Spain, also counteracts tetherin. Here, we show that LLOV GP antagonizes tetherin, indicating that tetherin may not impede LLOV spread in human cells. Moreover, we demonstrate that appropriate processing of N-glycans in tetherin/GP-coexpressing cells is required for tetherin counteraction by EBOV GP. Furthermore, we show that an intact receptor-binding domain (RBD) in the GP1 subunit of EBOV GP is a prerequisite for tetherin counteraction. In contrast, blockade of Niemann-Pick disease type C1 (NPC1), a cellular binding partner of the RBD, did not interfere with tetherin antagonism. Finally, we provide evidence that an antibody directed against GP1, which protects mice from a lethal EBOV challenge, may block GP-dependent tetherin antagonism. Our data, in conjunction with previous reports, indicate that tetherin antagonism is conserved among the GPs of all known filoviruses and demonstrate that the GP1 subunit of EBOV GP plays a central role in tetherin antagonism. IMPORTANCE Filoviruses are reemerging pathogens that constitute a public health threat. Understanding how Ebola virus (EBOV), a highly pathogenic filovirus responsible for the 2013-2016 Ebola virus disease epidemic in western Africa, counteracts antiviral effectors of the innate immune system might help to define novel targets for antiviral intervention. Similarly, determining whether Lloviu virus (LLOV), a filovirus detected in bats in northern Spain, is inhibited by innate antiviral effectors in human cells might help to determine whether the virus constitutes a threat to humans. The present study shows that LLOV, like EBOV, counteracts the antiviral effector protein tetherin via its glycoprotein (GP), suggesting that tetherin does not pose a defense against LLOV spread in humans. Moreover, our work identifies the GP1 subunit of EBOV GP, in particular an intact receptor-binding domain, as critical for tetherin counteraction and provides evidence that antibodies directed against GP1 can interfere with tetherin counteraction.

Tetherin Antagonism by HIV-1 Group M Nef Proteins

ABSTRACTAlthough 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 ofvpu-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.