90K, an interferon-stimulated gene product, reduces the infectivity of HIV-1

BACKGROUND: In response to viral infections, interferons induce the transcription of several hundred genes in mammalian cells. Specific antiviral functions, however, have only been attributed to a few of them. 90K/LGALS3BP has been reported to be an interferon-stimulated gene that is upregulated in individuals with cancer or HIV-1 infection. RESULTS: Here, we show that 90K expression dose-dependently decreased the particle infectivity of HIV-1 progeny. The lower infectivity of released particles correlated with reduced virion incorporation of mature envelope glycoproteins gp120 and gp41. Further, proteolytic processing of the gp160 precursor and surface expression of gp120 in the producer cell were impaired in the presence of 90K expression. In contrast, expression of Gag, Nef and Vpu, and virus release were not grossly affected by 90K expression. 90K-imposed restriction occurred in the absence of direct interaction of 90K with HIV-1 Env or entrapment of Env in the ER. The cell-associated, but not the secreted species of 90K, mediated the antiviral effect. A truncated version of human 90K, solely consisting of the two intermediate domains, displayed a similar antiviral activity as the full-length wildtype 90K, indicating that the N-terminal SRCR-like domain and the C-terminal domain are dispensable for 90K’s antiviral activity. The murine homolog of 90K, CypCAP (Cyclophilin C-associated protein), neither modulated particle infectivity of HIV-1 nor lowered the virion incorporation of mature gp120, suggesting a species-specific mode of action. 90K was expressed at basal levels in TZM-bl cells and in primary macrophages, and at low levels in CD4(+) T-cells and PBMCs. 90K’s susceptibility to IFN-mediated stimulation of expression was cell type-specific. siRNA-mediated knockdown of 90K in TZM-bl cells and primary macrophages enhanced the incorporation of Env glycoproteins into progeny virions, boosted the particle infectivity of released HIV-1, and accelerated HIV-1 spread. Conversely, treatment of HIV-1 infected macrophages with IFN-α induced 90K expression and lowered the particle infectivity of HIV-1. CONCLUSIONS: Thus, 90K constitutes a novel antiviral factor that reduces the particle infectivity of HIV-1, involving interference with the maturation and incorporation of HIV-1 Env molecules into virions

The efficiency of Vpx-mediated SAMHD1 antagonism does not correlate with the potency of viral control in HIV-2-infected individuals

Background: The presence of a vpx gene distinguishes HIV-2 from HIV-1, the main causative agent of AIDS. Vpx degrades the restriction factor SAMHD1 to boost HIV-2 infection of macrophages and dendritic cells and it has been suggested that the activation of antiviral innate immune responses after Vpx-dependent infection of myeloid cells may explain why most HIV-2-infected individuals efficiently control viral replication and become long-term survivors. However, the role of Vpx-mediated SAMHD1 antagonism in the virological and clinical outcome of HIV-2 infection remained to be investigated. Results: Here, we analyzed the anti-SAMHD1 activity of vpx alleles derived from seven viremic and four long-term aviremic HIV-2-infected individuals. We found that effective Vpx-mediated SAMHD1 degradation and enhancement of myeloid cell infection was preserved in most HIV-2-infected individuals including all seven that failed to control the virus and developed AIDS. The only exception were vpx alleles from an aviremic individual that predicted a M68K change in a highly conserved nuclear localization signal which disrupted the ability of Vpx to counteract SAMHD1. We also found that HIV-2 is less effective than HIV-1 in inducing innate immune activation in dendritic cells. Conclusions: Effective immune control of viral replication in HIV-2-infected individuals is not associated with increased Vpx-mediated degradation of SAMHD1

Structural Basis for Tetherin Antagonism as a Barrier to Zoonotic Lentiviral Transmission.

Tetherin is a host defense factor that physically prevents virion release from the plasma membrane. The Nef accessory protein of simian immunodeficiency virus (SIV) engages the clathrin adaptor AP-2 to downregulate tetherin via its DIWK motif. As human tetherin lacks DIWK, antagonism of tetherin by Nef is a barrier to simian-human transmission of non-human primate lentiviruses. To determine the molecular basis for tetherin counteraction, we reconstituted the AP-2 complex with a simian tetherin and SIV Nef and determined its structure by cryoelectron microscopy (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 most other Nef substrates, including MHC class I, CD3, and CD4 but overlaps with the site for the restriction factor SERINC5. This structure explains the dependence of SIVs on tetherin DIWK and consequent barrier to human transmission

HIV-1 Nefs Are Cargo-Sensitive AP-1 Trimerization Switches in Tetherin Downregulation

The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, HIV-1 Nef, and the cytosolic tail of the restriction factor tetherin suggested a mechanism for inactivating tetherin by Golgi retention. The 4.3 Å structure of a mutant Nef-induced dimer of AP-1 showed how the closed trimer is regulated by the dileucine loop of Nef. HDX-MS and mutational analysis were used to show how cargo dynamics leads to alternative Arf1 trimerization, directing Nef targets to be either retained at the trans-Golgi or sorted to lysosomes. Phosphorylation of the NL4-3 M-Nef was shown to regulate AP-1 trimerization, explaining how O-Nefs lacking this phosphosite counteract tetherin but most M-Nefs do not. These observations show how the higher-order organization of a vesicular coat can be allosterically modulated to direct cargoes to distinct fates

HIV-1 Nefs Are Cargo-Sensitive AP-1 Trimerization Switches in Tetherin Downregulation.

The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, HIV-1 Nef, and the cytosolic tail of the restriction factor tetherin suggested a mechanism for inactivating tetherin by Golgi retention. The 4.3 Å structure of a mutant Nef-induced dimer of AP-1 showed how the closed trimer is regulated by the dileucine loop of Nef. HDX-MS and mutational analysis were used to show how cargo dynamics leads to alternative Arf1 trimerization, directing Nef targets to be either retained at the trans-Golgi or sorted to lysosomes. Phosphorylation of the NL4-3 M-Nef was shown to regulate AP-1 trimerization, explaining how O-Nefs lacking this phosphosite counteract tetherin but most M-Nefs do not. These observations show how the higher-order organization of a vesicular coat can be allosterically modulated to direct cargoes to distinct fates

HIV-1 Nefs Are Cargo-Sensitive AP-1 Trimerization Switches in Tetherin Downregulation.

The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, HIV-1 Nef, and the cytosolic tail of the restriction factor tetherin suggested a mechanism for inactivating tetherin by Golgi retention. The 4.3 Å structure of a mutant Nef-induced dimer of AP-1 showed how the closed trimer is regulated by the dileucine loop of Nef. HDX-MS and mutational analysis were used to show how cargo dynamics leads to alternative Arf1 trimerization, directing Nef targets to be either retained at the trans-Golgi or sorted to lysosomes. Phosphorylation of the NL4-3 M-Nef was shown to regulate AP-1 trimerization, explaining how O-Nefs lacking this phosphosite counteract tetherin but most M-Nefs do not. These observations show how the higher-order organization of a vesicular coat can be allosterically modulated to direct cargoes to distinct fates

Convergent Evolution of HLA-C Downmodulation in HIV-1 and HIV-2

Genome-wide association studies suggest that HLA-C expression is a major determinant of viral load set points and CD4+ T cell counts in HIV-infected individuals. On the one hand, efficient HLA-C expression enables the killing of infected cells by cytotoxic T lymphocytes (CTLs). On the other hand, HLA-C sends inhibitory signals to natural killer (NK) cells and enhances the infectivity of newly produced HIV particles. HIV-1 group M viruses modulate HLA-C expression using the accessory protein Vpu, possibly to balance CTL- and NK cell-mediated immune responses. Here, we show that the second human immunodeficiency virus, HIV-2, can use its accessory protein Vif to evade HLA-C-mediated restriction. Furthermore, our mutational analyses provide insights into the underlying molecular mechanisms. In summary, our results reveal how the two human AIDS viruses modulate HLA-C, a key component of the antiviral immune response.HLA-C-mediated antigen presentation induces the killing of human immunodeficiency virus (HIV)-infected CD4+ T cells by cytotoxic T lymphocytes (CTLs). To evade killing, many HIV-1 group M strains decrease HLA-C surface levels using their accessory protein Vpu. However, some HIV-1 group M isolates lack this activity, possibly to prevent the activation of natural killer (NK) cells. Analyzing diverse primate lentiviruses, we found that Vpu-mediated HLA-C downregulation is not limited to pandemic group M but is also found in HIV-1 groups O and P as well as several simian immunodeficiency viruses (SIVs). We show that Vpu targets HLA-C primarily at the protein level, independently of its ability to suppress NF-κB-driven gene expression, and that in some viral lineages, HLA-C downregulation may come at the cost of efficient counteraction of the restriction factor tetherin. Remarkably, HIV-2, which does not carry a vpu gene, uses its accessory protein Vif to decrease HLA-C surface expression. This Vif activity requires intact binding sites for the Cullin5/Elongin ubiquitin ligase complex but is separable from its ability to counteract APOBEC3G. Similar to HIV-1 Vpu, the degree of HIV-2 Vif-mediated HLA-C downregulation varies considerably among different virus isolates. In agreement with opposing selection pressures in vivo, we show that the reduction of HLA-C surface levels by HIV-2 Vif is accompanied by increased NK cell-mediated killing. In summary, our results highlight the complex role of HLA-C in lentiviral infections and demonstrate that HIV-1 and HIV-2 have evolved at least two independent mechanisms to decrease HLA-C levels on infected cells

CpG Frequency in the 5′ Third of the env Gene Determines Sensitivity of Primary HIV-1 Strains to the Zinc-Finger Antiviral Protein

Evasion of the zinc-finger antiviral protein (ZAP) may drive CpG dinucleotide suppression in HIV-1 and many other viral pathogens but the viral determinants of ZAP sensitivity are poorly defined. Here, we examined CpG suppression and ZAP sensitivity in a large number of primate lentiviruses and demonstrate that their genomic frequency of CpGs varies substantially and does not correlate with ZAP sensitivity. We further show that the number of CpG residues in a defined region at the 5′ end of the env gene together with structural features plays a key role in HIV-1 susceptibility to ZAP and correlates with differences in clinical progression rates in HIV-1-infected individuals. Our identification of a specific part of env as a major determinant of HIV-1 susceptibility to ZAP restriction provides a basis for future studies of the underlying inhibitory mechanisms and their potential relevance in the pathogenesis of AIDS.CpG dinucleotide suppression has been reported to allow HIV-1 to evade inhibition by the zinc-finger antiviral protein (ZAP). Here, we show that primate lentiviruses display marked differences in CpG frequencies across their genome, ranging from 0.44% in simian immunodeficiency virus SIVwrc from Western red colobus to 2.3% in SIVmon infecting mona monkeys. Moreover, functional analyses of a large panel of human and simian immunodeficiency viruses revealed that the magnitude of CpG suppression does not correlate with their susceptibility to ZAP. However, we found that the number of CpG dinucleotides within a region of ∼700 bases at the 5′ end of the env gene determines ZAP sensitivity of primary HIV-1 strains but not of HIV-2. Increased numbers of CpGs in this region were associated with reduced env mRNA expression and viral protein production. ZAP sensitivity profiles of chimeric simian-human immunodeficiency viruses (SHIVs) expressing different HIV-1 env genes were highly similar to those of the corresponding HIV-1 strains. The frequency of CpGs in the identified env region correlated with differences in clinical progression rates. Thus, the CpG frequency in a specific part of env, rather than the overall genomic CpG content, governs the susceptibility of HIV-1 to ZAP and might affect viral pathogenicity in vivo

The efficiency of Vpx-mediated SAMHD1 antagonism does not correlate with the potency of viral control in HIV-2-infected individuals

textabstractBackground: The presence of a vpx gene distinguishes HIV-2 from HIV-1, the main causative agent of AIDS. Vpx degrades the restriction factor SAMHD1 to boost HIV-2 infection of macrophages and dendritic cells and it has been suggested that the activation of antiviral innate immune responses after Vpx-dependent infection of myeloid cells may explain why most HIV-2-infected individuals efficiently control viral replication and become long-term survivors. However, the role of Vpx-mediated SAMHD1 antagonism in the virological and clinical outcome of HIV-2 infection remained to be investigated. Results: Here, we analyzed the anti-SAMHD1 activity of vpx alleles derived from seven viremic and four long-term aviremic HIV-2-infected individuals. We found that effective Vpx-mediated SAMHD1 degradation and enhancement of myeloid cell infection was preserved in most HIV-2-infected individuals including all seven that failed to control the virus and developed AIDS. The only exception were vpx alleles from an aviremic individual that predicted a M68K change in a highly conserved nuclear localization signal which disrupted the ability of Vpx to counteract SAMHD1. We also found that HIV-2 is less effective than HIV-1 in inducing innate immune activation in dendritic cells. Conclusions: Effective immune control of viral replication in HIV-2-infected individuals is not associated with increased Vpx-mediated degradation of SAMHD1