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.

Natural cystatin C fragments inhibit GPR15-mediated HIV and SIV infection without interfering with GPR15L signaling.

GPR15 is a G protein-coupled receptor (GPCR) proposed to play a role in mucosal immunity that also serves as a major entry cofactor for HIV-2 and simian immunodeficiency virus (SIV). To discover novel endogenous GPR15 ligands, we screened a hemofiltrate (HF)-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2, and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4+ T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this GPCR.

SERINC5 Can Enhance Proinflammatory Cytokine Production by Primary Human Myeloid Cells in Response to Challenge with HIV-1 Particles.

HIV-1 has to overcome physical barriers posed by host cell restriction factors (RFs) for efficient replication. Some RFs, including Trim5α and tetherin, trigger antiviral signaling in addition to directly impairing HIV replication. SERINC5 (S5) is an RF that is incorporated into HIV-1 particles to potently impair their infectivity and is efficiently antagonized by the viral pathogenesis factor Nef. Since effects of S5 on HIV-1 infectivity were mostly studied in reporter cell lines, we analyzed the effects of S5 during infection of primary HIV-1 target cells. In activated CD4+ T lymphocytes, virion incorporation of S5 only moderately impaired virion infectivity and was not associated with altered innate immune recognition. In contrast, in monocyte-derived macrophages, S5 virion incorporation potentiated the production of proinflammatory cytokines with very potent but donor-dependent effects on virion infectivity. Nef counteracted effects of S5 on both cytokine production and virion infectivity. Similar S5-induced cytokine production was observed in immature monocyte-derived dendritic cells. Notably, S5-mediated enhancement of cytokine production was not linked to the efficacy of productive infection and could be overcome by using vesicular stomatitis virus glycoprotein (VSV-G) but not infectivity restriction-insensitive HIV-1 Env for cell entry. Moreover, inhibiting entry of S5-negative HIV-1 ΔNef particles increased proinflammatory cytokine production comparably to virion incorporation of S5. Together, these results describe the sensitization of noninfectious HIV-1 particles to proinflammatory cytokine production by myeloid target cells as an additional and Nef-sensitive activity of S5. Moreover, the study reveals important cell-type and donor-dependent differences in the sensitivity of HIV target cells for antiviral effects of S5.IMPORTANCE SERINC5 (S5) is a host cell restriction factor (RF) that impairs the infectivity of HIV-1 particles in target cell lines. To assess the potential physiological relevance of this restriction, we assessed the effects of S5 on HIV-1 infection of relevant primary human target cells. We found that effects of S5 on infection of CD4+ T lymphocytes were negligible. In myeloid target cells, however, virion incorporation of S5 potently suppressed infectivity and promoted innate immune recognition of HIV-1 particles characterized by proinflammatory cytokine production. Both effects were not observed in cells of all donors analyzed, were exerted independently of one another, and were counteracted by the HIV-1 pathogenesis factor Nef. These results identify the sensitization of HIV-1 particles for innate immune recognition by myeloid target cells as a novel activity of S5 and emphasize the need to study RF function in the context of primary target cells and taking donor variabilities into account.

APOBEC3F Constitutes a Barrier to Successful Cross-Species Transmission of Simian Immunodeficiency Virus SIVsmm to Humans.

Simian immunodeficiency virus infecting sooty mangabeys (SIVsmm) has been transmitted to humans on at least nine occasions, giving rise to human immunodeficiency virus type 2 (HIV-2) groups A to I. SIVsmm isolates replicate in human T cells and seem capable of overcoming major human restriction factors without adaptation. However, only groups A and B are responsible for the HIV-2 epidemic in sub-Saharan Africa, and it is largely unclear whether adaptive changes were associated with spread in humans. To address this, we examined the sensitivity of infectious molecular clones (IMCs) of five HIV-2 strains and representatives of five different SIVsmm lineages to various APOBEC3 proteins. We confirmed that SIVsmm strains replicate in human T cells, albeit with more variable replication fitness and frequently lower efficiency than HIV-2 IMCs. Efficient viral propagation was generally dependent on intact vif genes, highlighting the need for counteraction of APOBEC3 proteins. On average, SIVsmm was more susceptible to inhibition by human APOBEC3D, -F, -G, and -H than HIV-2. For example, human APOBEC3F reduced infectious virus yield of SIVsmm by ∼80% but achieved only ∼40% reduction in the case of HIV-2. Functional and mutational analyses of human- and monkey-derived alleles revealed that an R128T polymorphism in APOBEC3F contributes to species-specific counteraction by HIV-2 and SIVsmm Vifs. In addition, a T84S substitution in SIVsmm Vif increased its ability to counteract human APOBEC3F. Altogether, our results confirm that SIVsmm Vif proteins show intrinsic activity against human APOBEC3 proteins but also demonstrate that epidemic HIV-2 strains evolved an increased ability to counteract this class of restriction factors during human adaptation. IMPORTANCE Viral zoonoses pose a significant threat to human health, and it is important to understand determining factors. SIVs infecting great apes gave rise to HIV-1. In contrast, SIVs infecting African monkey species have not been detected in humans, with one notable exception. SIVsmm from sooty mangabeys has crossed the species barrier to humans on at least nine independent occasions and seems capable of overcoming many innate defense mechanisms without adaptation. Here, we confirmed that SIVsmm Vif proteins show significant activity against human APOBEC3 proteins. Our analyses also revealed, however, that different lineages of SIVsmm are significantly more susceptible to inhibition by various human APOBEC3 proteins than HIV-2 strains. Mutational analyses suggest that an R128T substitution in APOBEC3F and a T84S change in Vif contribute to species-specific counteraction by HIV-2 and SIVsmm. Altogether, our results support that epidemic HIV-2 strains acquired increased activity against human APOBEC3 proteins to clear this restrictive barrier.

Nuclear PYHIN proteins target the host transcription factor Sp1 thereby restricting HIV-1 in human macrophages and CD4+ T cells.

Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity.

Supramolecular Mechanism of Viral Envelope Disruption by Molecular Tweezers.

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the "phosphate tweezer" CLR01, a "carboxylate tweezer" CLR05, and a "phosphate clip" PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.

Primary HIV-1 Strains Use Nef To Downmodulate HLA-E Surface Expression.

Human immunodeficiency virus type 1 (HIV-1) has evolved elaborate ways to evade immune cell recognition, including downregulation of classical HLA class I (HLA-I) from the surfaces of infected cells. Recent evidence identified HLA-E, a nonclassical HLA-I, as an important part of the antiviral immune response to HIV-1. Changes in HLA-E surface levels and peptide presentation can prompt both CD8+ T-cell and natural killer (NK) cell responses to viral infections. Previous studies reported unchanged or increased HLA-E levels on HIV-1-infected cells. Here, we examined HLA-E surface levels following infection of CD4+ T cells with primary HIV-1 strains and observed that a subset downregulated HLA-E. Two primary strains of HIV-1 that induced the strongest reduction in surface HLA-E expression were chosen for further testing. Expression of single Nef or Vpu proteins in a T-cell line, as well as tail swap experiments exchanging the cytoplasmic tail of HLA-A2 with that of HLA-E, demonstrated that Nef modulated HLA-E surface levels and targeted the cytoplasmic tail of HLA-E. Furthermore, infection of primary CD4+ T cells with HIV-1 mutants showed that a lack of functional Nef (and Vpu to some extent) impaired HLA-E downmodulation. Taken together, the results of this study demonstrate for the first time that HIV-1 can downregulate HLA-E surface levels on infected primary CD4+ T cells, potentially rendering them less vulnerable to CD8+ T-cell recognition but at increased risk of NKG2A+ NK cell killing.IMPORTANCE For almost two decades, it was thought that HIV-1 selectively downregulated the highly expressed HLA-I molecules HLA-A and HLA-B from the cell surface in order to evade cytotoxic-T-cell recognition, while leaving HLA-C and HLA-E molecules unaltered. It was stipulated that HIV-1 infection thereby maintained inhibition of NK cells via inhibitory receptors that bind HLA-C and HLA-E. This concept was recently revised when a study showed that primary HIV-1 strains reduce HLA-C surface levels, whereas the cell line-adapted HIV-1 strain NL4-3 lacks this ability. Here, we demonstrate that infection with distinct primary HIV-1 strains results in significant downregulation of surface HLA-E levels. Given the increasing evidence for HLA-E as an important modulator of CD8+ T-cell and NKG2A+ NK cell functions, this finding has substantial implications for future immunomodulatory approaches aimed at harnessing cytotoxic cellular immunity against HIV.

SIVcol Nef counteracts SERINC5 by promoting its proteasomal degradation but does not efficiently enhance HIV-1 replication in human CD4+ T cells and lymphoid tissue.

SERINC5 is a host restriction factor that impairs infectivity of HIV-1 and other primate lentiviruses and is counteracted by the viral accessory protein Nef. However, the importance of SERINC5 antagonism for viral replication and cytopathicity remained unclear. Here, we show that the Nef protein of the highly divergent SIVcol lineage infecting mantled guerezas (Colobus guereza) is a potent antagonist of SERINC5, although it lacks the CD4, CD3 and CD28 down-modulation activities exerted by other primate lentiviral Nefs. In addition, SIVcol Nefs decrease CXCR4 cell surface expression, suppress TCR-induced actin remodeling, and counteract Colobus but not human tetherin. Unlike HIV-1 Nef proteins, SIVcol Nef induces efficient proteasomal degradation of SERINC5 and counteracts orthologs from highly divergent vertebrate species, such as Xenopus frogs and zebrafish. A single Y86F mutation disrupts SERINC5 and tetherin antagonism but not CXCR4 down-modulation by SIVcol Nef, while mutation of a C-proximal di-leucine motif has the opposite effect. Unexpectedly, the Y86F change in SIVcol Nef had little if any effect on viral replication and CD4+ T cell depletion in preactivated human CD4+ T cells and in ex vivo infected lymphoid tissue. However, SIVcol Nef increased virion infectivity up to 10-fold and moderately increased viral replication in resting peripheral blood mononuclear cells (PBMCs) that were first infected with HIV-1 and activated three or six days later. In conclusion, SIVcol Nef lacks several activities that are conserved in other primate lentiviruses and utilizes a distinct proteasome-dependent mechanism to counteract SERINC5. Our finding that evolutionarily distinct SIVcol Nefs show potent anti-SERINC5 activity supports a relevant role of SERINC5 antagonism for viral fitness in vivo. Our results further suggest this Nef function is particularly important for virion infectivity under conditions of limited CD4+ T cell activation.

The Antiviral Activity of the Cellular Glycoprotein LGALS3BP/90K Is Species Specific.

Cellular antiviral proteins interfere with distinct steps of replication cycles of viruses. The galectin 3 binding protein (LGALS3BP, also known as 90K) was previously shown to lower the infectivity of nascent human immunodeficiency virus type 1 (HIV-1) virions when expressed in virus-producing cells. This antiviral effect was accompanied by impaired gp160Env processing and reduced viral incorporation of mature Env glycoproteins. Here, we examined the ability of 90K orthologs from primate species to reduce the particle infectivity of distinct lentiviruses. We show that 90K's ability to diminish the infectivity of lentiviral particles is conserved within primate species, with the notable exception of 90K from rhesus macaque. Comparison of active and inactive 90K orthologs and variants uncovered the fact that inhibition of processing of the HIV-1 Env precursor and reduction of cell surface expression of HIV-1 Env gp120 are required, but not sufficient, for 90K-mediated antiviral activity. Rather, 90K-mediated reduction of virion-associated gp120 coincided with antiviral activity, suggesting that 90K impairs the incorporation of HIV-1 Env into budding virions. We show that a single "humanizing" amino acid exchange in the BTB (broad-complex, tramtrack, and bric-à-brac)/POZ (poxvirus and zinc finger) domain is sufficient to fully rescue the antiviral activity of a shortened version of rhesus macaque 90K, but not that of the full-length protein. Comparison of the X-ray structures of the BTB/POZ domains of 90K from rhesus macaques and humans point toward a slightly larger hydrophobic patch at the surface of the rhesus macaque BTB domain that may modulate a direct interaction with either a second 90K domain or a different protein.IMPORTANCE The cellular 90K protein has been shown to diminish the infectivity of nascent HIV-1 particles. When produced in 90K-expressing cells, particles bear smaller amounts of the HIV-1 Env glycoprotein, which is essential for attaching to and entering new target cells in the subsequent infection round. However, whether the antiviral function of 90K is conserved across primates is unknown. Here, we found that 90K orthologs from most primate species, but, surprisingly, not from rhesus macaques, inhibit HIV-1. The introduction of a single amino acid exchange into a short version of the rhesus macaque 90K protein, consisting of the two intermediate domains of 90K, resulted in full restoration of antiviral activity. Structural elucidation of the respective domain suggests that the absence of antiviral activity in the rhesus macaque factor may be linked to a subtle change in protein-protein interaction.

Mucosal stromal fibroblasts markedly enhance HIV infection of CD4+ T cells.

Understanding early events of HIV transmission within mucosal tissues is vital for developing effective prevention strategies. Here, we report that primary stromal fibroblasts isolated from endometrium, cervix, foreskin, male urethra, and intestines significantly increase HIV infection of CD4+ T cells-by up to 37-fold for R5-tropic HIV and 100-fold for X4-tropic HIV-without themselves becoming infected. Fibroblasts were more efficient than dendritic cells at trans-infection and mediate this response in the absence of the DC-SIGN and Siglec-1 receptors. In comparison, mucosal epithelial cells secrete antivirals and inhibit HIV infection. These data suggest that breaches in the epithelium allow external or luminal HIV to escape an antiviral environment to access the infection-favorable environment of the stromal fibroblasts, and suggest that resident fibroblasts have a central, but previously unrecognized, role in HIV acquisition at mucosal sites. Inhibiting fibroblast-mediated enhancement of HIV infection should be considered as a novel prevention strategy.

Efficient Vpu-Mediated Tetherin Antagonism by an HIV-1 Group O Strain.

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.

PEGylated Cationic Serum Albumin for Boosting Retroviral Gene Transfer.

Retroviral vectors are common tools for introducing genes into the genome of a cell. However, low transduction rates are a major limitation in retroviral gene transfer, especially in clinical applications. We generated cationic human serum albumin (cHSA) protected by a shell of poly(ethylene glycol) (PEG); this significantly enhanced retroviral gene transduction with potentially attractive pharmacokinetics and low immunogenicity. By screening a panel of chemically optimized HSA compounds, we identified a very potent enhancer that boosted the transduction rates of viral vectors. Confocal microscopy revealed a drastically increased number of viral particles attached to the surfaces of target cells. In accordance with the positive net charge of cationic and PEGylated HSA, this suggests a mechanism of action in which the repulsion of the negatively charged cellular and viral vector membranes is neutralized, thereby promoting attachment and ultimately transduction. Importantly, the transduction-enhancing PEGylated HSA derivative evaded recognition by HSA-specific antibodies and macrophage activation. Our findings hold great promise for facilitating improved retroviral gene transfer.

Prevention of contamination by xenotropic murine leukemia virus-related virus: susceptibility to alcohol-based disinfectants and environmental stability.

Xenotropic murine leukemia virus-related virus (XMRV) represents a novel γ-retrovirus that is capable of infecting human cells and has been classified as a biosafety level 2 (BSL-2) organism. Hence, XMRV represents a potential risk for personnel in laboratories worldwide. Here, we measured the stability of XMRV and its susceptibility to alcohol-based disinfectants. To this end, we exposed an infectious XMRV reporter virus encoding a secretable luciferase to different temperatures, pH values, and disinfectants and infected XMRV-permissive Raji B cells to measure residual viral infectivity. We found that 1 min treatment of XMRV particles at 60°C is sufficient to reduce infectivity by 99.9%. XMRV infectivity was maximal at a neutral pH but was reduced by 86% at pH 4 and 99.9% at pH 10. The common hand and surface disinfectants ethanol and isopropanol as well as the cell fixation reagent paraformaldehyde abrogated XMRV infectivity entirely, as indicated by a reduction of infectivity exceeding 99.99%. Our findings provide evidence of specific means to inactivate XMRV. Their application will help to prevent unintended XMRV contamination of cell cultures in laboratories and minimize the risk for laboratory personnel and health care workers to become infected with this biosafety level 2 organism.

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.

An additional NF-κB site allows HIV-1 subtype C to evade restriction by nuclear PYHIN proteins.

Subtype C is the most prevalent clade of human immunodeficiency virus type 1 (HIV-1) worldwide. The reasons for this are poorly understood. Here, we demonstrate that a characteristic additional third nuclear factor κB (NF-κB) binding site in the long terminal repeat (LTR) promoter allows subtype C HIV-1 strains to evade restriction by nuclear PYHIN proteins, which sequester the transcription factor Sp1. Further, other LTR alterations are responsible for rare PYHIN resistance of subtype B viruses. Resistance-conferring mutations generally reduce the dependency of HIV-1 on Sp1 for virus production and render LTR transcription highly responsive to stimulation by NF-κB/p65. A third NF-κB binding site increases infectious virus yield in primary CD4+ T cells in an γ-interferon-inducible protein 16 (IFI16)-dependent manner. Comprehensive sequence analyses suggest that the frequency of circulating PYHIN-resistant HIV-1 strains is increasing. Our finding that an additional NF-κB binding site in the LTR confers resistance to nuclear PYHIN proteins helps to explain the dominance of clade C HIV-1 strains.

SIV-induced terminally differentiated adaptive NK cells in lymph nodes associated with enhanced MHC-E restricted activity.

Natural killer (NK) cells play a critical understudied role during HIV infection in tissues. In a natural host of SIV, the African green monkey (AGM), NK cells mediate a strong control of SIVagm infection in secondary lymphoid tissues. We demonstrate that SIVagm infection induces the expansion of terminally differentiated NKG2alow NK cells in secondary lymphoid organs displaying an adaptive transcriptional profile and increased MHC-E-restricted cytotoxicity in response to SIV Env peptides while expressing little IFN-γ. Such NK cell differentiation was lacking in SIVmac-infected macaques. Adaptive NK cells displayed no increased NKG2C expression. This study reveals a previously unknown profile of NK cell adaptation to a viral infection, thus accelerating strategies toward NK-cell directed therapies and viral control in tissues.

IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro.

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.

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

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.IMPORTANCE 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.

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

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 vivoIMPORTANCE 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.