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Vertebrate genomes contain fewer CG dinucleotides than would be expected by chance, and this pattern is mimicked by many viruses; HIV-1 derivatives mutated to contain more CG dinucleotides are targeted by the human antiviral protein ZAP, suggesting that CG suppression has evolved in viruses to evade recognition. Viruses evade the ZAP Many virus genomes possess lower than expected numbers of 5′-CG-3′ dinucleotides, mimicking the CG suppression of their vertebrate hosts. The reason for this has been unclear, but Paul Bieniasz and colleagues now show that viruses use CG suppression to avoid recognition by the zinc-finger antiviral protein ZAP. They show that CG suppression is essential for HIV-1 replication; otherwise, the virus genome is recognized by ZAP and targeted for degradation. Vertebrate genomes exhibit marked CG suppression—that is, lower than expected numbers of 5′-CG-3′ dinucleotides 1 . This feature is likely to be due to C-to-T mutations that have accumulated over hundreds of millions of years, driven by CG-specific DNA methyl transferases and spontaneous methyl-cytosine deamination. Many RNA viruses of vertebrates that are not substrates for DNA methyl transferases mimic the CG suppression of their hosts 2 , 3 , 4 . This property of viral genomes is unexplained 4 , 5 , 6 . Here we show, using synonymous mutagenesis, that CG suppression is essential for HIV-1 replication. The deleterious effect of CG dinucleotides on HIV-1 replication was cumulative, associated with cytoplasmic RNA depletion, and was exerted by CG dinucleotides in both translated and non-translated exonic RNA sequences. A focused screen using small inhibitory RNAs revealed that zinc-finger antiviral protein (ZAP) 7 inhibited virion production by cells infected with CG-enriched HIV-1. Crucially, HIV-1 mutants containing segments whose CG content mimicked random nucleotide sequence were defective in unmanipulated cells, but replicated normally in ZAP-deficient cells. Crosslinking–immunoprecipitation–sequencing assays demonstrated that ZAP binds directly and selectively to RNA sequences containing CG dinucleotides. These findings suggest that ZAP exploits host CG suppression to identify non-self RNA. The dinucleotide composition of HIV-1, and perhaps other RNA viruses, appears to have adapted to evade this host defence.

HIV-1 accesses the nuclear DNA of interphase cells via a poorly defined process involving functional interactions between the capsid protein (CA) and nucleoporins (Nups). Here, we show that HIV-1 CA can bind multiple Nups, and that both natural and manipulated variation in Nup levels impacts HIV-1 infection in a manner that is strikingly dependent on cell-type, cell-cycle, and cyclophilin A (CypA). We also show that Nups mediate the function of the antiviral protein MX2, and that MX2 can variably inhibit non-viral NLS function. Remarkably, both enhancing and inhibiting effects of cyclophilin A and MX2 on various HIV-1 CA mutants could be induced or abolished by manipulating levels of the Nup93 subcomplex, the Nup62 subcomplex, NUP88, NUP214, RANBP2, or NUP153. Our findings suggest that several Nup-dependent ‘pathways’ are variably exploited by HIV-1 to target host DNA in a cell-type, cell-cycle, CypA and CA-sequence dependent manner, and are differentially inhibited by MX2.

The protective efficacy of serum antibodies results from the interplay of antigen-specific B cell clones of different affinities and specificities. These cellular dynamics underlie serum-level phenomena such as original antigenic sin (OAS)—a proposed propensity of the immune system to rely repeatedly on the first cohort of B cells engaged by an antigenic stimulus when encountering related antigens, in detriment to the induction of de novo responses 1 – 5 . OAS-type suppression of new, variant-specific antibodies may pose a barrier to vaccination against rapidly evolving viruses such as influenza and SARS-CoV-2 6 , 7 . Precise measurement of OAS-type suppression is challenging because cellular and temporal origins cannot readily be ascribed to antibodies in circulation; its effect on subsequent antibody responses therefore remains unclear 5 , 8 . Here we introduce a molecular fate-mapping approach with which serum antibodies derived from specific cohorts of B cells can be differentially detected. We show that serum responses to sequential homologous boosting derive overwhelmingly from primary cohort B cells, while later induction of new antibody responses from naive B cells is strongly suppressed. Such ‘primary addiction’ decreases sharply as a function of antigenic distance, allowing reimmunization with divergent viral glycoproteins to produce de novo antibody responses targeting epitopes that are absent from the priming variant. Our findings have implications for the understanding of OAS and for the design and testing of vaccines against evolving pathogens. Serum antibody responses to sequential homologous booster vaccines derive overwhelmingly from primary cohort B cells at the expense of de novo responses; this ‘primary addiction’ can be overcome by boosting with variant antigens.

Viruses employ a variety of strategies to escape or counteract immune responses, including depletion of cell surface major histocompatibility complex class I (MHC-I), that would ordinarily present viral peptides to CD8⁺ cytotoxic T cells. As part of a screen to elucidate biological activities associated with individual severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral proteins, we found that ORF7a reduced cell surface MHC-I levels by approximately fivefold. Nevertheless, in cells infected with SARS-CoV-2, surface MHC-I levels were reduced even in the absence of ORF7a, suggesting additional mechanisms of MHC-I down-regulation. ORF7a proteins from a sample of sarbecoviruses varied in their ability to induce MHC-I down-regulation and, unlike SARS-CoV-2, the ORF7a protein from SARS-CoV lacked MHC-I downregulating activity. A single amino acid at position 59 (T/F) that is variable among sarbecovirus ORF7a proteins governed the difference in MHC-I downregulating activity. SARS-CoV-2 ORF7a physically associated with the MHC-I heavy chain and inhibited the presentation of expressed antigen to CD8⁺ T cells. Specifically, ORF7a prevented the assembly of the MHC-I peptide loading complex and caused retention of MHC-I in the endoplasmic reticulum. The differential ability of ORF7a proteins to function in this way might affect sarbecovirus dissemination and persistence in human populations, particularly those with infection- or vaccineelicited immunity.

Human immunodeficiency virus 1 (HIV-1) is a retrovirus with a ten-kilobase single-stranded RNA genome. HIV-1 must express all of its gene products from a single primary transcript, which undergoes alternative splicing to produce diverse protein products that include structural proteins and regulatory factors 1 , 2 . Despite the critical role of alternative splicing, the mechanisms that drive the choice of splice site are poorly understood. Synonymous RNA mutations that lead to severe defects in splicing and viral replication indicate the presence of unknown cis -regulatory elements 3 . Here we use dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to investigate the structure of HIV-1 RNA in cells, and develop an algorithm that we name ‘detection of RNA folding ensembles using expectation–maximization’ (DREEM), which reveals the alternative conformations that are assumed by the same RNA sequence. Contrary to previous models that have analysed population averages 4 , our results reveal heterogeneous regions of RNA structure across the entire HIV-1 genome. In addition to confirming that in vitro characterized 5 alternative structures for the HIV-1 Rev responsive element also exist in cells, we discover alternative conformations at critical splice sites that influence the ratio of transcript isoforms. Our simultaneous measurement of splicing and intracellular RNA structure provides evidence for the long-standing hypothesis 6 – 8 that heterogeneity in RNA conformation regulates splice-site use and viral gene expression. Dimethyl sulfate mutational profiling with sequencing, combined with the newly developed DREEM algorithm, reveals that heterogeneity of RNA structure in HIV-1 regulates the use of splice sites and expression of viral genes.

Human cells possess an antiviral activity that inhibits the release of retrovirus particles, and other enveloped virus particles, and is antagonized by the HIV-1 accessory protein, Vpu. This antiviral activity can be constitutively expressed or induced by interferon-α, and it consists of protein-based tethers, which we term ‘tetherins’, that cause retention of fully formed virions on infected cell surfaces. Using deductive constraints and gene expression analyses, we identify CD317 (also called BST2 or HM1.24), a membrane protein of previously unknown function, as a tetherin. Specifically, CD317 expression correlated with, and induced, a requirement for Vpu during HIV-1 and murine leukaemia virus particle release. Furthermore, in cells where HIV-1 virion release requires Vpu expression, depletion of CD317 abolished this requirement. CD317 caused retention of virions on cell surfaces and, after endocytosis, in CD317-positive compartments. Vpu co-localized with CD317 and inhibited these effects. Inhibition of Vpu function and consequent mobilization of tetherin’s antiviral activity is a potential therapeutic strategy in HIV/AIDS. HIV/AIDS: ties that bind Studies of Vpu, an HIV-1 accessory protein required for efficient HIV-1 particle release in some human cells, pointed to the existence of a tether based in a cell surface protein inducible by interferon-α. That tether has now been identified as the host cell molecule CD317— renamed tetherin — a membrane protein with no previously known function. Tetherin is shown to be involved in the retention of HIV-1 virions at the cell surface. Vpu neutralizes its effect, allowing the release and propagation of virus particles. Inhibition of Vpu function is therefore a possible therapeutic strategy in HIV/AIDS. The HIV protein Vpu is required for the release of viral particles. This paper shows that it counteracts the host cell protein CD317, renamed as tetherin. Tetherin is involved in the retention of newly budded HIV-1 virions at the cell surface.

MX2 is shown to be an interferon-induced inhibitor of HIV-1 infection, and this antiviral activity may involve the inhibition of nuclear import of subviral complexes. Human MX2 protein is an HIV-1 resistance factor Two groups report in this issue of Nature that the human interferon-induced GTP-binding protein MX2 is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) and a number of other lentiviruses. For some years it had been known that the related protein MX1 can inhibit HIV-1 replication in humans, but MX2 was thought to be devoid of antiviral activity. The anti-HIV-1 action of MX2 is much less dependent on GTPase activity than is the broader antiviral activity of MX1, pointing to possible mechanistic differences between them. HIV-1 replication can be inhibited by type I interferon (IFN), and the expression of a number of gene products with anti-HIV-1 activity is induced by type I IFN 1 , 2 . However, none of the known antiretroviral proteins can account for the ability of type I IFN to inhibit early, preintegration phases of the HIV-1 replication cycle in human cells 3 , 4 . Here, by comparing gene expression profiles in cell lines that differ in their ability to support the inhibitory action of IFN-α at early steps of the HIV-1 replication cycle, we identify myxovirus resistance 2 (MX2) as an interferon-induced inhibitor of HIV-1 infection. Expression of MX2 reduces permissiveness to a variety of lentiviruses, whereas depletion of MX2 using RNA interference reduces the anti-HIV-1 potency of IFN-α. HIV-1 reverse transcription proceeds normally in MX2-expressing cells, but 2-long terminal repeat circular forms of HIV-1 DNA are less abundant, suggesting that MX2 inhibits HIV-1 nuclear import, or destabilizes nuclear HIV-1 DNA. Consistent with this notion, mutations in the HIV-1 capsid protein that are known, or suspected, to alter the nuclear import pathways used by HIV-1 confer resistance to MX2, whereas preventing cell division increases MX2 potency. Overall, these findings indicate that MX2 is an effector of the anti-HIV-1 activity of type-I IFN, and suggest that MX2 inhibits HIV-1 infection by inhibiting capsid-dependent nuclear import of subviral complexes.

The encapsidation of HIV-1 gRNA into virions is enabled by the binding of the nucleocapsid (NC) domain of the HIV-1 Gag polyprotein to the structured viral RNA packaging signal (Ψ) at the 5’ end of the viral genome. However, the subcellular location and oligomeric status of Gag during the initial Gag-Ψ encounter remain uncertain. Domains other than NC, such as capsid (CA), may therefore indirectly affect RNA recognition. To investigate the contribution of Gag domains to Ψ recognition in a cellular environment, we performed protein-protein crosslinking and protein-RNA crosslinking immunoprecipitation coupled with sequencing (CLIP-seq) experiments. We demonstrate that NC alone does not bind specifically to Ψ in living cells, whereas full-length Gag and a CANC subdomain bind to Ψ with high specificity. Perturbation of the Ψ RNA structure or NC zinc fingers affected CANC:Ψ binding specificity. Notably, CANC variants with substitutions that disrupt CA:CA dimer, trimer, or hexamer interfaces in the immature Gag lattice also affected RNA binding, and mutants that were unable to assemble a nascent Gag lattice were unable to specifically bind to Ψ. Artificially multimerized NC domains did not specifically bind Ψ. CA variants with substitutions in inositol phosphate coordinating residues that prevent CA hexamerization were also deficient in Ψ binding and second-site revertant mutants that restored CA assembly also restored specific binding to Ψ. Overall, these data indicate that the correct assembly of a nascent immature CA lattice is required for the specific interaction between Gag and Ψ in cells.

Background A critical step in the HIV-1 replication cycle is the assembly of Gag proteins to form virions at the plasma membrane. Virion assembly and maturation are facilitated by the cellular polyanion inositol hexaphosphate (IP 6 ), which is proposed to stabilize both the immature Gag lattice and the mature capsid lattice by binding to rings of primary amines at the center of Gag or capsid protein (CA) hexamers. The amino acids comprising these rings are critical for proper virion formation and their substitution results in assembly deficits or impaired infectiousness. To better understand the nature of the deficits that accompany IP 6 binding deficiency, we passaged HIV-1 mutants that had substitutions in IP 6 coordinating residues to select for compensatory mutations. Results We found a mutation, a threonine to isoleucine substitution at position 371 (T371I) in Gag, that restored replication competence to an IP 6 -binding-deficient HIV-1 mutant. Notably, unlike wild-type HIV-1, the assembly and infectiousness of resulting virus was not impaired when IP 6 biosynthetic enzymes were genetically ablated. Surprisingly, we also found that the maturation inhibitor Bevirimat (BVM) could restore the assembly and replication of an IP 6 -binding deficient mutant. Moreover, using BVM-dependent mutants we were able to image BVM-induced assembly of individual HIV-1 particles assembly in living cells. Conclusions Overall these results suggest that IP 6 -Gag and Gag-Gag contacts are finely tuned to generate a Gag lattice of optimal stability, and that under certain conditions BVM can rescue IP 6 deficiency. Additionally, our work identifies an inducible virion assembly system that can be utilized to visualize HIV-1 assembly events using live cell microscopy.

Tetherin (Bst2/CD317/HM1.24) is an interferon-induced antiviral host protein that inhibits the release of many enveloped viruses by tethering virions to the cell surface. The HIV-1 accessory protein, Vpu, antagonizes Tetherin through a variety of proposed mechanisms, including surface downregulation and degradation. Previous studies have demonstrated that mutation of the transmembrane domains (TMD) of both Vpu and Tetherin affect antagonism, but it is not known whether Vpu and Tetherin bind directly to each other. Here, we use cysteine-scanning mutagenesis coupled with oxidation-induced cross-linking to demonstrate that Vpu and Tetherin TMDs bind directly to each other in the membranes of living cells and to map TMD residues that contact each other. We also reveal a property of Vpu, namely the ability to displace Tetherin from sites of viral assembly, which enables Vpu to exhibit residual Tetherin antagonist activity in the absence of surface downregulation or degradation. Elements in the cytoplasmic tail domain (CTD) of Vpu mediate this displacement activity, as shown by experiments in which Vpu CTD fragments were directly attached to Tetherin in the absence of the TMD. In particular, the C-terminal α-helix (H2) of Vpu CTD is sufficient to remove Tetherin from sites of viral assembly and is necessary for full Tetherin antagonist activity. Overall, these data demonstrate that Vpu and Tetherin interact directly via their transmembrane domains enabling activities present in the CTD of Vpu to remove Tetherin from sites of viral assembly.