RESUMEN
The HIV-1 Gag protein orchestrates all steps of virion genesis, including membrane targeting and RNA recruitment into virions. Using crosslinking-immunoprecipitation (CLIP) sequencing, we uncover several dramatic changes in the RNA-binding properties of Gag that occur during virion genesis, coincident with membrane binding, multimerization, and proteolytic maturation. Prior to assembly, and after virion assembly and maturation, the nucleocapsid domain of Gag preferentially binds to psi and Rev Response elements in the viral genome, and GU-rich mRNA sequences. However, during virion genesis, this specificity transiently changes in a manner that facilitates genome packaging; nucleocapsid binds to many sites on the HIV-1 genome and to mRNA sequences with a HIV-1-like, A-rich nucleotide composition. Additionally, we find that the matrix domain of Gag binds almost exclusively to specific tRNAs in the cytosol, and this association regulates Gag binding to cellular membranes.
Asunto(s)
VIH-1/fisiología , ARN Viral/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Bases , Línea Celular , Inmunoprecipitación de Cromatina , Genes env , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN de Transferencia/metabolismo , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
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 responses1-5. OAS-type suppression of new, variant-specific antibodies may pose a barrier to vaccination against rapidly evolving viruses such as influenza and SARS-CoV-26,7. 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 unclear5,8. 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.
Asunto(s)
Formación de Anticuerpos , Linfocitos B , Inmunización Secundaria , Humanos , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Vacunas contra la Influenza/inmunología , SARS-CoV-2/inmunología , Vacunación , Linfocitos B/inmunología , Vacunas Virales/inmunologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
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 factors1,2. 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 elements3. 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 averages4, our results reveal heterogeneous regions of RNA structure across the entire HIV-1 genome. In addition to confirming that in vitro characterized5 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 hypothesis6-8 that heterogeneity in RNA conformation regulates splice-site use and viral gene expression.
Asunto(s)
Empalme Alternativo/genética , Regulación Viral de la Expresión Génica , VIH-1/genética , Mutación , Sitios de Empalme de ARN/genética , ARN Viral/química , ARN Viral/genética , Algoritmos , Secuencia de Bases , Células HEK293 , Humanos , Conformación de Ácido Nucleico , Pliegue del ARN , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN , Ésteres del Ácido Sulfúrico , TermodinámicaRESUMEN
Vaccination will likely be a key component of strategies to curtail or prevent future sarbecovirus pandemics and to reduce the prevalence of infection and disease by future SARS-CoV-2 variants. A "pan-sarbecovirus" vaccine, that provides maximum possible mitigation of human disease, should elicit neutralizing antibodies with maximum possible breadth. By positioning multiple different receptor binding domain (RBD) antigens in close proximity on a single immunogen, it is postulated that cross-reactive B cell receptors might be selectively engaged. Heteromultimeric vaccines could therefore elicit individual antibodies that neutralize a broad range of viral species. Here, we use model systems to investigate the ability of multimeric sarbecovirus RBD immunogens to expand cross-reactive B cells and elicit broadly reactive antibodies. Homomultimeric RBD immunogens generated higher serum neutralizing antibody titers than the equivalent monomeric immunogens, while heteromultimeric RBD immunogens generated neutralizing antibodies recognizing each RBD component. Moreover, RBD heterodimers elicited a greater fraction of cross-reactive germinal center B cells and cross-reactive RBD binding antibodies than did homodimers. However, when serum antibodies from RBD heterodimer-immunized mice were depleted using one RBD component, neutralization activity against the homologous viral pseudotype was removed, but neutralization activity against pseudotypes corresponding to the other RBD component was unaffected. Overall, simply combining divergent RBDs in a single immunogen generates largely separate sets of individual RBD-specific neutralizing serum antibodies that are mostly incapable of neutralizing viruses that diverge from the immunogen components.
Asunto(s)
Anticuerpos Neutralizantes , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Animales , Ratones , Humanos , Anticuerpos Antivirales , Pruebas de Neutralización , Vacunación , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
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 vaccine-elicited immunity.
Asunto(s)
Presentación de Antígeno , Linfocitos T CD8-positivos , COVID-19 , Antígenos de Histocompatibilidad Clase I , Proteínas Virales , Aminoácidos , Linfocitos T CD8-positivos/inmunología , COVID-19/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Complejo Mayor de Histocompatibilidad , Péptidos , SARS-CoV-2 , Proteínas Virales/inmunologíaRESUMEN
Tetherin is an interferon-induced protein whose expression blocks the release of HIV-1 and other enveloped viral particles. The underlying mechanism by which tetherin functions and whether it directly or indirectly causes virion retention are unknown. Here, we elucidate the mechanism by which tetherin exerts its antiviral activity. We demonstrate, through mutational analyses and domain replacement experiments, that tetherin configuration rather than primary sequence is critical for antiviral activity. These findings allowed the design of a completely artificial protein, lacking sequence homology with native tetherin, that nevertheless mimicked its antiviral activity. We further show that tetherin is incorporated into HIV-1 particles as a parallel homodimer using either of its two membrane anchors. These results indicate that tetherin functions autonomously and directly and that infiltration of virion envelopes by one or both of tetherin's membrane anchors is necessary, and likely sufficient, to tether enveloped virus particles that bud through the plasma membrane.
Asunto(s)
Antígenos CD/metabolismo , VIH-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Virión/metabolismo , Animales , Antígenos CD/química , Antígenos CD/genética , Línea Celular , Membrana Celular/metabolismo , Ebolavirus/metabolismo , Proteínas Ligadas a GPI , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Mutagénesis , Estructura Terciaria de Proteína , Ratas , Proteínas de la Matriz Viral/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación ViralRESUMEN
Vertebrate genomes exhibit marked CG suppression-that is, lower than expected numbers of 5'-CG-3' dinucleotides. 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. This property of viral genomes is unexplained. 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) 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.
Asunto(s)
Fosfatos de Dinucleósidos/genética , Secuencia Rica en GC/genética , VIH-1/genética , VIH-1/inmunología , ARN Viral/genética , ARN Viral/inmunología , Línea Celular , Citoplasma/genética , Citoplasma/virología , VIH-1/crecimiento & desarrollo , Humanos , Inmunoprecipitación , Mutagénesis , Mutación , Unión Proteica , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Replicación Viral/genéticaRESUMEN
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 (IP6), 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 IP6 binding deficiency, we passaged HIV-1 mutants that had substitutions in IP6 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 IP6-binding-deficient HIV-1 mutant. Notably, unlike wild-type HIV-1, the assembly and infectiousness of resulting virus was not impaired when IP6 biosynthetic enzymes were genetically ablated. Surprisingly, we also found that the maturation inhibitor Bevirimat (BVM) could restore the assembly and replication of an IP6-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 IP6-Gag and Gag-Gag contacts are finely tuned to generate a Gag lattice of optimal stability, and that under certain conditions BVM can rescue IP6 deficiency. Additionally, our work identifies an inducible virion assembly system that can be utilized to visualize HIV-1 assembly events using live cell microscopy.
Asunto(s)
VIH-1/genética , VIH-1/metabolismo , Mutación , Ácido Fítico/metabolismo , Virión/metabolismo , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Sustitución de Aminoácidos , Proteínas de la Cápside/metabolismo , Células HEK293 , HumanosRESUMEN
Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs), many of which are responsible for the cellular antiviral state in which the replication of numerous viruses is blocked. How the majority of individual ISGs inhibit the replication of particular viruses is unknown. We conducted a loss-of-function screen to identify genes required for the activity of alpha interferon (IFN-α) against vesicular stomatitis virus, Indiana serotype (VSVIND), a prototype negative-strand RNA virus. Our screen revealed that TRIM69, a member of the tripartite motif (TRIM) family of proteins, is a VSVIND inhibitor. TRIM69 potently inhibited VSVIND replication through a previously undescribed transcriptional inhibition mechanism. Specifically, TRIM69 physically associates with the VSVIND phosphoprotein (P), requiring a specific peptide target sequence encoded therein. P is a cofactor for the viral polymerase and is required for viral RNA synthesis, as well as the assembly of replication compartments. By targeting P, TRIM69 inhibits pioneer transcription of the incoming virion-associated minus-strand RNA, thereby preventing the synthesis of viral mRNAs, and consequently impedes all downstream events in the VSVIND replication cycle. Unlike some TRIM proteins, TRIM69 does not inhibit viral replication by inducing degradation of target viral proteins. Rather, higher-order TRIM69 multimerization is required for its antiviral activity, suggesting that TRIM69 functions by sequestration or anatomical disruption of the viral machinery required for VSVIND RNA synthesis.IMPORTANCE Interferons are important antiviral cytokines that work by inducing hundreds of host genes whose products inhibit the replication of many viruses. While the antiviral activity of interferon has long been known, the identities and mechanisms of action of most interferon-induced antiviral proteins remain to be discovered. We identified gene products that are important for the antiviral activity of interferon against vesicular stomatitis virus (VSV), a model virus that whose genome consists of a single RNA molecule with negative-sense polarity. We found that a particular antiviral protein, TRIM69, functions by a previously undescribed molecular mechanism. Specifically, TRIM69 interacts with and inhibits the function of a particular phosphoprotein (P) component of the viral transcription machinery, preventing the synthesis of viral messenger RNAs.
Asunto(s)
Interferón-alfa/farmacología , Proteínas de Motivos Tripartitos/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Virus de la Estomatitis Vesicular Indiana/efectos de los fármacos , Vesiculovirus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Antivirales/farmacología , Línea Celular , Citocinas/farmacología , Humanos , Modelos Moleculares , Fosfoproteínas/genética , Conformación Proteica , Dominios Proteicos , ARN Mensajero/metabolismo , ARN Viral/biosíntesis , Proteínas de Motivos Tripartitos/química , Ubiquitina-Proteína Ligasas/química , Estomatitis Vesicular/virología , Virus de la Estomatitis Vesicular Indiana/genética , Vesiculovirus/genética , Proteínas ViralesRESUMEN
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. 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. 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.
Asunto(s)
Infecciones por VIH/prevención & control , VIH-1/fisiología , Interferón-alfa/inmunología , Proteínas de Resistencia a Mixovirus/metabolismo , Transporte Activo de Núcleo Celular , Cápside/metabolismo , División Celular , Línea Celular , Núcleo Celular/metabolismo , Núcleo Celular/virología , Células Cultivadas , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/metabolismo , VIH-1/inmunología , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas de Resistencia a Mixovirus/genética , Interferencia de ARN , Transcripción Reversa , Transcriptoma , Replicación ViralRESUMEN
All retroviruses package cellular RNAs into virions. Studies of murine leukemia virus (MLV) revealed that the major host cell RNAs encapsidated by this simple retrovirus were LTR retrotransposons and noncoding RNAs (ncRNAs). Several classes of ncRNAs appeared to be packaged by MLV shortly after synthesis, as precursors to tRNAs, small nuclear RNAs, and small nucleolar RNAs were all enriched in virions. To determine the extent to which the human immunodeficiency virus (HIV-1) packages similar RNAs, we used high-throughput sequencing to characterize the RNAs within infectious HIV-1 virions produced in CEM-SS T lymphoblastoid cells. We report that the most abundant cellular RNAs in HIV-1 virions are 7SL RNA and transcripts from numerous divergent and truncated members of the long interspersed element (LINE) and short interspersed element (SINE) families of retrotransposons. We also detected precursors to several tRNAs and small nuclear RNAs as well as transcripts derived from the ribosomal DNA (rDNA) intergenic spacers. We show that packaging of a pre-tRNA requires the nuclear export receptor Exportin 5, indicating that HIV-1 recruits at least some newly made ncRNAs in the cytoplasm. Together, our work identifies the set of RNAs packaged by HIV-1 and reveals that early steps in HIV-1 assembly intersect with host cell ncRNA biogenesis pathways.
Asunto(s)
VIH-1/genética , ARN Viral/genética , Línea Celular , HumanosRESUMEN
Human antibodies to human immunodeficiency virus-1 (HIV-1) can neutralize a broad range of viral isolates in vitro and protect non-human primates against infection. Previous work showed that antibodies exert selective pressure on the virus but escape variants emerge within a short period of time. However, these experiments were performed before the recent discovery of more potent anti-HIV-1 antibodies and their improvement by structure-based design. Here we re-examine passive antibody transfer as a therapeutic modality in HIV-1-infected humanized mice. Although HIV-1 can escape from antibody monotherapy, combinations of broadly neutralizing antibodies can effectively control HIV-1 infection and suppress viral load to levels below detection. Moreover, in contrast to antiretroviral therapy, the longer half-life of antibodies led to control of viraemia for an average of 60 days after cessation of therapy. Thus, combinations of potent monoclonal antibodies can effectively control HIV-1 replication in humanized mice, and should be re-examined as a therapeutic modality in HIV-1-infected individuals.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Anti-VIH/inmunología , Anticuerpos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Especificidad de Anticuerpos/inmunología , Modelos Animales de Enfermedad , Infecciones por VIH/virología , VIH-1/genética , VIH-1/crecimiento & desarrollo , VIH-1/inmunología , VIH-1/aislamiento & purificación , Semivida , Humanos , Inmunización Pasiva , Ratones , Ratones Endogámicos NOD , Factores de Tiempo , Carga Viral/efectos de los fármacosRESUMEN
Myxovirus resistance 2 (Mx2/MxB) has recently been uncovered as an effector of the anti-HIV-1 activity of type I interferons (IFNs) that inhibits HIV-1 at an early stage postinfection, after reverse transcription but prior to proviral integration into host DNA. The mechanistic details of Mx2 antiviral activity are not yet understood, but a few substitutions in the HIV-1 capsid have been shown to confer resistance to Mx2. Through a combination of in vitro evolution and unbiased mutagenesis, we further map the determinants of sensitivity to Mx2 and reveal that multiple capsid (CA) surfaces define sensitivity to Mx2. Intriguingly, we reveal an unanticipated sensitivity determinant within the C-terminal domain of capsid. We also report that Mx2s derived from multiple primate species share the capacity to potently inhibit HIV-1, whereas selected nonprimate orthologs have no such activity. Like TRIM5α, another CA targeting antiretroviral protein, primate Mx2s exhibit species-dependent variation in antiviral specificity against at least one extant virus and multiple HIV-1 capsid mutants. Using a combination of chimeric Mx2 proteins and evolution-guided approaches, we reveal that a single residue close to the N terminus that has evolved under positive selection can determine antiviral specificity. Thus, the variable N-terminal region can define the spectrum of viruses inhibited by Mx2. Importance: Type I interferons (IFNs) inhibit the replication of most mammalian viruses. IFN stimulation upregulates hundreds of different IFN-stimulated genes (ISGs), but it is often unclear which ISGs are responsible for inhibition of a given virus. Recently, Mx2 was identified as an ISG that contributes to the inhibition of HIV-1 replication by type I IFN. Thus, Mx2 might inhibit HIV-1 replication in patients, and this inhibitory action might have therapeutic potential. The mechanistic details of how Mx2 inhibits HIV-1 are currently unclear, but the HIV-1 capsid protein is the likely viral target. Here, we determine the regions of capsid that specify sensitivity to Mx2. We demonstrate that Mx2 from multiple primates can inhibit HIV-1, whereas Mx2 from other mammals (dogs and sheep) cannot. We also show that primate variants of Mx2 differ in the spectrum of lentiviruses they inhibit and that a single residue in Mx2 can determine this antiviral specificity.
Asunto(s)
Proteína p24 del Núcleo del VIH/inmunología , VIH-1/inmunología , Proteínas de Resistencia a Mixovirus/inmunología , Animales , Análisis Mutacional de ADN , Evolución Molecular , Proteína p24 del Núcleo del VIH/genética , VIH-1/genética , Humanos , MutagénesisRESUMEN
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.
Asunto(s)
Antígenos CD/metabolismo , VIH-1/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Ensamble de Virus , Liberación del Virus , Antígenos CD/química , Sitios de Unión , Línea Celular Tumoral , Membrana Celular/metabolismo , Regulación hacia Abajo , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/metabolismo , Células HEK293 , Infecciones por VIH/virología , VIH-1/genética , Células HeLa , Proteínas del Virus de la Inmunodeficiencia Humana/química , Proteínas del Virus de la Inmunodeficiencia Humana/genética , Humanos , Proteínas de la Membrana/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/genéticaRESUMEN
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-alpha, 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.
Asunto(s)
Antígenos CD/metabolismo , VIH-1/metabolismo , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Antígenos CD/genética , Línea Celular , Membrana Celular/virología , Endocitosis , Proteínas Ligadas a GPI , Perfilación de la Expresión Génica , Infecciones por VIH/metabolismo , Infecciones por VIH/terapia , Infecciones por VIH/virología , Células HeLa , Proteínas del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Humanos , Interferón-alfa/farmacología , Virus de la Leucemia Murina/metabolismo , Glicoproteínas de Membrana/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Transporte de Proteínas , Proteínas Reguladoras y Accesorias Virales/antagonistas & inhibidores , Virión/metabolismo , Replicación Viral , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The morphogenesis of retroviral particles is driven by Gag and GagPol proteins that provide the major structural component and enzymatic activities required for particle assembly and maturation. In addition, a number of cellular proteins are found in retrovirus particles; some of these are important for viral replication, but many lack a known functional role. One such protein is clathrin, which is assumed to be passively incorporated into virions due to its abundance at the plasma membrane. We found that clathrin is not only exceptionally abundant in highly purified HIV-1 particles but is recruited with high specificity. In particular, the HIV-1 Pol protein was absolutely required for clathrin incorporation and point mutations in reverse transcriptase or integrase domains of Pol could abolish incorporation. Clathrin was also specifically incorporated into other retrovirus particles, including members of the lentivirus (simian immunodeficiency virus, SIVmac), gammaretrovirus (murine leukemia virus, MLV) and betaretrovirus (Mason-Pfizer monkey virus, M-PMV) genera. However, unlike HIV-1, these other retroviruses recruited clathrin primarily using peptide motifs in their respective Gag proteins that mimicked motifs found in cellular clathrin adaptors. Perturbation of clathrin incorporation into these retroviruses, via mutagenesis of viral proteins, siRNA based clathrin depletion or adaptor protein (AP180) induced clathrin sequestration, had a range of effects on the accuracy of particle morphogenesis. These effects varied according to which retrovirus was examined, and included Gag and/or Pol protein destabilization, inhibition of particle assembly and reduction in virion infectivity. For each retrovirus examined, clathrin incorporation appeared to be important for optimal replication. These data indicate that a number of retroviruses employ clathrin to facilitate the accurate morphogenesis of infectious particles. We propose a model in which clathrin contributes to the spatial organization of Gag and Pol proteins, and thereby regulates proteolytic processing of virion components during particle assembly.
Asunto(s)
Clatrina/metabolismo , VIH-1/metabolismo , Ensamble de Virus , Betaretrovirus/enzimología , Línea Celular , Clatrina/genética , Gammaretrovirus/enzimología , Productos del Gen gag/metabolismo , Productos del Gen pol/genética , Productos del Gen pol/metabolismo , Humanos , Lentivirus/enzimología , Proteínas de Ensamble de Clatrina Monoméricas/genética , Proteínas de Ensamble de Clatrina Monoméricas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Ensamble de Virus/genéticaRESUMEN
Tetherin/BST2 is a type-II membrane protein that inhibits the release of a range of enveloped viruses, including HIV-1. Here we report three crystal structures of human tetherin, including the full-length ectodomain, a triple cysteine mutant and an ectodomain truncation. These structures show that tetherin forms a continuous alpha helix encompassing almost the entire ectodomain. Tetherin helices dimerize into parallel coiled coils via interactions throughout the C-terminal portion of the ectodomain. A comparison of the multiple structures of the tetherin dimer reveals inherent constrained flexibility at two hinges positioned at residues A88 and G109. In the crystals, two tetherin ectodomain dimers associate into a tetramer by forming an antiparallel four-helix bundle at their N termini. However, mutagenesis studies suggest that the tetrametric form of tetherin, although potentially contributing to, is not essential for its antiviral activity. Nonetheless, the structural and chemical properties of the N terminus of the ectodomain are important for optimal tethering function. This study provides detailed insight into the mechanisms by which this broad-spectrum antiviral restriction factor can function.
Asunto(s)
Antígenos CD/química , Antígenos CD/fisiología , VIH-1/fisiología , Antígenos CD/genética , Antígenos CD/inmunología , Secuencia de Bases , Línea Celular , Cristalografía por Rayos X , Cartilla de ADN/genética , Dimerización , Proteínas Ligadas a GPI/química , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/fisiología , VIH-1/inmunología , Humanos , Inmunidad Innata , Técnicas In Vitro , Modelos Moleculares , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad Estática , Liberación del VirusRESUMEN
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.
Asunto(s)
VIH-1 , Empaquetamiento del Genoma Viral , ARN Viral/genética , VIH-1/genética , Ensamble de Virus/genética , Nucleocápside/metabolismo , Proteínas de la Cápside/metabolismo , Genoma ViralRESUMEN
Tetherin (CD317/BST2) is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) is sensitive to tetherin restriction and its activity is counteracted by the KSHV encoded RING-CH E3 ubiquitin ligase K5. Tetherin expression in KSHV-infected cells inhibits viral particle release, as does depletion of K5 protein using RNA interference. K5 induces a species-specific downregulation of human tetherin from the cell surface followed by its endosomal degradation. We show that K5 targets a single lysine (K18) in the cytoplasmic tail of tetherin for ubiquitination, leading to relocalization of tetherin to CD63-positive endosomal compartments. Tetherin degradation is dependent on ESCRT-mediated endosomal sorting, but does not require a tyrosine-based sorting signal in the tetherin cytoplasmic tail. Importantly, we also show that the ability of K5 to substitute for Vpu in HIV-1 release is entirely dependent on K18 and the RING-CH domain of K5. By contrast, while Vpu induces ubiquitination of tetherin cytoplasmic tail lysine residues, mutation of these positions has no effect on its antagonism of tetherin function, and residual tetherin is associated with the trans-Golgi network (TGN) in Vpu-expressing cells. Taken together our results demonstrate that K5 is a mechanistically distinct viral countermeasure to tetherin-mediated restriction, and that herpesvirus particle release is sensitive to this mode of antiviral inhibition.