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1.
Proc Natl Acad Sci U S A ; 120(51): e2317367120, 2023 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-38096415

RESUMO

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.


Assuntos
Anticorpos Neutralizantes , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Animais , Camundongos , Humanos , Anticorpos Antivirais , Testes de Neutralização , Vacinação , Glicoproteína da Espícula de Coronavírus/química
2.
Nature ; 615(7952): 482-489, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36646114

RESUMO

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.


Assuntos
Formação de Anticorpos , Linfócitos B , Imunização Secundária , Humanos , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Vacinas contra Influenza/imunologia , SARS-CoV-2/imunologia , Vacinação , Linfócitos B/imunologia , Vacinas Virais/imunologia
3.
Elife ; 122023 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-36688533

RESUMO

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.


Assuntos
HIV-1 , Empacotamento do Genoma Viral , RNA Viral/genética , HIV-1/genética , Montagem de Vírus/genética , Nucleocapsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Genoma Viral
4.
bioRxiv ; 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36093344

RESUMO

The ability of serum antibody to protect against pathogens arises from the interplay of antigen-specific B cell clones of different affinities and fine specificities. These cellular dynamics are ultimately responsible for serum-level phenomena such as antibody imprinting or "Original Antigenic Sin" (OAS), a proposed propensity of the immune system to rely repeatedly on the first cohort of B cells that responded to a stimulus upon exposure to related antigens. Imprinting/OAS is thought to pose a barrier to vaccination against rapidly evolving viruses such as influenza and SARS-CoV-2. Precise measurement of the extent to which imprinting/OAS inhibits the recruitment of new B cell clones by boosting is challenging because cellular and temporal origins cannot readily be assigned to antibodies in circulation. Thus, the extent to which imprinting/OAS impacts the induction of new responses in various settings remains unclear. To address this, we developed a "molecular fate-mapping" approach in which serum antibodies derived from specific cohorts of B cells can be differentially detected. We show that, upon sequential homologous boosting, the serum antibody response strongly favors reuse of the first cohort of B cell clones over the recruitment of new, naÏve-derived B cells. This "primary addiction" decreases as a function of antigenic distance, allowing secondary immunization with divergent influenza virus or SARS-CoV-2 glycoproteins to overcome imprinting/OAS by targeting novel epitopes absent from the priming variant. Our findings have implications for the understanding of imprinting/OAS, and for the design and testing of vaccines aimed at eliciting antibodies to evolving antigens.

5.
Proc Natl Acad Sci U S A ; 119(41): e2209042119, 2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36136978

RESUMO

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.


Assuntos
Apresentação de Antígeno , Linfócitos T CD8-Positivos , COVID-19 , Antígenos de Histocompatibilidade Classe I , Proteínas Virais , Aminoácidos , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Complexo Principal de Histocompatibilidade , Peptídeos , SARS-CoV-2 , Proteínas Virais/imunologia
6.
bioRxiv ; 2022 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-35665005

RESUMO

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 SARS-CoV-2 viral proteins, we found that ORF7a reduced cell surface MHC-I levels by approximately 5-fold. 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 downregulation. ORF7a proteins from a sample of sarbecoviruses varied in their ability to induce MHC-I downregulation 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. Speficially, ORF7a prevented the assembly of the MHC-I peptide loading complex and causing 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.

7.
Retrovirology ; 18(1): 25, 2021 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-34454514

RESUMO

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.


Assuntos
HIV-1/genética , HIV-1/metabolismo , Mutação , Ácido Fítico/metabolismo , Vírion/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Substituição de Aminoácidos , Proteínas do Capsídeo/metabolismo , Células HEK293 , Humanos
9.
Nature ; 582(7812): 438-442, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555469

RESUMO

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.


Assuntos
Processamento Alternativo/genética , Regulação Viral da Expressão Gênica , HIV-1/genética , Mutação , Sítios de Splice de RNA/genética , RNA Viral/química , RNA Viral/genética , Algoritmos , Sequência de Bases , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Dobramento de RNA , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Ésteres do Ácido Sulfúrico , Termodinâmica
10.
Elife ; 82019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31644426

RESUMO

Numerous challenges have impeded HIV-1 vaccine development. Among these is the lack of a convenient small animal model in which to study antibody elicitation and efficacy. We describe a chimeric Rhabdo-Immunodeficiency virus (RhIV) murine model that recapitulates key features of HIV-1 entry, tropism and antibody sensitivity. RhIVs are based on vesicular stomatitis viruses (VSV), but viral entry is mediated by HIV-1 Env proteins from diverse HIV-1 strains. RhIV infection of transgenic mice expressing human CD4 and CCR5, exclusively on mouse CD4+ cells, at levels mimicking those on human CD4+ T-cells, resulted in acute, resolving viremia and CD4+ T-cell depletion. RhIV infection elicited protective immunity, and antibodies to HIV-1 Env that were primarily non-neutralizing and had modest protective efficacy following passive transfer. The RhIV model enables the convenient in vivo study of HIV-1 Env-receptor interactions, antiviral activity of antibodies and humoral responses against HIV-1 Env, in a genetically manipulatable host.


Assuntos
Anticorpos Antivirais/biossíntese , Linfócitos T CD4-Positivos/imunologia , HIV-1/genética , Vírus Reordenados/genética , Vesiculovirus/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Animais , Especificidade de Anticorpos , Antígenos CD4/genética , Antígenos CD4/imunologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/virologia , Modelos Animais de Doenças , Efeito Fundador , Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Camundongos , Camundongos Transgênicos , Vírus Reordenados/imunologia , Receptores CCR5/genética , Receptores CCR5/imunologia , Vesiculovirus/imunologia , Tropismo Viral/genética , Tropismo Viral/imunologia , Internalização do Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
11.
J Virol ; 93(24)2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31578292

RESUMO

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.


Assuntos
Interferon-alfa/farmacologia , Proteínas com Motivo Tripartido/antagonistas & inibidores , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vesiculovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Linhagem Celular , Citocinas/farmacologia , Humanos , Modelos Moleculares , Fosfoproteínas/genética , Conformação Proteica , Domínios Proteicos , RNA Mensageiro/metabolismo , RNA Viral/biossíntese , Proteínas com Motivo Tripartido/química , Ubiquitina-Proteína Ligases/química , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/genética , Vesiculovirus/genética , Proteínas Virais
12.
Elife ; 72018 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-30084827

RESUMO

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.


Assuntos
Antivirais/metabolismo , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Proteínas de Resistência a Myxovirus/metabolismo , Poro Nuclear/metabolismo , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ligação Proteica
13.
Nature ; 550(7674): 124-127, 2017 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-28953888

RESUMO

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.


Assuntos
Fosfatos de Dinucleosídeos/genética , Sequência Rica em GC/genética , HIV-1/genética , HIV-1/imunologia , RNA Viral/genética , RNA Viral/imunologia , Linhagem Celular , Citoplasma/genética , Citoplasma/virologia , HIV-1/crescimento & desenvolvimento , Humanos , Imunoprecipitação , Mutagênese , Mutação , Ligação Proteica , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/genética
14.
Cell Host Microbe ; 20(3): 392-405, 2016 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-27631702

RESUMO

Interferons (IFNs) exert their anti-viral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). The activity of known ISGs is insufficient to account for the antiretroviral effects of IFN, suggesting that ISGs with antiretroviral activity are yet to be described. We constructed an arrayed library of ISGs from rhesus macaques and tested the ability of hundreds of individual macaque and human ISGs to inhibit early and late replication steps for 11 members of the retroviridae from various host species. These screens uncovered numerous ISGs with antiretroviral activity at both the early and late stages of virus replication. Detailed analyses of two antiretroviral ISGs indicate that indoleamine 2,3-dioxygenase 1 (IDO1) can inhibit retroviral replication by metabolite depletion while tripartite motif-56 (TRIM56) accentuates ISG induction by IFNα and inhibits the expression of late HIV-1 genes. Overall, these studies reveal numerous host proteins that mediate the antiretroviral activity of IFNs.


Assuntos
Antivirais/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Interferons/metabolismo , Retroviridae/imunologia , Retroviridae/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Replicação Viral , Animais , Biblioteca Gênica , Testes Genéticos , Humanos , Macaca mulatta
15.
RNA ; 22(8): 1228-38, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27247436

RESUMO

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.


Assuntos
HIV-1/genética , RNA Viral/genética , Linhagem Celular , Humanos
16.
Cell ; 159(5): 1096-1109, 2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25416948

RESUMO

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.


Assuntos
HIV-1/fisiologia , RNA Viral/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Bases , Linhagem Celular , Imunoprecipitação da Cromatina , Genes env , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA de Transferência/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química
17.
J Virol ; 88(14): 7738-52, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24760893

RESUMO

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.


Assuntos
Proteína do Núcleo p24 do HIV/imunologia , HIV-1/imunologia , Proteínas de Resistência a Myxovirus/imunologia , Animais , Análise Mutacional de DNA , Evolução Molecular , Proteína do Núcleo p24 do HIV/genética , HIV-1/genética , Humanos , Mutagênese
18.
Nature ; 502(7472): 563-6, 2013 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-24121441

RESUMO

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.


Assuntos
Infecções por HIV/prevenção & controle , HIV-1/fisiologia , Interferon-alfa/imunologia , Proteínas de Resistência a Myxovirus/metabolismo , Transporte Ativo do Núcleo Celular , Capsídeo/metabolismo , Divisão Celular , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Células Cultivadas , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/imunologia , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas de Resistência a Myxovirus/genética , Interferência de RNA , Transcrição Reversa , Transcriptoma , Replicação Viral
19.
PLoS Pathog ; 9(4): e1003299, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23633949

RESUMO

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.


Assuntos
Antígenos CD/metabolismo , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Montagem de Vírus , Liberação de Vírus , Antígenos CD/química , Sítios de Ligação , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Regulação para Baixo , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Células HEK293 , Infecções por HIV/virologia , HIV-1/genética , Células HeLa , Proteínas do Vírus da Imunodeficiência Humana/química , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Proteínas de Membrana/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética
20.
Nature ; 492(7427): 118-22, 2012 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-23103874

RESUMO

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.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Especificidade de Anticorpos/imunologia , Modelos Animais de Doenças , Infecções por HIV/virologia , HIV-1/genética , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , HIV-1/isolamento & purificação , Meia-Vida , Humanos , Imunização Passiva , Camundongos , Camundongos Endogâmicos NOD , Fatores de Tempo , Carga Viral/efeitos dos fármacos
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