RESUMO
The early appearance of broadly neutralizing antibodies (bNAbs) in serum is associated with spontaneous hepatitis C virus (HCV) clearance, but to date, the majority of bNAbs have been isolated from chronically infected donors. Most of these bNAbs use the VH1-69 gene segment and target the envelope glycoprotein E2 front layer. Here, we performed longitudinal B cell receptor (BCR) repertoire analysis on an elite neutralizer who spontaneously cleared multiple HCV infections. We isolated 10,680 E2-reactive B cells, performed BCR sequencing, characterized monoclonal B cell cultures, and isolated bNAbs. In contrast to what has been seen in chronically infected donors, the bNAbs used a variety of VH genes and targeted at least three distinct E2 antigenic sites, including sites previously thought to be non-neutralizing. Diverse front-layer-reactive bNAb lineages evolved convergently, acquiring breadth-enhancing somatic mutations. These findings demonstrate that HCV clearance-associated bNAbs are genetically diverse and bind distinct antigenic sites that should be the target of vaccine-induced bNAbs.
Assuntos
Hepacivirus , Hepatite C , Humanos , Anticorpos Amplamente Neutralizantes , Epitopos , Anticorpos Neutralizantes , Proteínas do Envelope Viral/genéticaRESUMO
The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.
Assuntos
Anticorpos Amplamente Neutralizantes/genética , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/genética , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/química , Anticorpos Amplamente Neutralizantes/imunologia , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , Epitopos , Feminino , Genótipo , Hepacivirus/genética , Hepatite C/imunologia , Anticorpos Anti-Hepatite C/química , Anticorpos Anti-Hepatite C/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologiaRESUMO
Human IGHV1-69-encoded broadly neutralizing antibodies (bnAbs) that target the hepatitis C virus (HCV) envelope glycoprotein (Env) E2 are important for protection against HCV infection. An IGHV1-69 ortholog gene, VH1.36, is preferentially used for bnAbs isolated from HCV Env-immunized rhesus macaques (RMs). Here, we studied the genetic, structural, and functional properties of VH1.36-encoded bnAbs generated by vaccination, in comparison to IGHV1-69-encoded bnAbs from HCV patients. Global B cell repertoire analysis confirmed the expansion of VH1.36-derived B cells in immunized animals. Most E2-specific, VH1.36-encoded antibodies cross-neutralized HCV. Crystal structures of two RM bnAbs with E2 revealed that the RM bnAbs engaged conserved E2 epitopes using similar molecular features as human bnAbs but with a different binding mode. Longitudinal analyses of the RM antibody repertoire responses during immunization indicated rapid lineage development of VH1.36-encoded bnAbs with limited somatic hypermutation. Our findings suggest functional convergence of a germline-encoded bnAb response to HCV Env with implications for vaccination in humans.
Assuntos
Anticorpos Neutralizantes/imunologia , Células Germinativas/imunologia , Glicoproteínas/imunologia , Hepacivirus/imunologia , Hepatite C/imunologia , Macaca mulatta/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Linfócitos B/imunologia , Células CHO , Linhagem Celular , Cricetulus , Epitopos/imunologia , Células HEK293 , Hepatite C/virologia , Humanos , Estudos Longitudinais , Macaca mulatta/virologia , Receptores de Antígenos de Linfócitos B/imunologia , Vacinação/métodosRESUMO
Translation initiation of hepatitis C virus (HCV) genomic RNA is induced by an internal ribosome entry site (IRES). Our cryoelectron microscopy (cryo-EM) analysis revealed that the HCV IRES binds to the solvent side of the 40S platform of the cap-dependently translating 80S ribosome. Furthermore, we obtained the cryo-EM structures of the HCV IRES capturing the 40S subunit of the IRES-dependently translating 80S ribosome. In the elucidated structures, the HCV IRES "body," consisting of domain III except for subdomain IIIb, binds to the 40S subunit, while the "long arm," consisting of domain II, remains flexible and does not impede the ongoing translation. Biochemical experiments revealed that the cap-dependently translating ribosome becomes a better substrate for the HCV IRES than the free ribosome. Therefore, the HCV IRES is likely to efficiently induce the translation initiation of its downstream mRNA with the captured translating ribosome as soon as the ongoing translation terminates.
Assuntos
Fatores de Iniciação em Eucariotos/química , Hepacivirus/genética , Iniciação Traducional da Cadeia Peptídica , RNA Viral/química , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Sítios de Ligação , Microscopia Crioeletrônica , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Células HEK293 , Hepacivirus/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Sítios Internos de Entrada Ribossomal , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Viral/genética , RNA Viral/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismoRESUMO
It is known that pre-mRNAs in eukaryotic cells can be processed to circular RNAs by a backsplicing mechanism. Circular RNAs have great stability and can sequester proteins or small RNAs to exert functions on cellular pathways. Because viruses often exploit host pathways, we explored whether the RNA genome of the cytoplasmic hepatitis C virus is processed to yield virus-derived circRNAs (vcircRNAs). Computational analyses of RNA-seq experiments predicted that the viral RNA genome is fragmented to generate hundreds of vcircRNAs. More than a dozen of them were experimentally verified by rolling-circle amplification. VcircRNAs that contained the viral internal ribosome entry site were found to be translated into proteins that displayed proviral functions. Furthermore, two highly abundant, nontranslated vcircRNAs were shown to enhance viral RNA abundance. These findings argue that novel vcircRNA molecules modulate viral amplification in cells infected by a cytoplasmic RNA virus.
Assuntos
Hepatite C , RNA Circular , Humanos , Hepacivirus/genética , RNA Viral/genética , Provírus/genéticaRESUMO
Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Biochemical studies revealed that direct binding of the IRES to the 40S ribosomal subunit places the initiation codon into the P site, where it base pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Subsequently, eIF5 and eIF5B mediate subunit joining, yielding an elongation-competent 80S ribosome. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. However, the structures of initiation complexes assembled on the HCV IRES, the transitions between different states, and the accompanying conformational changes have remained unknown. To fill these gaps, we now obtained cryo-EM structures of IRES initiation complexes, at resolutions up to 3.5 Å, that cover all major stages from the initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role of IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes and prepare them for subunit joining.
Assuntos
Hepacivirus , Hepatite C , Fator de Iniciação 2 em Eucariotos/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/metabolismo , Humanos , Sítios Internos de Entrada Ribossomal , Biossíntese de Proteínas , RNA Viral/genética , RNA Viral/metabolismo , Ribossomos/metabolismoRESUMO
The 3' end of the hepatitis C virus genome is terminated by a highly conserved, 98 nt sequence called 3'X. This untranslated structural element is thought to regulate several essential RNA-dependent processes associated with infection. 3'X has two proposed conformations comprised of either three or two stem-loop structures that result from the different base-pairing interactions within the first 55 nt. Here, we used single-molecule Förster resonance energy transfer spectroscopy to monitor the conformational status of fluorescently labeled constructs that isolate this region of the RNA (3'X55). We observed that 3'X55 can adopt both proposed conformations and the relative abundance of them can be modulated by either solution conditions or nucleotide deletions. Furthermore, interconversion between the two conformations takes place over the course of several hours. The simultaneous existence of two slowly interconverting conformations may help prime individual copies of the viral genome for either viral protein or RNA synthesis, thereby minimizing conflicts between these two competing processes.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Hepacivirus , Conformação de Ácido Nucleico , RNA Viral , Hepacivirus/genética , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Pareamento de Bases , Regiões 3' não Traduzidas , Genoma Viral , Sequência de BasesRESUMO
Distinct molecular pathways govern the differentiation of CD8+ effector T cells into memory or exhausted T cells during acute and chronic viral infection, but these are not well studied in humans. Here, we employed an integrative systems immunology approach to identify transcriptional commonalities and differences between virus-specific CD8+ T cells from patients with persistent and spontaneously resolving hepatitis C virus (HCV) infection during the acute phase. We observed dysregulation of metabolic processes during early persistent infection that was linked to changes in expression of genes related to nucleosomal regulation of transcription, T cell differentiation, and the inflammatory response and correlated with subject age, sex, and the presence of HCV-specific CD4+ T cell populations. These early changes in HCV-specific CD8+ T cell transcription preceded the overt establishment of T cell exhaustion, making this signature a prime target in the search for the regulatory origins of T cell dysfunction in chronic viral infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Transcrição Gênica/imunologia , Doença Aguda , Imunidade Adaptativa/genética , Imunidade Adaptativa/imunologia , Adulto , Idoso , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/imunologia , Variação Genética/imunologia , Hepacivirus/fisiologia , Hepatite C Crônica/genética , Hepatite C Crônica/virologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Fatores de Tempo , Adulto JovemRESUMO
A hepatitis C virus (HCV) vaccine is urgently needed. Vaccine development has been hindered by HCV's genetic diversity, particularly within the immunodominant hypervariable region 1 (HVR1). Here, we developed a strategy to elicit broadly neutralizing antibodies to HVR1, which had previously been considered infeasible. We first applied a unique information theory-based measure of genetic distance to evaluate phenotypic relatedness between HVR1 variants. These distances were used to model the structure of HVR1's sequence space, which was found to have five major clusters. Variants from each cluster were used to immunize mice individually, and as a pentavalent mixture. Sera obtained following immunization neutralized every variant in a diverse HCVpp panel (n = 10), including those resistant to monovalent immunization, and at higher mean titers (1/ID50 = 435) than a glycoprotein E2 (1/ID50 = 205) vaccine. This synergistic immune response offers a unique approach to overcoming antigenic variability and may be applicable to other highly mutable viruses.
Assuntos
Hepacivirus , Hepatite C , Animais , Camundongos , Proteínas do Envelope Viral/genética , Imunização , Imunidade , Anticorpos Anti-Hepatite C , Anticorpos NeutralizantesRESUMO
Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments, ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.
Assuntos
Anexina A3 , Hepacivirus , Hepatite C , Antígeno SS-B , Internalização do Vírus , Humanos , Anexina A3/metabolismo , Anexina A3/genética , Autoantígenos/metabolismo , Autoantígenos/genética , Células HEK293 , Hepacivirus/metabolismo , Hepacivirus/fisiologia , Hepatite C/metabolismo , Hepatite C/virologia , Hepatite C/genética , Interações Hospedeiro-Patógeno , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/virologia , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Proteínas do Core Viral/metabolismo , Proteínas do Core Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/genéticaRESUMO
Spontaneous clearance of acute hepatitis C virus (HCV) infection is associated with single nucleotide polymorphisms (SNPs) on the MHC class II. We fine-mapped the MHC region in European (n = 1,600; 594 HCV clearance/1,006 HCV persistence) and African (n = 1,869; 340 HCV clearance/1,529 HCV persistence) ancestry individuals and evaluated HCV peptide binding affinity of classical alleles. In both populations, HLA-DQß1Leu26 (p valueMeta = 1.24 × 10-14) located in pocket 4 was negatively associated with HCV spontaneous clearance and HLA-DQß1Pro55 (p valueMeta = 8.23 × 10-11) located in the peptide binding region was positively associated, independently of HLA-DQß1Leu26. These two amino acids are not in linkage disequilibrium (r2 < 0.1) and explain the SNPs and classical allele associations represented by rs2647011, rs9274711, HLA-DQB1∗03:01, and HLA-DRB1∗01:01. Additionally, HCV persistence classical alleles tagged by HLA-DQß1Leu26 had fewer HCV binding epitopes and lower predicted binding affinities compared to clearance alleles (geometric mean of combined IC50 nM of persistence versus clearance; 2,321 nM versus 761.7 nM, p value = 1.35 × 10-38). In summary, MHC class II fine-mapping revealed key amino acids in HLA-DQß1 explaining allelic and SNP associations with HCV outcomes. This mechanistic advance in understanding of natural recovery and immunogenetics of HCV might set the stage for much needed enhancement and design of vaccine to promote spontaneous clearance of HCV infection.
Assuntos
Cadeias beta de HLA-DQ/genética , Hepacivirus/patogenicidade , Hepatite C/genética , Interações Hospedeiro-Patógeno/genética , Polimorfismo de Nucleotídeo Único , Doença Aguda , Alelos , Substituição de Aminoácidos , População Negra , Feminino , Expressão Gênica , Estudo de Associação Genômica Ampla , Genótipo , Cadeias beta de HLA-DQ/imunologia , Hepacivirus/crescimento & desenvolvimento , Hepacivirus/imunologia , Hepatite C/etnologia , Hepatite C/imunologia , Hepatite C/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Leucina/imunologia , Leucina/metabolismo , Masculino , Prolina/imunologia , Prolina/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Remissão Espontânea , População BrancaRESUMO
BACKGROUND AND AIMS: Second-generation direct-acting antivirals (2G DAA) to cure HCV have led to dramatic clinical improvements. HCV-associated hepatocellular carcinoma (HCC), however, remains common. Impaired immune tumor surveillance may play a role in HCC development. Our cohort evaluated the effects of innate immune types and clinical variables on outcomes including HCC. METHODS: Participants underwent full HLA class I/KIR typing and long-term HCV follow-up. RESULTS: A total of 353 HCV+ participants were followed for a mean of 7 years. Cirrhosis: 25% at baseline, developed in 12% during follow-up. 158 participants received 2G DAA therapy. HCC developed without HCV therapy in 20 subjects, 24 HCC after HCV therapy, and 10 of these after 2G DAA. Two predictors of HCC among 2G DAA-treated patients: cirrhosis (OR, 10.0, p = 0.002) and HLA/KIR profiles predicting weak natural killer (NK) cell-mediated immunity (NK cell complementation groups 6, 9, 11, 12, OR of 5.1, p = 0.02). Without 2G DAA therapy: cirrhosis was the main clinical predictor of HCC (OR, 30.8, p < 0.0001), and weak NK-cell-mediated immunity did not predict HCC. CONCLUSION: Cirrhosis is the main risk state predisposing to HCC, but weak NK-cell-mediated immunity may predispose to post-2G DAA HCC more than intermediate or strong NK-cell-mediated immunity.
Assuntos
Antivirais , Carcinoma Hepatocelular , Hepacivirus , Células Matadoras Naturais , Neoplasias Hepáticas , Receptores KIR , Humanos , Carcinoma Hepatocelular/imunologia , Neoplasias Hepáticas/imunologia , Células Matadoras Naturais/imunologia , Masculino , Antivirais/uso terapêutico , Feminino , Pessoa de Meia-Idade , Receptores KIR/imunologia , Idoso , Hepacivirus/imunologia , Hepatite C/imunologia , Hepatite C/tratamento farmacológico , Hepatite C/complicações , Antígenos HLA/imunologia , Adulto , Imunidade Celular , Seguimentos , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/imunologia , Hepatite C Crônica/complicaçõesRESUMO
Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE: Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.
Assuntos
Aptidão Genética , Hepacivirus , Hepatócitos , Interações entre Hospedeiro e Microrganismos , Imunidade Inata , Mutação , Humanos , Células Cultivadas , Estresse do Retículo Endoplasmático , Aptidão Genética/genética , Aptidão Genética/imunologia , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Hepacivirus/imunologia , Hepacivirus/fisiologia , Hepatite C/imunologia , Hepatite C/virologia , Hepatócitos/imunologia , Hepatócitos/virologia , Interações entre Hospedeiro e Microrganismos/imunologia , MicroRNAs/metabolismo , Inoculações Seriadas , Resposta a Proteínas não Dobradas , Tropismo Viral , Vírion/crescimento & desenvolvimento , Vírion/metabolismo , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
Hepatitis C virus (HCV) is a member of the Flaviviridae family; however, unlike other family members, the HCV virion has an unusually high lipid content. HCV has two envelope glycoproteins, E1 and E2. E2 contributes to receptor binding, cell membrane attachment, and immune evasion. In contrast, the functions of E1 are poorly characterized due, in part, to challenges in producing the protein. This manuscript describes the expression and purification of a soluble E1 ectodomain (eE1) that is recognized by conformational, human monoclonal antibodies. eE1 forms a complex with apolipoproteins AI and AII, cholesterol, and phospholipids by recruiting high-density lipoprotein (HDL) from the extracellular media. We show that HDL binding is a function specific to eE1 and HDL hinders recognition of E1 by a neutralizing monoclonal antibody. Either low-density lipoprotein or HDL increases the production and infectivity of cell culture-produced HCV, but E1 preferentially selects HDL, influencing both viral life cycle and antibody evasion.IMPORTANCEHepatitis C virus (HCV) infection is a significant burden on human health, but vaccine candidates have yet to provide broad protection against this infection. We have developed a method to produce high quantities of soluble E1 or E2, the viral proteins located on the surface of HCV. HCV has an unusually high lipid content due to the recruitment of apolipoproteins. We found that E1 (and not E2) preferentially recruits host high-density lipoprotein (HDL) extracellularly. This recruitment of HDL by E1 prevents binding of E1 by a neutralizing antibody and furthermore prevents antibody-mediated neutralization of the virus. By comparison, low-density lipoprotein does not protect the virus from antibody-mediated neutralization. Our findings provide mechanistic insight into apolipoprotein recruitment, which may be critical for vaccine development.
Assuntos
Hepacivirus , Hepatite C , Evasão da Resposta Imune , Lipoproteínas HDL , Proteínas do Envelope Viral , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Apolipoproteínas/metabolismo , Hepacivirus/patogenicidade , Hepatite C/imunologia , Hepatite C/virologia , Anticorpos Anti-Hepatite C/imunologia , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Proteínas do Envelope Viral/metabolismo , Células HEK293RESUMO
The 3' untranslated region (3'UTR) of the hepatitis C virus (HCV) RNA genome, which contains a highly conserved 3' region named the 3'X-tail, plays an essential role in RNA replication and promotes viral IRES-dependent translation. Although our previous work has found a cis-acting element for genome encapsidation within 3'X, there is limited information on the involvement of the 3'UTR in particle formation. In this study, proteomic analyses identified host cell proteins that bind to the 3'UTR containing the 3'X region but not to the sequence lacking the 3'X. Further characterization showed that RNA-binding proteins, ribosomal protein L17 (RPL17), and Y-box binding protein 1 (YBX1) facilitate the efficient production of infectious HCV particles in the virus infection cells. Using small interfering RNA (siRNA)-mediated gene silencing in four assays that distinguish between the various stages of the HCV life cycle, RPL17 and YBX1 were found to be most important for particle assembly in the trans-packaging assay with replication-defective subgenomic RNA. In vitro assays showed that RPL17 and YBX1 bind to the 3'UTR RNA and deletion of the 3'X region attenuates their interaction. Knockdown of RPL17 or YBX1 resulted in reducing the amount of HCV RNA co-precipitating with the viral Core protein by RNA immunoprecipitation and increasing the relative distance in space between Core and double-stranded RNA by confocal imaging, suggesting that RPL17 and YBX1 potentially affect HCV RNA-Core interaction, leading to efficient nucleocapsid assembly. These host factors provide new clues to understanding the molecular mechanisms that regulate HCV particle formation. IMPORTANCE: Although basic research on the HCV life cycle has progressed significantly over the past two decades, our understanding of the molecular mechanisms that regulate the process of particle formation, in particular encapsidation of the genome or nucleocapsid assembly, has been limited. We present here, for the first time, that two RNA-binding proteins, RPL17 and YBX1, bind to the 3'X in the 3'UTR of the HCV genome, which potentially acts as a packaging signal, and facilitates the viral particle assembly. Our study revealed that RPL17 and YBX1 exert a positive effect on the interaction between HCV RNA and Core protein, suggesting that the presence of both host factors modulate an RNA structure or conformation suitable for packaging the viral genome. These findings help us to elucidate not only the regulatory mechanism of the particle assembly of HCV but also the function of host RNA-binding proteins during viral infection.
Assuntos
Regiões 3' não Traduzidas , Genoma Viral , Hepacivirus , RNA Viral , Proteínas Ribossômicas , Montagem de Vírus , Proteína 1 de Ligação a Y-Box , Regiões 3' não Traduzidas/genética , Hepacivirus/genética , Hepacivirus/fisiologia , Hepacivirus/metabolismo , Humanos , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Proteína 1 de Ligação a Y-Box/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Montagem de Vírus/genética , RNA Viral/metabolismo , RNA Viral/genética , Replicação Viral , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteômica/métodosRESUMO
ATPase family AAA domain-containing protein 1 (ATAD1) maintains mitochondrial homeostasis by removing mislocalized tail-anchored (TA) proteins from the mitochondrial outer membrane (MOM). Hepatitis C virus (HCV) infection induces mitochondrial fragmentation, and viral NS5B protein is a TA protein. Here, we investigate whether ATAD1 plays a role in regulating HCV infection. We find that HCV infection has no effect on ATAD1 expression, but knockout of ATAD1 significantly enhances HCV infection; this enhancement is suppressed by ATAD1 complementation. NS5B partially localizes to mitochondria, dependent on its transmembrane domain (TMD), and induces mitochondrial fragmentation, which is further enhanced by ATAD1 knockout. ATAD1 interacts with NS5B, dependent on its three internal domains (TMD, pore-loop 1, and pore-loop 2), and induces the proteasomal degradation of NS5B. In addition, we provide evidence that ATAD1 augments the antiviral function of MAVS upon HCV infection. Taken together, we show that the mitochondrial quality control exerted by ATAD1 can be extended to a novel antiviral function through the extraction of the viral TA-protein NS5B from the mitochondrial outer membrane.
Assuntos
Hepacivirus , Hepatite C , Humanos , Hepacivirus/metabolismo , Proteínas Virais/metabolismo , Hepatite C/metabolismo , Mitocôndrias/metabolismo , Antivirais , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
The liver-specific microRNA, miR-122, plays an essential role in the propagation of hepatitis C virus (HCV) by binding directly to the 5'-end of its genomic RNA. Despite its significance for HCV proliferation, the host factors responsible for regulating miR-122 remain largely unknown. In this study, we identified the cellular RNA-binding protein, ELAVL1/HuR (embryonic lethal-abnormal vision-like 1/human antigen R), as critically contributing to miR-122 biogenesis by strong binding to the 3'-end of miR-122. The availability of ELAVL1/HuR was highly correlated with HCV proliferation in replicon, infectious, and chronically infected patient conditions. Furthermore, by screening a kinase inhibitor library, we identified rigosertib, an anticancer agent under clinical trials, as having both miR-122-modulating and anti-HCV activities that were mediated by its ability to target polo-like kinase 1 (PLK1) and subsequently modulate ELAVL1/HuR-miR-122 signaling. The expression of PLK1 was also highly correlated with HCV proliferation and the HCV positivity of HCC patients. ELAVL1/HuR-miR-122 signaling and its mediation of PLK1-dependent HCV proliferation were demonstrated by performing various rescue experiments and utilizing an HCV mutant with low dependency on miR-122. In addition, the HCV-inhibitory effectiveness of rigosertib was validated in various HCV-relevant conditions, including replicons, infected cells, and replicon-harboring mice. Rigosertib was highly effective in inhibiting the proliferation of not only wild-type HCVs, but also sofosbuvir resistance-associated substitution-bearing HCVs. Our study identifies PLK1-ELAVL1/HuR-miR-122 signaling as a regulatory axis that is critical for HCV proliferation, and suggests that a therapeutic approach targeting this host cell signaling pathway could be useful for treating HCV and HCV-associated diseases.
Assuntos
Carcinoma Hepatocelular , Hepatite C , Neoplasias Hepáticas , MicroRNAs , Animais , Humanos , Camundongos , Carcinoma Hepatocelular/genética , Proliferação de Células , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Hepacivirus/fisiologia , Hepatite C/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Transdução de Sinais , Quinase 1 Polo-LikeRESUMO
SignificanceHepatitis C virus chronically infects approximately 1% of the world's population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.
Assuntos
Anticorpos Amplamente Neutralizantes , Anticorpos Anti-Hepatite C , Hepatite C , Imunogenicidade da Vacina , Proteínas do Envelope Viral , Vacinas contra Hepatite Viral , Animais , Anticorpos Amplamente Neutralizantes/biossíntese , Anticorpos Amplamente Neutralizantes/sangue , Hepatite C/prevenção & controle , Anticorpos Anti-Hepatite C/biossíntese , Anticorpos Anti-Hepatite C/sangue , Camundongos , Multimerização Proteica , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Vacinas contra Hepatite Viral/química , Vacinas contra Hepatite Viral/imunologiaRESUMO
More than 2 million adults have hepatitis C virus (HCV) infection in the United States, and new infections continue to increase. Without treatment, HCV infection can lead to advanced liver disease and death. Treatment is recommended for nearly everyone with hepatitis C, resulting in a cure in >95% of people treated and raising the possibility of hepatitis C elimination. Testing is the first step to accessing life-saving treatment. The Centers for Disease Control and Prevention recommends hepatitis C screening for all adults, all pregnant persons, and anyone with risk; yet about one-third of people with hepatitis C remain unaware of their infection. Testing begins with a hepatitis C antibody test, followed, when reactive, by a nucleic acid test to detect HCV RNA. This antibody-first, 2-step testing strategy misses early infections and can result in incomplete diagnoses. Advancements in hepatitis C diagnostics and the US regulatory landscape have created an opportunity to include viral-first testing strategies and improve hepatitis C diagnosis. This journal supplement features 8 articles detailing challenges and opportunities for improving hepatitis C diagnostics in support of advancing hepatitis C elimination in the United States.
Assuntos
Hepacivirus , Hepatite C , Humanos , Estados Unidos/epidemiologia , Hepatite C/diagnóstico , Hepatite C/epidemiologia , Hepatite C/prevenção & controle , Hepacivirus/genética , Programas de Rastreamento/métodos , Erradicação de Doenças , Anticorpos Anti-Hepatite C/sangue , Centers for Disease Control and Prevention, U.S. , RNA ViralRESUMO
Access to direct acting antivirals (DAAs) may be associated with reductions in hepatitis C virus (HCV) viremia prevalence among people with human immunodeficiency virus (PWH). Among 3755 PWH, estimated HCV viremia prevalence decreased by 94.0% from 36% (95% confidence interval [CI], 27%-46%) in 2009 (pre-DAA era) to 2% (95% CI, 0%-4%) in 2021 (DAA era). Male sex, black race, and older age were associated with HCV viremia in 2009 but not in 2021. Injection drug use remained associated with HCV viremia in 2009 and 2021. Targeted interventions are needed to meet the HCV care needs of PWH who use drugs.