RESUMO
The reality of hepatitis C is inescapable for the estimated 130 million people worldwide chronically infected with the virus. Yet this pathogen has been notoriously difficult to move from the infected individual into experimental systems, and each advance--from the identification of the infectious agent to its culture and study--has been a significant challenge. As a result of unrelenting technical hurdles, preventative and therapeutic options have been slow to reach hepatitis C patients. More than 35 years since the recognition of the disease, there is no vaccine available, and the only approved treatment, a combination of pegylated interferon-alpha (IFN-α) and ribavirin, is frequently ineffective. Decades of research, however, have resulted in systematic progress and much is now known about this once elusive pathogen. Most importantly, key breakthroughs have stimulated drug discovery, and the first generation of specifically targeted antiviral inhibitors is poised to enter the market. This review provides a look back at progress in developing tractable model systems for this important agent of chronic hepatitis.
Assuntos
Hepacivirus/fisiologia , Hepatite C/virologia , Animais , Antivirais/uso terapêutico , Descoberta de Drogas , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepatite C/tratamento farmacológico , HumanosRESUMO
Hepatitis C virus (HCV) is a liver-tropic pathogen with severe health consequences for infected individuals. Chronic HCV infection can progress to cirrhosis and hepatocellular carcinoma and is a leading indicator for liver transplantation. The HCV core protein is an essential component of the infectious virus particle, but many aspects of its role remain undefined. The C-terminal region of the core protein acts as a signal sequence for the E1 glycoprotein and undergoes dual processing events during infectious virus assembly. The exact C terminus of the mature, virion-associated core protein is not known. Here, we performed genetic analyses to map the essential determinants of the HCV core C-terminal region, as well as to define the minimal length of the protein that can function for infectious virus production in trans.
Assuntos
Hepacivirus/genética , Proteínas do Core Viral/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular , Genes Virais , Teste de Complementação Genética , Hepacivirus/patogenicidade , Hepacivirus/fisiologia , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sinais Direcionadores de Proteínas/genética , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Transfecção , Proteínas do Core Viral/química , Virulência , Montagem de Vírus/genética , Montagem de Vírus/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Viruses of the Flaviviridae family, including hepatitis C, dengue and bovine viral diarrhoea, are responsible for considerable morbidity and mortality worldwide. Recent advances in our understanding of virion assembly have uncovered commonalities among distantly related members of this family. We discuss the emerging hypothesis that physical virion components are not alone in forming the infectious particle, but that non-structural proteins are intimately involved in orchestrating morphogenesis. Pinpointing the roles of Flaviviridae proteins in virion production could reveal new avenues for antiviral therapeutics.
Assuntos
Flaviviridae/metabolismo , Proteínas não Estruturais Virais/metabolismo , Vírion/metabolismo , Flaviviridae/crescimento & desenvolvimento , Modelos Biológicos , Proteínas não Estruturais Virais/fisiologia , Vírion/crescimento & desenvolvimentoAssuntos
Antivirais/farmacologia , Antivirais/uso terapêutico , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Ensaios Clínicos como Assunto , Farmacorresistência Viral/genética , Humanos , Mutação/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Pestiviruses, including bovine viral diarrhea virus (BVDV), are important animal pathogens and close relatives of hepatitis C virus. Pestivirus particles are composed of an RNA genome, a host-derived lipid envelope, and four virion-encoded structural proteins, core (C), E(rns), E1, and E2. Core is a small, highly basic polypeptide that is processed by three enzymatic cleavages before its incorporation into virions. Little is known about its biological properties or its role in virion assembly and structure. We have purified BVDV core protein and characterized it biochemically. We have determined that the processed form of core lacks significant secondary structure and is instead intrinsically disordered. Consistent with its highly basic sequence, we observed that core binds to RNA, although with low affinity and little discernible specificity. We found that BVDV core protein was able to functionally replace the nonspecific RNA binding and condensing region of an unrelated viral capsid protein. Together these results suggest that the in vitro properties of core may reflect its mechanism of action in RNA packaging and virion morphogenesis.
Assuntos
Vírus da Diarreia Viral Bovina/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/química , Proteínas do Core Viral/metabolismo , Montagem de Vírus , Sequência de Aminoácidos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas do Core Viral/isolamento & purificaçãoRESUMO
Hepatitis C virus (HCV) is an important human pathogen affecting an estimated 3% of the world's population. Recent advances have enabled in vitro propagation of the virus and allow assembly and egress to be investigated for the first time. As a component of the virion, the HCV core protein likely functions primarily in infectious virus production, although little is known about the determinants of this activity. To investigate the roles of core in the viral life cycle, we performed a comprehensive deletion and alanine scanning mutagenesis study of this protein in the context of a genotype 2a reporter virus. We have confirmed that core protein is essential for infectious virion production and have identified numerous residues required for this role. The infectivity of several assembly-defective core mutants could be rescued by compensatory mutations identified in p7 and NS2, suggesting genetic interactions with core and highlighting the importance of these nonstructural proteins in infectious virion morphogenesis.
Assuntos
Hepacivirus/fisiologia , Proteínas do Core Viral/fisiologia , Vírion/fisiologia , Replicação Viral/genética , Alanina/química , Alanina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Linhagem Celular , Genoma Viral/genética , Hepacivirus/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Serina/química , Serina/genética , Proteínas do Core Viral/genética , Vírion/genéticaRESUMO
Hepatitis C virus (HCV) infection is a global health concern affecting an estimated 3% of the world's population. Recently, cell culture systems have been established, allowing recapitulation of the complete virus life cycle for the first time. Since the HCV proteins p7 and NS2 are not predicted to be major components of the virion, nor are they required for RNA replication, we investigated whether they might have other roles in the viral life cycle. Here we utilize the recently described infectious J6/JFH chimera to establish that the p7 and NS2 proteins are essential for HCV infectivity. Furthermore, unprocessed forms of p7 and NS2 were not required for this activity. Mutation of two conserved basic residues, previously shown to be important for the ion channel activity of p7 in vitro, drastically impaired infectious virus production. The protease domain of NS2 was required for infectivity, whereas its catalytic active site was dispensable. We conclude that p7 and NS2 function at an early stage of virion morphogenesis, prior to the assembly of infectious virus.
Assuntos
Hepacivirus/crescimento & desenvolvimento , Canais Iônicos/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Células Cultivadas , Sequência Conservada , Genes Reporter/genética , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Canais Iônicos/genética , Luciferases/análise , Luciferases/genética , Mutação , Proteínas não Estruturais Virais/genética , Proteínas Virais/genéticaRESUMO
Despite increasing characterization of pestivirus-encoded proteins, functions for nonstructural (NS) proteins NS2, NS2-3, NS4B, and NS5A have not yet been reported. Here we investigated the function of bovine viral diarrhea virus (BVDV) uncleaved NS2-3. To test whether NS2-3 has a discrete function, the uncleaved protein was specifically abolished in two ways: first by inserting a ubiquitin monomer between NS2 and NS3, and second by placing an internal ribosome entry site between the two proteins (a bicistronic genome). In both cases, complete processing of NS2-3 prevented infectious virion formation without affecting RNA replication. We tested the hypothesis that uncleaved NS2-3 was involved in morphogenesis by creating a bicistronic genome in which NS2-3 was restored in the second cistron. With this genome, both uncleaved NS2-3 expression and particle production returned. We then investigated the minimal regions of the polyprotein that could rescue an NS2-3 defect by developing a trans-complementation assay. We determined that the expression of NS4A in cis with NS2-3 markedly increased its activity, while p7 could be supplied in trans. Based on these data, we propose a model for NS2-3 action in virion morphogenesis.