Catherine L. Murray

Hepatitis C Virus p7 and NS2 Proteins Are Essential for Production of Infectious Virus

ABSTRACT Hepatitis C virus (HCV) infection is a global health concern affecting an estimated 3% of the worlds 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.

Architects of assembly: roles of Flaviviridae non-structural proteins in virion morphogenesis

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.

Alanine Scanning of the Hepatitis C Virus Core Protein Reveals Numerous Residues Essential for Production of Infectious Virus

ABSTRACT Hepatitis C virus (HCV) is an important human pathogen affecting an estimated 3% of the worlds 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.

Uncleaved NS2-3 Is Required for Production of Infectious Bovine Viral Diarrhea Virus

ABSTRACT 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.

Turning Hepatitis C into a Real Virus

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.

Genetic Analysis of the Carboxy-Terminal Region of the Hepatitis C Virus Core Protein

ABSTRACT 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.

Bovine Viral Diarrhea Virus Core Is an Intrinsically Disordered Protein That Binds RNA

ABSTRACT 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), Erns, 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.

Hepatitis C: An unsuspected drug target

Infection with hepatitis C is one of the main causes of liver disease, yet there are no broadly effective treatments. Discovery of a potent inhibitor of this virus shows that researchers must think outside the box.

Keeping Track of Viruses

Publisher Summary This chapter reviews methods of isolating, identifying, and tracking viruses with potential applications to microbial forensic investigations. Viruses are the most abundant biological entities on earth. These obligate parasites infect every form of life, from archaea and eubacteria to fungi, plants, and animals. Viruses play key roles in global ecology—they form a vast reservoir of genetic diversity, influence the composition and evolution of host populations, and affect the cycling of chemical compounds through the environment. Research has focused on the tiny fraction that causes disease in humans, domestic animals, and crops; sequencing surveys have suggested that the majority of viruses are completely unknown. The ability of viruses to jump species barriers, move between habitats, and circle the globe rapidly underscores the importance of continued vigilance for naturally emerging or deliberately engineered outbreaks. Viruses are extremely simple “life” forms without metabolic capacity, organelles, translational machinery, or autonomous replicative potential. Virus particles constitute a minimal set of components, primarily those required to deliver the genome to the target cell and initiate replication. Consequently, virus particles (or virions) are extremely small, most in the range of 20 to 200 nm in diameter. Virions are diverse not only in size but also in composition, morphology, and genome characteristics. Virus particles may be irregular in shape or possess a distinct symmetry, such as helical or icosahedral. Particles may be surrounded by a host-derived membrane, termed “enveloped,” or a tight protein shell, termed “nonenveloped.”

End game: Getting the most out of microRNAs

Viruses are notorious for their ability to usurp cellular pathways for their own benefit, often by exploiting host factors in new and unusual ways. Hepatitis C virus (HCV), a causative agent of chronic liver disease, is no exception. As well as using host proteins and lipids, the virus is known to enlist an abundant liver-specific microRNA, miR-122, to aid in its replication (1). MicroRNAs are short (≈22-nt) sequences that typically bind, through complementary ≈6-nt “seed” sites, to the 3′ noncoding regions (NCRs) of certain cellular mRNAs. By recruiting the RNA-induced silencing factor complex (RISC), microRNA binding generally leads to translational suppression and/or degradation of the target transcript. HCV interactions with microRNAs, however, seem to defy all conventions. miR-122 binds the viral genome at not one but two sites. These tandem target sequences are located in the 5′ NCR rather than at the 3′ end, and recruitment of miR-122 does not repress translation but enhances viral replication through as-yet-unclear mechanisms. In PNAS, Machlin et al. (2) uncover a further unique feature of the miR-122–HCV association. Their work reveals that miR-122 binds the viral genome in a complex structure that requires not only the seed sequence but also functionally important associations beyond the seed site. The complex RNA interaction seems to be indispensable for HCV replication but is not required for the actions of miR-122 on cellular mRNAs. This model opens up new potential mechanisms for miR-122 in the HCV life cycle.