A Genetic Interaction between the Core and NS3 Proteins of Hepatitis C Virus Is Essential for Production of Infectious Virus

Within the polyprotein encoded by hepatitis C virus (HCV), the minimum components required for viral RNA replication lie in the NS3-5B region, while virion assembly requires expression of all virus components. Here, we have employed complementation systems to examine the role that HCV polyprotein precursors play in RNA replication and virion assembly. In a trans-complementation assay, an HCV NS3-5A polyprotein precursor was required to facilitate efficient complementation of a replicationdefective mutation in NS5A. However, this requirement for precursor expression was partially alleviated when a second functional copy of NS5A was expressed from an additional upstream cistron within the RNA to be rescued. In contrast, rescue of a virion assembly mutation in NS5A was more limited but exhibited little or no requirement for expression of functional NS5A as a precursor, even when produced in the context of a second replicating helper RNA. Furthermore, expression of NS5A alone from an additional cistron within a replicon construct gave greater rescue of virion assembly in cis than in trans. Combined with the findings of confocal microscope analysis examining the extent to which the two copies of NS5A from the various expression systems colocalize, the results point to NS3-5A playing a role in facilitating the integration of nonstructural (NS) proteins into viral membrane-associated foci, with this representing an early stage in the steps leading to replication complex formation. The data further imply that HCV employs a minor virion assembly pathway that is independent of replication. IMPORTANCEIn hepatitis C virus-infected cells, replication is generally considered an absolute prerequisite for virus particle formation. Here we investigated the role that the viral protein NS5A has in both replication and particle assembly using complementation assays and microscopy. We found that efficient rescue of replication required NS5A to be expressed as part of a larger polyprotein, and this correlated with detection of NS5A at sites where replication occurred. In contrast, rescue of particle assembly did not require expression of NS5A within the context of a polyprotein. Interestingly, although only partial restoration of particle assembly was possible by complementation, that proportion that could be rescued benefitted from expressing NS5A from the same RNA being packaged. Collectively, these findings provide new insight into aspects of polyprotein function. They also support the existence of a minor virion assembly pathway that bypasses replication.

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Genetic Complementation of Hepatitis C Virus Nonstructural Protein Functions Associated with Replication Exhibits Requirements That Differ from Those for Virion Assembly

Herod

Schregel

Hinds

et al. 2014

“…Protein NS3 has been implicated in genome encapsidation as an RNP complex with the core protein to allow efficient budding of infectious particles. Both in HCV (20,21,56) and in yellow fever virus (22,23), the NS3 mutations rescuing impaired genomic RNP formation map to D1 and D2. In contrast, the mutations that rescue infectious pestivirus particle formation in the absence of a functional core protein (24) all map to D3 (Fig.…”

Section: Biochemical Characterization Of Csfv Ns3mentioning

confidence: 99%

“…In addition, NS3 was shown to play other functions in the virus cycle, such as interfering with the innate immune system in the case of HCV (18) and modulating fatty acid synthesis in the case of dengue virus (DENV) (19). It was also shown to participate in genome packaging and particle assembly in HCV (20,21), yellow fever virus (22,23), and pestiviruses (24). Indeed, an apparent interplay between genomic RNP formation and interactions with NS3 has been reported: CSFV virions lacking the core protein were found to be viable only when NS3 is mutated at specific locations.…”

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confidence: 99%

X-Ray Structure of the Pestivirus NS3 Helicase and Its Conformation in Solution

Tortorici

Duquerroy

Kwok

et al. 2015

Pestiviruses form a genus in the Flaviviridae family of small enveloped viruses with a positive-sense single-stranded RNA genome. Viral replication in this family requires the activity of a superfamily 2 RNA helicase contained in the C-terminal domain of nonstructural protein 3 (NS3). NS3 features two conserved RecA-like domains (D1 and D2) with ATPase activity, plus a third domain (D3) that is important for unwinding nucleic acid duplexes. We report here the X-ray structure of the pestivirus NS3 helicase domain (pNS3h) at a 2.5-Å resolution. The structure deviates significantly from that of NS3 of other genera in the Flaviviridae family in D3, as it contains two important insertions that result in a narrower nucleic acid binding groove. We also show that mutations in pNS3h that rescue viruses from which the core protein is deleted map to D3, suggesting that this domain may be involved in interactions that facilitate particle assembly. Finally, structural comparisons of the enzyme in different crystalline environments, together with the findings of small-angle X-ray-scattering studies in solution, show that D2 is mobile with respect to the rest of the enzyme, oscillating between closed and open conformations. Binding of a nonhydrolyzable ATP analog locks pNS3h in a conformation that is more compact than the closest apo-form in our crystals. Together, our results provide new insight and bring up new questions about pNS3h function during pestivirus replication. IMPORTANCEAlthough pestivirus infections impose an important toll on the livestock industry worldwide, little information is available about the nonstructural proteins essential for viral replication, such as the NS3 helicase. We provide here a comparative structural and functional analysis of pNS3h with respect to its orthologs in other viruses of the same family, the flaviviruses and hepatitis C virus. Our studies reveal differences in the nucleic acid binding groove that could have implications for understanding the unwinding specificity of pNS3h, which is active only on RNA duplexes. We also show that pNS3h has a highly dynamic behavior-a characteristic probably shared with NS3 helicases from all Flaviviridae members-that could be targeted for drug design by using recent algorithms to specifically block molecular motion. Compounds that lock the enzyme in a single conformation or limit its dynamic range of conformations are indeed likely to block its helicase function. P estiviruses infect a wide range of cloven-hoofed animals, wild and domestic, causing serious disease. The most studied are the classical swine fever virus (CSFV) (1) and the bovine viral diarrhea virus (BVDV), which impose important economic losses to the livestock industry worldwide (2). They form a genus within the Flaviviridae family of single-stranded RNA viruses, which also includes medically important pathogens in the flavi-and hepacivirus genera. Recent studies also revealed that hepaci-and pegiviruses (the fourth genus in the family) cause infections in horses and other d...

“…Furthermore, subdomains I and II of the NS3 helicase seemed to work together to promote HCV assembly because combination of an adaptive mutation, Q221N or I286V, in subdomain I and a second mutation, I399V, in subdomain II rescued virus production (26). Another study found that an adaptive mutation, K272R, in NS3 helicase could rescue virus production of defective HCVcc that has mutations in core protein (27). Recently, Kohlway and colleagues suggested that amino acids 171 to 194, located in a linker between the helicase and protease domains, also participate in HCV assembly (28).…”

Section: Discussionmentioning

confidence: 99%

A Point Mutation in the N-Terminal Amphipathic Helix α ₀ in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

Boson

et al. 2017

The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic ␣ helix (designated helix ␣ 0 ), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix ␣ 0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix ␣ 0 , which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix ␣ 0 and aid understanding of the role of NS3 in HCV virion morphogenesis.IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix ␣ 0 domain of NS3 that significantly enhanced virus assembly while having no effect on viral genome replication. The mechanistic studies suggested that this mutation promoted the relocation of core proteins from LD to the ER, leading to a more efficient envelopment of viral nucleocapsids. Our results revealed a possible new function of helix ␣ 0 in the HCV life cycle and provided new clues to understanding the molecular mechanisms for the action of NS3 in HCV assembly.KEYWORDS HCV, NS3, core, helix ␣ 0 , assembly, hepatitis C virus