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

Herod, Morgan R., Shregel, Vera, Hinds, Christopher, Liu, Mengya, McLauchlan, John and McCormick, Christopher J. (2014) Genetic Complementation of Hepatitis C Virus Nonstructural Protein Functions Associated with Replication Exhibits Requirements That Differ from Those for Virion Assembly. Journal of Virology, 88, 2748-2762. (doi:10.1128/JVI.03588-13). (PMID:24352463)

Abstract

Within the polyprotein encoded by hepatitis C virus (HCV), the minimum components 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, a HCV NS3-5A polyprotein precursor was required to facilitate efficient complementation of a replication-defective 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 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 as compared to in trans. Combined with confocal microscope analysis examining the extent to which the two copies of NS5A from the various expression systems co-localize, the results point to NS3-5A playing role in facilitating the integration of 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.

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Contributors

Author: Christopher J. McCormick

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