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

Abstract

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.

NS2 is dispensable for efficient assembly of hepatitis C virus-like particles in a bipartite trans-encapsidation system

“…Taken together, these observations support the notion that distinct pools of replicated/translated/packaged HCV RNA might exist within the cell, and that NS2 may regulate their composition via its multiple protein–protein interactions, in particular the HCV RNA-binding protein, NS5A. Accordingly, NS5A trans -complementation studies discriminate between requirements for particle assembly and replication ( Herod et al , 2014 ), and the pharmacokinetics of the NS5Ai, Daclatasvir, support distinct RNA pools for packaging and replication ( McGivern et al , 2014 ).…”

Hepatitis C Virus RNA Replication Depends on Specific Cis- and Trans-Acting Activities of Viral Nonstructural Proteins

“…Complementation of NS5A also requires expression of an intact NS3-5A polyprotein [57,62] or an active replicon [57][58][59][60][61]. Similar results were observed for NS5A complementation with bovine viral diarrhea virus-1 (BVDV-1), a related member of the Flaviviridae [13].…”

“…Mutations in the HCV core-NS2 genes, including large in-frame deletions, can be complemented in trans [53][54][55][56]. However, among the genes required for RNA replication, only NS4B and NS5A have been shown to be trans-complemented [57][58][59][60][61][62]. These observations have led to the suggestion that the other NS genes work only in cis, although the underlying basis of these cis requirements remains unclear.…”

Hepatitis C Virus RNA Replication and Assembly: Living on the Fat of the Land

“…These data illustrate the complexity of HCV assembly since virtually all viral proteins seem to participate in this process. Novel experimental approaches, including Review trans-complementation assays, as recently established for NS5A (Herod et al, 2014), are needed to resolve the individual steps of HCV virion formation. HCV particle production is tightly linked to apolipoproteins E, A1, C1, and B.…”