Polyprotein driven formation of two interdependent sets of complexes supporting hepatitis C virus genome replication

Gomes, R., Isken, O., Tautz, N., McLauchlan, J. and McCormick, C. J. (2016) Polyprotein driven formation of two interdependent sets of complexes supporting hepatitis C virus genome replication. Journal of Virology, 90(6), pp. 2868-2883. (doi:10.1128/JVI.01931-15) (PMID:26719260)

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Hepatitis C virus (HCV) requires proteins from the NS3-NS5B polyprotein to create a replicase unit for replication of its genome. The replicase proteins form membranous compartments in cells to facilitate replication, but little is known about their functional organization within these structures. We recently reported on intragenomic replicons, bicistronic viral transcripts expressing an authentic replicase from ORF2 and a second duplicate NS polyprotein from ORF1. Using these constructs and other methods, we have assessed polyprotein requirements needed for rescue of different lethal point mutations across NS3-5B. Mutations readily tractable to rescue broadly fell into two groupings; those requiring expression of a minimum NS3-5A and those requiring expression of a minimum NS3-5B polyprotein. A cis-acting mutation that blocked NS3 helicase activity, T1299A, was tolerated when introduced into either ORF within the intragenomic replicon, but unlike many other mutations required the other ORF to express a functional NS3-5B. Three mutations were identified as more refractile to rescue; one that blocked cleavage of the NS4B5A boundary (S1977P), another in the NS3 helicase (K1240N) and a third in NS4A (V1665G). Introduced into ORF1, these exhibited a dominant negative phenotype, but with K1240N inhibiting replication as a minimum NS3-5A polyprotein whereas V1665G and S1977P only impaired replication as a NS3-5B polyprotein. Furthermore, a S1977P mutated NS3-5A polyprotein complemented other defects shown to be dependent on NS3-5A for rescue. Overall, our findings suggest the existence of two inter-dependent sets of protein complexes supporting RNA replication, distinguishable by the minimum polyprotein requirement needed for their formation.

Item Type:Articles
Glasgow Author(s) Enlighten ID:McLauchlan, Professor John
Authors: Gomes, R., Isken, O., Tautz, N., McLauchlan, J., and McCormick, C. J.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Journal of Virology
Publisher:American Society for Microbiology
ISSN (Online):1098-5514
Published Online:30 December 2015
Copyright Holders:Copyright © 2015 Gomes et al.
First Published:First published in Journal of Virology 90(6):2868-2883
Publisher Policy:Reproduced under a Creative Commons License

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