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

Polyprotein driven formation of two independent sets of complexes supporting hepatitis C virus genome replication
Polyprotein driven formation of two independent sets of complexes supporting hepatitis C virus genome replication
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.
IMPORTANCE:
Positive strand RNA viruses reshape the intracellular membranes of cells to form a compartment within which to replicate their genome, but little is known about functional organization of viral proteins within this structure. We have complemented protein-encoded defects in HCV by constructing sub-genomic HCV transcripts capable of simultaneously expressing both a mutated and functional polyprotein precursor needed for RNA genome replication (intragenomic replicons). Our results reveal that HCV relies on two interdependent sets of protein complexes to support viral replication. They also show that the intragenomic replicon offers a unique way to study replication complex assembly as it enables improved composite polyprotein complex formation compared to traditional trans-complementation systems. Finally, the differential behaviour of distinct NS3 helicase knock-out mutations hints that certain conformations of this enzyme might be particularly deleterious for replication.
0022-538X
1-53
Gomes, Rafael
89b428e0-c16e-4a34-9ba2-69ace35865a1
Isken, Olaf
b1650323-404c-4831-855a-6f9be3ee232d
Tautz, Norbert
59c7b97a-5ff9-4100-b7db-63eae756d688
McLauchlan, John
bbd0b652-5cfb-40ea-afac-1ed7cd85a238
McCormick, Christoper J.
0fce14bf-2f67-4d08-991f-114dd1e7f0bd
Gomes, Rafael
89b428e0-c16e-4a34-9ba2-69ace35865a1
Isken, Olaf
b1650323-404c-4831-855a-6f9be3ee232d
Tautz, Norbert
59c7b97a-5ff9-4100-b7db-63eae756d688
McLauchlan, John
bbd0b652-5cfb-40ea-afac-1ed7cd85a238
McCormick, Christoper J.
0fce14bf-2f67-4d08-991f-114dd1e7f0bd

Gomes, Rafael, Isken, Olaf, Tautz, Norbert, McLauchlan, John and McCormick, Christoper J. (2015) Polyprotein driven formation of two independent sets of complexes supporting hepatitis C virus genome replication. Journal of Virology, 1-53. (doi:10.1128/JVI.01931-15). (PMID:26719260)

Record type: Article

Abstract

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.
IMPORTANCE:
Positive strand RNA viruses reshape the intracellular membranes of cells to form a compartment within which to replicate their genome, but little is known about functional organization of viral proteins within this structure. We have complemented protein-encoded defects in HCV by constructing sub-genomic HCV transcripts capable of simultaneously expressing both a mutated and functional polyprotein precursor needed for RNA genome replication (intragenomic replicons). Our results reveal that HCV relies on two interdependent sets of protein complexes to support viral replication. They also show that the intragenomic replicon offers a unique way to study replication complex assembly as it enables improved composite polyprotein complex formation compared to traditional trans-complementation systems. Finally, the differential behaviour of distinct NS3 helicase knock-out mutations hints that certain conformations of this enzyme might be particularly deleterious for replication.

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JVI preprint on website.pdf - Accepted Manuscript
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More information

Accepted/In Press date: 21 December 2015
e-pub ahead of print date: 30 December 2015
Organisations: Clinical & Experimental Sciences

Identifiers

Local EPrints ID: 385782
URI: https://eprints.soton.ac.uk/id/eprint/385782
ISSN: 0022-538X
PURE UUID: e18f17b1-c35a-42e7-b65c-55d8fe42f3c9
ORCID for Christoper J. McCormick: ORCID iD orcid.org/0000-0002-6155-9161

Catalogue record

Date deposited: 22 Jan 2016 12:53
Last modified: 19 Jul 2019 00:54

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