A conserved proline between domains II and III of hepatitis C virus NS5A influences both RNA replication and virus assembly
A conserved proline between domains II and III of hepatitis C virus NS5A influences both RNA replication and virus assembly
We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence
Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3
domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal
motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to
be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role
of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues
in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone,
JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and,
furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for
genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated
genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was
not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both
a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical
roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against
extrapolation of data from culture-adapted replicons to infectious virus
10788-10796
Hughes, Mair
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Gretton, Sarah
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Shelton, Holly
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Brown, David D.
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McCormick, Christopher J.
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Angus, Allan G.
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Patel, Arvind H.
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Griffin, Stephen
49c409b7-ee50-4809-b6b8-d5e192ff7424
Harris, Mark
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October 2009
Hughes, Mair
bfd89a3c-6b8f-4180-9874-dddaa8d2ca7c
Gretton, Sarah
954fc9c0-93dd-4c71-a609-2b135322271c
Shelton, Holly
269d8190-81d2-4a6c-a2eb-8803e2a8c81a
Brown, David D.
0a7bc156-1c2a-4995-8a62-39e58c3164a5
McCormick, Christopher J.
0fce14bf-2f67-4d08-991f-114dd1e7f0bd
Angus, Allan G.
7ddff847-c53a-4eb5-9d7a-ad50ccf4920c
Patel, Arvind H.
27de6e99-34a8-407d-80d9-b42fc9e1a41f
Griffin, Stephen
49c409b7-ee50-4809-b6b8-d5e192ff7424
Harris, Mark
065415f3-96a7-443c-91b4-f75a5945f82b
Hughes, Mair, Gretton, Sarah, Shelton, Holly, Brown, David D., McCormick, Christopher J., Angus, Allan G., Patel, Arvind H., Griffin, Stephen and Harris, Mark
(2009)
A conserved proline between domains II and III of hepatitis C virus NS5A influences both RNA replication and virus assembly.
Journal of Virology, 83 (20), .
(doi:10.1128/JVI.02406-08).
Abstract
We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence
Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3
domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal
motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to
be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role
of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues
in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone,
JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and,
furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for
genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated
genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was
not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both
a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical
roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against
extrapolation of data from culture-adapted replicons to infectious virus
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Published date: October 2009
Organisations:
Infection Inflammation & Immunity
Identifiers
Local EPrints ID: 72618
URI: http://eprints.soton.ac.uk/id/eprint/72618
ISSN: 0022-538X
PURE UUID: 89d37fb9-b91d-4ff7-8dd2-c902e44bb94a
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Date deposited: 18 Feb 2010
Last modified: 14 Mar 2024 02:50
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Contributors
Author:
Mair Hughes
Author:
Sarah Gretton
Author:
Holly Shelton
Author:
David D. Brown
Author:
Allan G. Angus
Author:
Arvind H. Patel
Author:
Stephen Griffin
Author:
Mark Harris
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