Hughes, Mair, Gretton, Sarah, Shelton, Holly, Brown, David D., McCormick, Christopher J., Angus, Allan G., Patel, Arvind H., Griffin, Stephen and Harris, Mark
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).
Full text not available from this repository.
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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