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Journal of Virology, October 2009, p. 10788-10796, Vol. 83, No. 20
0022-538X/09/$08.00+0     doi:10.1128/JVI.02406-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly{triangledown}

Mair Hughes,1 Sarah Gretton,1 Holly Shelton,1,{dagger} David D. Brown,1,{ddagger} Christopher J. McCormick,1,§ Allan G. N. Angus,2 Arvind H. Patel,2 Stephen Griffin,1 and Mark Harris1*

Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom,1 MRC Virology Unit, Church Street, Glasgow G11 5JR, United Kingdom2

Received 21 November 2008/ Accepted 28 July 2009

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.

* Corresponding author. Mailing address: Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Mount Preston St., Leeds LS2 9JT, United Kingdom. Phone: 44-113-343-5632. Fax: 44-113-343-5638. E-mail: m.harris{at}leeds.ac.uk

{triangledown} Published ahead of print on 5 August 2009.

{dagger} Present address: Department of Virology, Faculty of Medicine, Imperial College London, St. Marys, Norfolk Place, London W2 1PG, United Kingdom.

{ddagger} Present address: Health and Safety Executive, Biological Agents Unit, Bootle L20 7HS, United Kingdom.

§ Present address: Molecular Microbiology and Infection, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, United Kingdom.

Journal of Virology, October 2009, p. 10788-10796, Vol. 83, No. 20
0022-538X/09/$08.00+0     doi:10.1128/JVI.02406-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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