Generation and characterization of a recombinant Rift Valley fever virus expressing a V5 epitope-tagged RNA-dependent RNA polymerase

Brennan, B. , Li, P. and Elliott, R. M. (2011) Generation and characterization of a recombinant Rift Valley fever virus expressing a V5 epitope-tagged RNA-dependent RNA polymerase. Journal of General Virology, 92(12), pp. 2906-2913. (doi: 10.1099/vir.0.036749-0) (PMID:21900422)

Abstract

The viral RNA-dependent RNA polymerase (RdRp; L protein) of Rift Valley fever virus (RVFV; family <i>Bunyaviridae</i>) is a 238 kDa protein that is crucial for the life cycle of the virus, as it catalyses both transcription of viral mRNAs and replication of the tripartite genome. Despite its importance, little is known about the intracellular distribution of the polymerase or its other roles during infection, primarily because of lack of specific antibodies that recognize L protein. To begin to address these questions we investigated whether the RVFV (MP12 strain) polymerase could tolerate insertion of the V5 epitope, as has been previously demonstrated for the Bunyamwera virus L protein. Insertion of the 14 aa epitope into the polymerase sequence at aa 1852 resulted in a polymerase that retained functionality in a minigenome assay, and we were able to rescue recombinant viruses that expressed the modified L protein by reverse genetics. The L protein could be detected in infected cells by Western blotting with anti-V5 antibodies. Examination of recombinant virus-infected cells by immunofluorescence revealed a punctate perinuclear or cytoplasmic distribution of the polymerase that co-localized with the nucleocapsid protein. The generation of RVFV expressing a tagged RdRp will allow detailed examination of the role of the viral polymerase in the virus life cycle.

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Elliott, Professor Richard and Brennan, Dr Benjamin and Li, Dr Ping
• Authors: Brennan, B., Li, P., and Elliott, R. M.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity; College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
• Journal Name: Journal of General Virology
• Publisher: Society for General Microbiology
• ISSN: 0022-1317
• ISSN (Online): 1465-2099
• Published Online: 01 December 2011
• Copyright Holders: Copyright © 2011 SGM
• First Published: First published in Journal of General Virology 92(12):2906-2913
• Publisher Policy: Reproduced under a Creative Commons License