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

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Brennanetal2011JGV92Generation.pdf (833.0Kb)
Date
12/2011
Author
Brennan, Benjamin
Li, Ping
Elliott, Richard M.
Keywords
Viral-RNA
L-protein
Bunyamwera virus
L-segment
Replication
Rescue
Genome
Transcription
Sequence
identification
QR355 Virology
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Abstract
The viral RNA-dependent RNA polymerase (RdRp; L protein) of Rift Valley fever virus (RVFV; family Bunyaviridae) 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.
Citation
Brennan , B , Li , P & 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 , vol. 92 , no. 12 , pp. 2906-2913 . https://doi.org/10.1099/vir.0.036749-0
Publication
Journal of General Virology
Status
Peer reviewed
DOI
https://doi.org/10.1099/vir.0.036749-0
ISSN
0022-1317
Type
Journal article
Rights
(c) 2011 SGM. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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  • University of St Andrews Research
URI
http://hdl.handle.net/10023/2191

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