title: Generation and characterization of a recombinant Rift Valley fever virus expressing a V5 epitope-tagged RNA-dependent RNA polymerase.
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.
authors: Biomedical Sciences Research Centre, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK.
authors: Benjamin
authors: B
authors: Brennan
authors: Biomedical Sciences Research Centre, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK.
authors: Ping
authors: P
authors: Li
authors: Biomedical Sciences Research Centre, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK.
authors: Richard M
authors: RM
authors: Elliott
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D000939:Epitopes / Q000235:genetics; D005455:Fluorescent Antibody Technique; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D016254:Mutagenesis, Insertional; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D059386:Reverse Genetics; D012296:Rift Valley fever virus / Q000235:genetics*; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
chemical_list: D000939:Epitopes; C069882:L protein, Bunyamwera virus; D019590:Nucleocapsid Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase