title: Generation and analysis of recombinant Bunyamwera orthobunyaviruses expressing V5 epitope-tagged L proteins.
abstract: The L protein of Bunyamwera virus (BUNV; family Bunyaviridae) is an RNA-dependent RNA polymerase, 2238 aa in length, that catalyses transcription and replication of the negative-sense, tripartite RNA genome. To learn more about the molecular interactions of the L protein and to monitor its intracellular distribution we inserted a 14 aa V5 epitope derived from parainfluenza virus type 5, against which high-affinity antibodies are available, into different regions of the protein. Insertion of the epitope at positions 1935 or 2046 resulted in recombinant L proteins that retained functionality in a minireplicon assay. Two viable recombinant viruses, rBUNL4V5 and rBUNL5V5, expressing the tagged L protein were rescued by reverse genetics, and characterized with respect to their plaque size, growth kinetics and protein synthesis profile. The recombinant viruses behaved similarly to wild-type (wt) BUNV in BHK-21 cells, but formed smaller plaques and grew to lower titres in Vero E6 cells compared with wt BUNV. Immunofluorescent staining of infected cells showed the L protein to have a punctate to reticular distribution in the cytoplasm, and cell fractionation studies indicated that the L protein was present in both soluble and microsomal fractions. Co-immunoprecipitation and confocal microscopic assays confirmed an interaction between BUNV L and N proteins. The recombinant viruses expressing tagged L protein will be highly valuable reagents for the detailed dissection of the role of the BUNV L protein in virus replication.
authors.affiliation: Centre for Biomolecular Sciences, School of Biology, University of St Andrews, North Haugh, St Andrews, Scotland KY16 9ST, UK.
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Shi
authors.affiliation: Centre for Biomolecular Sciences, School of Biology, University of St Andrews, North Haugh, St Andrews, Scotland KY16 9ST, UK.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Elliott
mesh_terms: D000818:Animals; D002042:Bunyamwera virus / Q000201:enzymology / Q000235:genetics*; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D000939:Epitopes / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D007668:Kidney; D019590:Nucleocapsid Proteins / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D011995:Recombination, Genetic; D012093:Replicon / Q000235:genetics; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D000939:Epitopes; C069882:L protein, Bunyamwera virus; C423924:N protein, Bunyamwera bunyavirus; D019590:Nucleocapsid Proteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase