Details

Autor(en) / Beteiligte

• Shi, Xiaohong
• Elliott, Richard M

Titel

Generation and analysis of recombinant Bunyamwera orthobunyaviruses expressing V5 epitope-tagged L proteins

Ist Teil von

• Journal of general virology, 2009-02, Vol.90 (2), p.297-306

Ort / Verlag

Reading: Soc General Microbiol

Erscheinungsjahr

2009

Link zum Volltext

Quelle

EZB Electronic Journals Library

Beschreibungen/Notizen

• Centre for Biomolecular Sciences, School of Biology, University of St Andrews, North Haugh, St Andrews, Scotland KY16 9ST, UK Correspondence Richard M. Elliott rme1{at}st-andrews.ac.uk 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. Published online ahead of print on 31 October 2008 as DOI 10.1099/vir.0.007567-0. Supplementary material is available with the online version of this paper.