1887

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Volume 90, Issue 2

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

Abstract

The L protein of Bunyamwera virus (BUNV; family ) 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.

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2009-02-01
2024-05-11
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Generation and analysis of recombinant Bunyamwera orthobunyaviruses expressing V5 epitope-tagged L proteins
J. Gen. Virol. 90, 297 (2009); https://doi.org/10.1099/vir.0.007567-0
/content/journal/jgv/10.1099/vir.0.007567-0
/content/journal/jgv/10.1099/vir.0.007567-0
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