Shi, X. and Elliott, R.M. (2009) Generation and analysis of recombinant Bunyamwera orthobunyaviruses expressing V5 epitope-tagged L proteins. Journal of General Virology, 90(2), pp. 297-306. (doi: 10.1099/vir.0.007567-0)
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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.
| Item Type: | Articles |
|---|---|
| Status: | Published |
| Refereed: | Yes |
| Glasgow Author(s) Enlighten ID: | Elliott, Professor Richard and Shi, Dr Xiaohong |
| Authors: | Shi, X., and Elliott, R.M. |
| College/School: | College of Medical Veterinary and Life Sciences > School of Infection & Immunity |
| Journal Name: | Journal of General Virology |
| Publisher: | Society for General Microbiology |
| ISSN: | 0022-1317 |
| ISSN (Online): | 1098-5514 |
| Copyright Holders: | Copyright © 2009 SGM |
| First Published: | First published in Journal of General Virology 90(2):297-306 |
| Publisher Policy: | Reproduced in accordance with the copyright policy of the publisher |
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