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

J Gen Virol. 2009 Feb;90(Pt 2):297-306. doi: 10.1099/vir.0.007567-0.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bunyamwera virus / enzymology
  • Bunyamwera virus / genetics*
  • Cell Line
  • Chlorocebus aethiops
  • Cricetinae
  • Epitopes / genetics
  • Gene Expression Regulation, Viral
  • Kidney
  • Nucleocapsid Proteins / genetics*
  • RNA Replicase / genetics
  • Recombination, Genetic
  • Replicon / genetics
  • Vero Cells
  • Viral Proteins / genetics*

Substances

  • Epitopes
  • L protein, Bunyamwera virus
  • N protein, Bunyamwera bunyavirus
  • Nucleocapsid Proteins
  • Viral Proteins
  • RNA Replicase