Rift valley fever virus L protein forms a biologically active oligomer.
Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) causes mosquito-borne epidemic diseases in humans and livestock. The virus carries three RNA segments, L, M, and S, of negative or ambisense polarity. L protein, an RNA-dependent RNA polymerase, encoded in the L segment, and N protein, encoded in the S segment, exert viral RNA replication and transcription. Coexpression of N, hemagglutinin (HA)-tagged L, and viral minigenome resulted in minigenome replication and transcription, a finding that demonstrated HA-tagged L was biologically active. Likewise L tagged with green fluorescent protein (GFP) was biologically competent. Coimmunoprecipitation analysis using extracts from cells coexpressing HA-tagged L and GFP-tagged L showed the formation of an L oligomer. Bimolecular fluorescence complementation analysis and coimmunoprecipitation studies demonstrated the formation of an intermolecular L-L interaction through its N-terminal and C-terminal regions and also suggested an intramolecular association between the N-terminal and C-terminal regions of L protein. A biologically inactive L mutant, in which the conserved signature SDD motif was replaced by the amino acid residues GNN, exhibited a dominant negative phenotype when coexpressed with wild-type L in the minigenome assay system. Expression of this mutant L also inhibited viral gene expression in virus-infected cells. These data provided compelling evidence for the importance of oligomerization of RVFV L protein for its polymerase activity.
Zamoto-Niikura
Aya
A
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-1019, USA.
Terasaki
Kaori
K
Ikegami
Tetsuro
T
Peters
C J
CJ
Makino
Shinji
S
D002478:Cells, Cultured; D006801:Humans; D009154:Mutation; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000502:physiology; D012296:Rift Valley fever virus / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry*
D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase