Reconstitution of bluetongue virus polymerase activity from isolated domains based on a three-dimensional structural model.
Bluetongue virus (BTV) is a double-stranded RNA virus of the Reoviridae family. The VP1 protein of BTV is the viral RNA-dependent RNA polymerase (RdRp), which is responsible for the replication of the viral genome. Currently there is no structural information available for VP1. By manual alignment of BTV, Reovirus and other viral RdRps we have generated a model for the structure of VP1, the RdRp of BTV. The structure can be divided into three domains: an N-terminal domain, a C-terminal domain, and a central polymerase domain. Mutation of the putative catalytic site in the central polymerase domain by site-directed mutagenesis abrogated in vitro replicase activity. Each of the domains was expressed individually and subsequently partially purified to obtain direct evidence for the location of polymerase activity and the nucleoside triphosphate binding site. The nucleoside triphosphate binding site was located by showing that CTP only bound to the full-length protein or to the polymerase domain and not to either of the other two domains. None of the domains had catalytic activity when tested individually or in tandem but when all three domains were mixed together the RdRp activity was reconstituted. This is the first report of the reconstitution of a functional viral RdRp in vitro from individual domains.
Wehrfritz
Josa-marie
JM
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK.
Boyce
Mark
M
Mirza
Sahdia
S
Roy
Polly
P
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001820:Bluetongue virus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014764:Viral Proteins; C091731:lambda3 protein, Reovirus; D012324:RNA-Dependent RNA Polymerase