RNA-dependent RNA polymerase complex of Brome mosaic virus: analysis of the molecular structure with monoclonal antibodies.
Viral RNA-dependent RNA polymerase (RdRp) plays crucial roles in the genomic replication and subgenomic transcription of Brome mosaic virus (BMV), a positive-stranded RNA plant virus. BMV RdRp is a complex of virus-encoded 1a and 2a proteins and some cellular factors, and associates with the endoplasmic reticulum at an infection-specific structure in the cytoplasm of host cells. In this study, we investigate the gross structure of the active BMV RdRp complex using monoclonal antibodies raised against the 1a and 2a proteins. Immunoprecipitation experiments showed that the intermediate region between the N-terminal methyltransferase-like domain and the C-terminal helicase-like domain of 1a protein, and the N terminus region of 2a protein are exposed on the surface of the solubilized RdRp complex. Inhibition assays for membrane-bound RdRp suggested that the intermediate region between the methyltransferase-like and the helicase-like domains of 1a protein is located at the border of the region buried within a membrane structure or with membrane-associated material.
Dohi
Koji
K
Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan1.
Mise
Kazuyuki
K
Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan1.
Furusawa
Iwao
I
Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan1.
Okuno
Tetsuro
T
Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan1.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D017795:Bromovirus / Q000201:enzymology*; D005591:Chemical Fractionation; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000235:genetics / Q000276:immunology; D004983:Ethanolamines; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011233:Precipitin Tests; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D018985:Epitopes, B-Lymphocyte; D004983:Ethanolamines; D012324:RNA-Dependent RNA Polymerase; C007369:2-diethylaminoethanol