Identification of the rubella virus nonstructural proteins.
Five segments of the rubella virus (RUB) nonstructural protein open reading frame (NSP-ORF) were cloned into pATH (trpE) bacterial fusion protein expression plasmid vectors. Antisera raised in rabbits against these fusion proteins were used to identify RUB nonstructural polypeptides in lysates from RUB-infected Vero cells and from BHK cells transfected with pTM3/nsRUB, a vector from which the RUB NSP-ORF is expressed. In both systems, three polypeptides were immunoprecipitated. A 200-kDa polypeptide (P200) was immunoprecipitated by all of the antisera and therefore is the primary translation product of the ORF. A 150-kDa polypeptide (P150) was immunoprecipitated by antisera against fusion proteins containing N-terminal regions of the ORF, and a 90-kDa polypeptide (P90) was immunoprecipitated by sera against fusion proteins containing C-terminal regions of the ORF. The order of these polypeptides within the NSP-ORF is thus NH2-P150-P90-COOH. It was shown in a previous study that a protease within P200 catalyzes cleavage of P200 (L. D. Marr et al., Virology 198, 586-592, 1994). When hypertonic block was used to synchronize initiation of translation in RUB-infected cells, P200 was detected within 10 min following release of the block, while P150 and P90 were not detected until 20 min, indicating that translation of the precursor is completed before proteolytic cleavage occurs. Pulse-chase experiments showed that cleavage of the P200 precursor was complete within 90 min of synthesis. P150 was stable for 24 hr following processing, while turnover of P90 was detected within 90 min. In immunofluorescence experiments on RUB-infected cells, antisera that recognized P150 stained a perinuclear focus, a thread-like cytoplasmic structure, and the nuclear membrane.
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Department of Biology, Georgia State University, Atlanta 30302-4010.
Frey
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D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007668:Kidney; D007700:Kinetics; D008970:Molecular Weight; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D012412:Rubella virus / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins