Identification of a tenth mRNA of respiratory syncytial virus and assignment of polypeptides to the 10 viral genes

Abstract

Nine mRNAs, their cDNA clones, and a genome transcriptional map have been reported previously for respiratory syncytial virus (P. L. Collins and G. W. Wertz, Proc. Natl. Acad. Sci. U.S.A. 80:3208-3212, 1983). We report here the identification of a 10th viral mRNA, designated mRNA 2b (molecular weight [MW] ca. 0.39 X 10(6)), that was detected by RNA (Northern) blot hybridization with cDNA clones. Analysis of a polycistronic readthrough transcript was used to deduce the position in the viral transcriptional map of the gene encoding the newly identified mRNA. The polypeptide coding assignments of 9 of the 10 respiratory syncytial virus mRNAs were determined. Individual viral mRNAs were purified by hybridization selection with nine unique, nonoverlapping cDNA clones and analyzed by translation in vitro. Each of the nine mRNAs encoded a single polypeptide chain. The coding assignments were as follows: RNA 1a (MW ca. 0.24 X 10(6)), a 9,500-dalton (9.5K) protein; RNA 1b (MW 0.26 X 10(6)), an 11K protein; RNA 1c (MW 0.26 X 10(6)), a 14K protein; RNA 2a (MW 0.38 X 10(6)), the 34K phosphorylated (P) protein; RNA 2b (MW 0.39 X 10(6)), a 36K protein; RNA 3a (MW 0.40 X 10(6)), the 26K matrix (M) protein; RNA 3b (MW 0.40 X 10(6)), a 24K protein; RNA 4 (MW 0.47 X 10(6)), the 42K major nucleocapsid (N) protein; and RNA 5 (MW 0.74 X 10(6)), a 59K protein. The cDNA clones used for the hybridization selections were respiratory syncytial virus specific and did not hybridize with uninfected-cell mRNA; therefore the proteins synthesized with the selected mRNAs were virus specific. The 9.5K, 11K, 14K, 24K, M, P, 36K, N, and 59K proteins were encoded by different mRNAs; therefore these nine proteins are all unique. The 9.5K, 11K, 14K, 24K, M, P, and N proteins synthesized in vitro with hybrid-selected mRNAs each had counterparts with the same electrophoretic mobilities in extracts of virus-infected cells. The in vitro polypeptides and their authentic counterparts were shown to be closely related by limited digest peptide mapping. The 36K and 59K polypeptides lacked counterparts with the same electrophoretic mobilities in infected cells and therefore are candidates for the unprocessed precursors of the viral F and G glycoproteins. The 10th viral mRNA, the 2,500K RNA 7, was not tested directly but is the only known mRNA of the appropriate size to encode the 200K large (L) protein of the viral nucleocapsid. These assignments account for all 10 of the reported viral mRNAs and bring to 10 the number of known unique viral proteins.