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 106), 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 la (MW ca. 0.24 x 106), a 9,500-dalton (9.5K) protein; RNA lb (MW 0.26 x 106), an 11K protein; RNA lc (MW 0.26 x 106), a 14K protein; RNA 2a (MW 0.38 x 106), the 34K phosphorylated (P) protein; RNA 2b (MW 0.39 x 106), a 36K protein; RNA 3a (MW 0.40 x 106), the 26K matrix (M) protein; RNA 3b (MW 0.40 x 106), a 24K protein; RNA 4 (MW 0.47 x 106), the 42K major nucleocapsid (N) protein; and RNA 5 (MW 0.74 x 106), 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.

Cloning and sequencing of the matrix protein (M) gene of turkey rhinotracheitis virus reveal a gene order different from that of respiratory syncytial virus

“…In order to identify the gene upstream (3'proximal) from the F gene, we have used an oligonucleotide, based on the F gene sequence, to prime cDNA synthesis in the hope that some clones would be generated from polycistronic mRNA which comprised F and the 3'proximal gene. This was indeed the case, although the gene adjacent to the 3' terminus of the F gene was not that which had been expected from knowledge of the gene order of RS virus and pneumonia virus of mice (PVM) (Collins et a/., 1984;Chambers et al, 1990).…”

“…All paramyxoviruses and morbilliviruses examined to date have the partial gene order 3'-M-F-(SH)-HN/H-5'; SH is in parentheses to indicate that this gene is not possessed by all viruses in these genera. In contrast two pneumoviruses, human RS virus and PVM, have the equivalent genes in the order 3'-M-SH-G-F-5'; i.e., they have, unlike the other two genera, the SH and cell attachment protein gene(G) on the 3'side of the F gene (Collins et a/., 1984;Chambers et a/., 1990). It was a surprise, therefore, when we found that TRTV had the M gene immediately adjacent to the 3' side of the F gene.…”

“…The subfamily Pneumovirinae is of particular interest because, based on the analysis of the genomes of human RS virus and PVM, the member viruses contain several genes that are not present in species of the Paramyxovirinae subfamily (Collins et a/., 1984;Chambers et al, 1990). Given that TRTV has some pneumovirus characteristics but appears to have diverged a considerable time ago from the other known members of this genus, further analysis of the number, nature, and order of TRTV genes will be instructive with regard to whether those genes currently known to be unique to the pneumovirus genus are essential for replication.…”

Beyond Channel Activity: Protein-Protein Interactions Involving Viroporins

“…SH has potential glycosylation sites in both the C-and N-terminal domains (Collins et al 1990). In infected cells, the SH protein of strain A2 accumulates in four different forms (Olmsted and Collins 1989;Collins et al 1984;Collins and Mottet 1993), but the most abundant is a full-length unglycosylated form. The G protein forms G-F and G-SH complexes, but direct interactions between SH and F have not been observed (Low et al 2008).…”

Applications of nucleic acid probes in veterinary infectious diseases

“…Transcriptional maps for specific genes and identification of microbespecific RNAs can be identified by Northern blot hybridization. Additionally, coding assignments for genes can be established by a combination of Northern blot hybridization and in vitro translation (Collins et al, 1984). Probes have also been useful for screening libraries for specific genes (Rigas et al, 1986 ) and cloning genes by oligonucleotide hybridization (Mayaux et al, 1987).…”