The major surface glycoprotein (G) of human respiratory syncytial (RS) virus has an estimated mature Mr of 84,000-90,000. Among a library of cDNA clones prepared from RS virus mRNAs, we identified clones that hybridized to a message that encoded a M, 36,000 polypeptide that was specifically immunoprecipitated with anti-G antiserum. The amino acid sequence of the G protein backbone was determined by nucleotide sequence analysis of several of the cDNA clones. It contains a combination of structural features that make it unique among the known viral glycoproteins. The G mRNA is 918 nucleotides long and contains a single major open reading frame that encodes a polypeptide having 298 amino acid residues with a Mr of 32,587, a finding consistent with the Mr 36,000 estimate for the in vitro translation product of the G mRNA. This suggests that >50% of the molecular weight ofthe mature glycoprotein may be contributed by carbohydrate. Glycosylation of G is largely resistant to tunicamycin, an inhibitor of the attachment of N-linked oligosaccharides, suggesting that the majority of the carbohydrate residues are attached via O-glycosidic bonds. In accordance with this, serine and threonine residues, the acceptor sites for O-linked oligosaccharides, comprise 30.6% of the total amino acid composition. There are also four potential acceptor sites for N-linked oligosaccharides. The amino acid sequence lacks both an

The human microbiome encodes vast numbers of uncharacterized enzymes, limiting our functional understanding of this community and its effects on host health and disease. By incorporating information about enzymatic chemistry into quantitative metagenomics, we determined the abundance and distribution of individual members of the glycyl radical enzyme superfamily among the microbiomes of healthy humans. We identified many uncharacterized family members, including a universally distributed enzyme that enables commensal gut microbes and human pathogens to dehydrate trans-4-hydroxy-L-proline, the product of the most abundant human posttranslational modification. This ‘chemically-guided functional profiling’ workflow can therefore use ecological context to facilitate the discovery of enzymes in microbial communities.

The nucleotide sequence of the mRNA encoding the F protein of respiratory syncytial (RS) virus (strain A2) was determined from cDNA clones that contain the complete mRNA sequence. The mRNA is 1899 nudeotides long exclusive of polyadenylylate. The single major open reading frame encodes a protein of 574 amino acids, with a calculated molecular weight of 63,453. Major structural features predicted from the amino acid sequence include an NH2-terminal signal sequence (residues 1-22), hydrophobic transmembrane anchor sequence (residues 525-550), five potential acceptor sites for asparagine-linked carbohydrate, and a potential site (residues 131-136) for the proteolytic cleavage that generates the disulfide-linked F1 and F2 subunits, which, by analogy to other paramyxoviruses, constitute the biologically active form of the F protein. The sequence also contains an internal hydrophobic domain (residues 137-154) that, as a consequence of the activating proteolytic cleavage described above, would become the NH2 terminus of the larger, F1 subunit. The amino acid sequence of the hydrophobic terminus of the F1 subunit is known to be highly conserved among several paramyxoviruses but is markedly dissimilar for RS virus. The F2 subunit is relatively hydrophilic and contains four of the five potential carbohydrate acceptor sites. The subunit order is NH2-F2-F1-COOH.

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Respiratory syncytial virus (RSV) is a significant cause of lower respiratory tract disease in children and individuals with cell-mediated immunodeficiencies. Airway epithelial cells may be infected with RSV, but it is unknown whether other cells within the lung permit viral replication. We studied whether human alveolar macrophages supported RSV replication in vitro. Alveolar macrophages exposed to RSV demonstrated expression of RSV fusion gene, which increased in a time-dependent manner and correlated with RSV protein expression. RSV-exposed alveolar macrophages produced and released infectious virus into supernatants for at least 25 d after infection. Viral production per alveolar macrophage declined from 0.053 plaque-forming units (pfu)/cell at 24 h after infection to 0.003 pfu/cell by 10 d after infection and then gradually increased. The capability of alveolar macrophages to support prolonged RSV replication may have a role in the pulmonary response to RSV infection. (J. Clin. Invest. 1990.86:113-119.)

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.

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Background-Coronaviruses infect numerous animal species causing a variety of illnesses including respiratory, neurologic and enteric disease. Human coronaviruses (HCoV) are mainly associated with respiratory tract disease but have been implicated in enteric disease.