Human respiratory syncytial (RS) virus is an RNA-containing enveloped virus that is the major cause of serious lower respiratory tract infections in young children. Despite its importance as a pathogen, the basic molecular biology of RS virus has not been defined and there is no effective vaccine for this virus. We propose a study of the molecular biology of RS virus that will have two interdependent objectives: (I.) Characterization of the viral genome and viral specified gene products by direct analysis of RNAs and proteins and by molecular cloning of cDNA copies of the viral mRNAs and (II.) Use of the information and the cDNA clones produced in Part I to carry out a detailed study of the structure and function of the viral surface glycoproteins in RS virus infection and immunity. Specifically, we plan to enumerate a complete catalogue of the RS virus genes and gene products. This will be done by in vitro translation of each of the individual RS virus-specific mRNAs and correlation of proteins synthesized in vitro with authentic virus structural and nonstructural proteins by gel electrophoresis, peptide mapping using HPLC and immunoprecipitation. A transcriptional map of the genes will be made by UV inactivation studies. Molecular clones of cDNAs to each of the viral mRNAs will be identified by Northern blot analysis and hybrid-select translation. These studies will confirm and complete the mRNA coding assignments and directly correlate cDNA clones with viral mRNAs and viral polypeptides. The cDNA clones will be used to construct a physical map of the genes by heteroduplex analysis using EM. The nucleotide sequence of cDNAs encoding the two viral surface glycoproteins will be determined. Both procaryotic and eucaryotic expression vectors will be constructed from the cDNAs of the two surface proteins to (i) provide a source of protein for production of monospecific antibodies in rabbits and cotton rats (sigmoden hispidus) that will be used to analyze the roles of the two surface proteins in RS virus infection and immunity and (ii) provide a means for analyzing important functional domains of the surface glycoproteins by recombinant DNA rearrangements.
NIH ExPorter
NIH ExPorter