The major object of this proposal is to further develop and apply a genetic system for the study of coronaviruses. Coronoaviruses are a family of single-stranded, positive-sense RNA viruses whoe helical nucleocapsids are packaged within host-derived membrane envelopes containing a small complement of viral membrane glycoproteins. The RNA genomes of coronaviruses are the largest mature RNA molecules yet discovered, making their genetic manipulation unapproachable by the techniques that have been used with other RNA viruses. We have established a system for the site-specific mutagenesis of the prototype coronavirus mouse hepatitis virus (HMV) which takes advantage of the high rate of RNA-RNA recombination in MHV by transducing a site-specific mutation into the viral genome by recombination with a synthetic RNA introduced into infected cells. Single point mutations and extensive substitutions have been engineered into all of the structural protein genes of MHV in this fashion. This procedure will be enhanced further by the advantages offered by a powerful host-range-based selection system and by efforts to obtain an infectious full-length cDNA of the MHV genome. The genetic system will be used to answer basic questions about the structure and function of the components of MHV virions: the spike glycoprotein, the matrix glycoprotein, the small envelope protein, and the nucleocapsid protein. This will provide valuable insights into the roles these proteins play in viral replication and how they interact with both viral and host components. Coronaviruses are important respiratory, neurologic and enteric pathogens, and an understanding of their molecular biology is critical for their control and prophylaxis. The studies proposed will provide fundamental insights into the coronavirus life cycle and assembly, potential targets for antiviral chemotherapy, and a possible means to manipulate these infectious agents for vaccine design.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Virology Study Section (VR)
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Beisel, Christopher E
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Wadsworth Center
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Masters, Paul S; Kuo, Lili; Ye, Rong et al. (2006) Genetic and molecular biological analysis of protein-protein interactions in coronavirus assembly. Adv Exp Med Biol 581:163-73
Goebel, Scott J; Hsue, Bilan; Dombrowski, Todd F et al. (2004) Characterization of the RNA components of a putative molecular switch in the 3' untranslated region of the murine coronavirus genome. J Virol 78:669-82
Goebel, Scott J; Taylor, Jill; Masters, Paul S (2004) The 3' cis-acting genomic replication element of the severe acute respiratory syndrome coronavirus can function in the murine coronavirus genome. J Virol 78:7846-51
Ye, Rong; Montalto-Morrison, Cynthia; Masters, Paul S (2004) Genetic analysis of determinants for spike glycoprotein assembly into murine coronavirus virions: distinct roles for charge-rich and cysteine-rich regions of the endodomain. J Virol 78:9904-17
Trifilo, Matthew J; Montalto-Morrison, Cynthia; Stiles, Linda N et al. (2004) CXC chemokine ligand 10 controls viral infection in the central nervous system: evidence for a role in innate immune response through recruitment and activation of natural killer cells. J Virol 78:585-94
Kuo, Lili; Masters, Paul S (2003) The small envelope protein E is not essential for murine coronavirus replication. J Virol 77:4597-608
de Haan, Cornelis A M; de Wit, Marel; Kuo, Lili et al. (2003) The glycosylation status of the murine hepatitis coronavirus M protein affects the interferogenic capacity of the virus in vitro and its ability to replicate in the liver but not the brain. Virology 312:395-406
Kuo, Lili; Masters, Paul S (2002) Genetic evidence for a structural interaction between the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus. J Virol 76:4987-99
Shen, X; Masters, P S (2001) Evaluation of the role of heterogeneous nuclear ribonucleoprotein A1 as a host factor in murine coronavirus discontinuous transcription and genome replication. Proc Natl Acad Sci U S A 98:2717-22
Ontiveros, E; Kuo, L; Masters, P S et al. (2001) Inactivation of expression of gene 4 of mouse hepatitis virus strain JHM does not affect virulence in the murine CNS. Virology 289:230-8

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