The coronavirus mouse hepatitis virus, strain A59 (MHV-A59), causes acute encephalitis and hepatitis followed by chronic demyelination in mice. Infection of mice by MHV provides a model system for the study of human demyelinating diseases such as multiple sclerosis. The long term goal of this project is to understand the molecular basis of the biological properties of MHV-A59. The focus of this proposal is on the activities encoded in gene 1 of MHV-A59, the putative polymerase gene. This 21 kb gene encodes three predicted proteinase domains as well as polymerase, NTPase/helicase and zinc finger domains. The gene contains two large open reading frames (ORF 1a and ORF 1b); ORF 1b is thought to be expressed via a translational frame shift at the end of ORF 1a. The presence of large ORFs and proteinase motifs suggests that synthesis and processing of the viral replicase will involve a cascade of proteolytic events. The goal of this project is to study the structure and function as well as the mechanism of genesis of the polypeptides encoded in the putative RNA polymerase gene.The focus of this proposal will be on the three predicted proteinase domains in ORF 1a (1) cDNA clones containing portions of ORF 1a will be used to analyze the two predicted papain-like proteinase domains, PLP-1 and PLP-2. N-terminal protein sequencing will be carried out to precisely map the recognition/cleavage site for p28 in vivo and to map second cleavage site observed in vitro. We will determine whether the second predicted papain-like domain does indeed have proteinase activity. We will begin to explore a possible role for intracellular factors in the MHV papain-like activities.We will express in E. coli high levels of PLP-1 and begin to characterize its enzymatic properties on peptide substrates. (2) The cDNA clone containing the predicted """"""""picornavirus 3C-like"""""""" proteinase domain will be analyzed both by in vitro translation and in E. coli to verify its catalytic amino acids, and to determine its specificities for cleavage both of itself in cis and predicted cleavage of ORF 1b polypeptides. Using antibodies, intracellular forms of the 3C-like proteinase will be examined. A possible association of these polypeptides with membranes will also be investigated. The understanding of these activities will contribute greatly to understanding the molecular basis for replication and pathogenesis of MHV and furthermore, will aid in the design of antiviral strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI017418-14A2
Application #
2060516
Study Section
Experimental Virology Study Section (EVR)
Project Start
1989-05-01
Project End
2000-06-30
Budget Start
1995-07-01
Budget End
1996-06-30
Support Year
14
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Navas-Martin, Sonia; Brom, Maarten; Chua, Ming-Ming et al. (2007) Replicase genes of murine coronavirus strains A59 and JHM are interchangeable: differences in pathogenesis map to the 3'one-third of the genome. J Virol 81:1022-6
De Albuquerque, Nadine; Baig, Ehtesham; Ma, Xuezhong et al. (2006) Murine hepatitis virus strain 1 produces a clinically relevant model of severe acute respiratory syndrome in A/J mice. J Virol 80:10382-94
Navas-Martin, Sonia; Brom, Maarten; Weiss, Susan R (2006) Role of the replicase gene of murine coronavirus JHM strain in hepatitis. Adv Exp Med Biol 581:415-20
Zuo, Xun; Mattern, Michael R; Tan, Robin et al. (2005) Expression and purification of SARS coronavirus proteins using SUMO-fusions. Protein Expr Purif 42:100-10
Sperry, Steven M; Kazi, Lubna; Graham, Rachel L et al. (2005) Single-amino-acid substitutions in open reading frame (ORF) 1b-nsp14 and ORF 2a proteins of the coronavirus mouse hepatitis virus are attenuating in mice. J Virol 79:3391-400
Kazi, Lubna; Lissenberg, Arjen; Watson, Richard et al. (2005) Expression of hemagglutinin esterase protein from recombinant mouse hepatitis virus enhances neurovirulence. J Virol 79:15064-73
Weiss, Susan R; Navas-Martin, Sonia (2005) Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. Microbiol Mol Biol Rev 69:635-64
Navas-Martin, Sonia; Hingley, Susan T; Weiss, Susan R (2005) Murine coronavirus evolution in vivo: functional compensation of a detrimental amino acid substitution in the receptor binding domain of the spike glycoprotein. J Virol 79:7629-40
Chua, Ming Ming; MacNamara, Katherine C; San Mateo, Lani et al. (2004) Effects of an epitope-specific CD8+ T-cell response on murine coronavirus central nervous system disease: protection from virus replication and antigen spread and selection of epitope escape mutants. J Virol 78:1150-9
Navas-Martin, Sonia R; Weiss, Susan (2004) Coronavirus replication and pathogenesis: Implications for the recent outbreak of severe acute respiratory syndrome (SARS), and the challenge for vaccine development. J Neurovirol 10:75-85

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