Genetic approaches to the study of coronavirus replication and pathogenesis Public Deposited

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  • March 21, 2019
Creator
  • Deming, Damon Joshua
    • Affiliation: School of Medicine, Department of Microbiology and Immunology
Abstract
  • The recently developed coronavirus reverse genetic systems have been a tremendous asset for improving our understanding of the viruses' complex replication strategy, pathogenesis, mechanisms of host-range expansion, and in the development of anti-viral therapies. We completed two studies using coronavirus infectious clones. The first evaluated a severe acute respiratory syndrome coronavirus (SARS-CoV) vaccine to protect against an antigenically divergent strain. The second study determined the requirement for proteolytic processing of a highly conserved region of the replicase polyprotein for efficient replication. Ideally, a SARS-CoV vaccine should confer long-term protection, especially in vulnerable senescent populations, against both the 2003 epidemic strains and zoonotic strains that may yet emerge from animal reservoirs. Using Venezuelan equine encephalitis virus replicon particles (VRP) expressing the 2003 epidemic Urbani SARS-CoV strain spike (S) glycoprotein (VRP-S) or the nucleocapsid (N) protein from the same strain (VRP-N) as candidate vaccines, we tested their ability to protect young and senescent mice when challenged with homologous and heterologous SARS-CoV strains. The novel heterologous SARS-CoV strain (icGDO3-S) was constructed using synthetic biology and reverse genetics to generate a chimeric virus encoding a synthetic S glycoprotein gene of the most genetically divergent human strain, GDO3, which clusters among the zoonotic SARS-CoV, and represents a strain of SARS-CoV that emerged into the human population independently of the epidemic strain. VRP-S, but not VRP-N, provided protection for both young and senescent mice when challenged with the epidemic strain. When challenged with icGDO3-S, VRP-S protected young mice but only partially protected senescent animals. VRP-N vaccinated mice demonstrated enhanced pulmonary inflammation, which included eosinophils among the cellular infiltrates, following SARS-CoV or icGDO3-S challenge. The highly conserved region at the carboxy-terminus of the coronavirus replicase ORF1a polyprotein is processed by the main proteinase (Mpro) into mature products including nsp7, nsp8, nsp9 and nsp10, proteins with predicted or identified activities involved with RNA synthesis. Mpro continuous translation and processing of ORF1ab polyproteins is required for replication, but specific cleavage events may be dispensable. We determined the requirement for the nsp7-10 proteins and their proteolytic processing during the replication of murine hepatitis virus (MHV), which is phylogenetically grouped with the human coronaviruses OC43 and SARS-CoV. Using the MHV reverse genetics system, in frame deletions of the coding sequences for nsp7, 8, 9, and 10 were either deleted, or the flanking cleavage sites ablated, and the effect upon replication determined. Viable viruses were characterized through analysis of Mpro processing, subgenomic RNA transcription, and in vitro growth fitness. Deletion of any of the four regions encoding nsp7 through 10 was lethal. Disruption of the cleavage sites flanking the protein domains were lethal with the exception of the nsp9/10 cleavage site, which resulted in a mutant virus with severely attenuated replication. In order to determine if a distinct function could be attributed to preprocessed forms of the replicase polyprotein including nsp7-10, the genes encoding nsp7 and nsp8 were rearranged. The mutant virus MHV8/7 was not viable, suggesting that the noncleaved intermediate protein may be essential for replication or proteolytic processing.
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  • In Copyright
Advisor
  • Baric, Ralph S.
Degree granting institution
  • University of North Carolina at Chapel Hill
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  • Open access
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