The major objective of this proposal is to establish a genetic system for the study of the structural proteins of coronaviruses, a family of single-stranded, positive sense RNA viruses whose nucleocapsids are assembled into helical structures. The RNA genomes of members of this family are the largest mature RNA molecules yet discovered, which has made the study of coronaviruses unapproachable by the techniques that have been used for the genetic manipulation of other RNA viruses. A system has been developed for the site-specific mutagenesis of the nucleocapsid (N) protein gene of the prototype coronavirus, mouse hepatitis virus (MHV). This method takes advantage of the high rate of RNA-RNA recombination characteristic of these viruses by transducing a site-specific mutation into the viral genome by means of recombination with a synthetic RNA that has been introduced into infected cells. Single point mutations, as well as extensive substitutions, have been engineered into MHV in this fashion. This procedure will be extended to study the other structural proteins of the virus: the spike glycoprotein (S), the membrane glycoprotein (M), and the small membrane protein (sM), to provide tools for elucidating structural features of these proteins and for helping to determine the roles they play in viral replication and how they interact with both viral and host components. In addition to in vivo recombination based approaches to MHV mutagenesis, multiple methods for the in vitro manipulation of the MHV genome will be developed. These will enable the construction of a wider range of mutant types and will allow more efficient and standardized production of recombinant viruses. The proposed studies will provide 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.
Showing the most recent 10 out of 20 publications
