Coronaviruses cause disease in man and animals. This group of single-stranded, positive-sense, enveloped RNA viruses causes a range of serious gastrointestinal and upper respiratory tract infections. Our overall objective is to learn how the parts of this viral pathogen assemble, using mouse hepatitis virus (MHV) as a coronavirus model system. Some experiments will ask whether a virus protein if not involved in assembly exerts an effect on infectivity. The packaging signal (PS) of MHV is a short RNA sequence that is necessary and sufficient for MHV to package RNA into virion. We observed a specific interaction between MHV envelope protein M and the MHV PS, which occurred in the absence of N protein. This observation suggested a previously unforeseen possibility that specific binding of the PS to the M membrane glycoprotein is the initiator of MHV's precise RNA packaging. We propose to describe the MHV viral RNA packaging initiation mechanism, and will start by determining whether or not the PS directly binds M. Assuming M directly binds the PS, then we will identify exactly which M region binds to the PS. We will investigate the possibilities that RNA with the PS drives N protein-M protein interaction, and that binding of M to multiple regions outside of the PS on the genome-length MHV mRNA occurs in infected cells. Coexpression of E and M proteins results in the production of virus-like particles (VLP), and this was shown to be temperature sensitive (ts). This ts phenotype of VLP production will be used to prove that M interacts with E protein and to determine whether or not M oligomerizes at MHV budding sites during VLP production; the biological importance of these putative processes will be investigated. We will also study whether N is needed for RNA packaging, because we speculate that it is dispensable for packaging and central to infectivity. If our speculation is correct, then whether or not VLPs lacking the N are infectious will be determined. The case that VLPs lacking the N are not infectious would mean that an N domain(s) is important for viral infectivity, and these will be identified. How packaged RNA and M interact in the presence of N will be studied. If, however, the case turns out to be that N is necessary for RNA packaging, then we will look at the importance of N protein-M protein interaction in RNA packaging. Any N domain(s) affecting viral RNA packaging will be investigated. Finally, we will study whether homotypic interaction of N exists in the viral nucleocapsid, and if it does, we will look at whether the PS or M protein triggers this.
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