This grant application proposes to study murine coronavirus (MHV) maturation. Previously, using a cloned MHV DI cDNA, an MHV """"""""packaging signal"""""""" (PS) was mapped to within 61 nucleotides. In addition, another system, which synthesizes a subgenomic DI RNA when an intergenic region of MHV genome RNA is inserted into the DI RNA, has been established. These two DI systems and, a self-replicating """"""""helper"""""""" MHV DI RNA, DIssA, and a vaccinia virus T7 expression system will be used to study MHV maturation. I. Identify sequence(s) required for nucleocapsid formation and virion packaging. MHV RNA fragments will be expressed by the vaccinia virus T7 expression system in MHV-infected cells. Nucleocapsid formation and packaging of the expressed RNA will be noted. Through analysis of deletion mutants, the MHV sequence(s) that is necessary and sufficient for nucleocapsid formation and RNA packaging will be determined. II. Identify MHV-specific proteins necessary for nucleocapsid formation and virus assembly. Cells infected with DI particles containing DIssA and the """"""""complementary"""""""" DI RNA, which subgenomically expresses three structural envelope protein genes, will produce the same protein products found in a wild-type MHV infection. Coinfection of these two DI particles should allow for synthesis of both DI RNAs and the subgenomic DI RNAs, thereby, leading to mature particle formation and viral release. This system is called the complementary DI system. MHV structural proteins and MHV DI RNA will be coexpressed by the vaccinia virus T7 expression system in cells not infected with MHV, to see which component(s) is necessary for nucleocapsid formation without MHV infection. If nucleocapsid is not formed, then the role of gene 1-derived functions in nucleocapsid formation will be studied using DIssA RNA. If gene 1 proteins and N protein are insufficient for nucleocapsid formation, then the role of the envelope proteins in nucleocapsid formation will be examined using the complementary DI system. The role of SM, M and S proteins in virus assembly will be examined using the complementary DI system. Region(s) of the M protein endodomain that function in MHV assembly will be identified.
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