Functional analysis of RNA interacting domains of brome mosiac virus coat protein involved in genome packaging.

摘要

The work presented in this dissertation is divided into the following individual but related topics. In Chapter 1, four predicted RNA interacting domains (RIDs) in BMV CP were characterized. In particular one mutation in RID 1, referred to as Delta7aa, showed discernible effects to RNA packaging. Virions of Delta7aa remained non-infectious and failed to package full-length RNA 1. Additionally, the virions were found to contain large amounts of truncated RNAs derived from BMV genomic RNAs.{09}The Delta7aa mutation was also engineered into CCMV CP to examine if it could elicit similar defects in CCMV. Surprisingly, the CC3/Delta7aa mutant behaved like Wt CCMV with respect to biology and RNA packaging. Collectively these studies revealed an amino acid region within the BMV CP that is important for RNA interaction and packaging and exemplified how the same mutation introduced into two structurally similar viruses can elicit quite different effects. In Chapter 2, the Delta7aa mutant was examined in more detail. In vitro assembly studies and gel retardation assays showed that the Delta7aa variant CP is incompetent to assemble and bind RNA in vitro, respectively. It was found that Delta7aa forms polymorphic virions in vivo. CD spectral analysis of the Delta7aa CP revealed a secondary structure different than that of Wt BMV CP. Lastly, MALDI-TOF analysis of Delta7aa virions showed a trypsin digestion pattern that differed from that of Wt BMV virions, indicating that the deletion affected capsid assembly. Consequently, the peptide region that was deleted in Delta7aa is proposed to function as a "molecular switch" necessary for maintaining optimal conformation of CP subunits. Based on the inability of Delta7aa to assemble in vitro, but not in vivo, a chaperon mediated assembly mechanism is proposed. In Chapter 3, the mechanism of N- and C-terminal interactions leading to aberrant particles in vivo was examined, by constructing chimeras involving the exchange of CP regions between BMV and CCMV. Comparative electrophoretic mobility patterns of wild type and chimeric progeny virions allowed me to classify five distinct virion forms: BMV-like, CCMV-like, a mixture containing both parental types, a population distinct from both parents, and finally a non-infectious form.