Study of the role of viral coat protein and host factor HSP70 homologs in the assembly and disassembly of Cucumber necrosis virus particles

Virion assembly and disassembly are crucial aspects of the virus multiplication cycle, however, relatively little is known about these processes in plant viruses. While the former helps to produce multiple copies of stable infectious progeny virions, the latter is required for release of the encapsidated viral genome into a host cell for initiating new rounds of virus multiplication. In this thesis, I aimed to study Cucumber necrosis virus (CNV) particle assembly and disassembly and the role of CNV coat protein (CP) and host HSP70 homologs in these processes. It was found that CNV infection of Nicotiana benthamiana causes a significant upregulation of HSP70 homologs, and that, in turn, HSP70 is co-opted by the virus at several stages of the multiplication cycle to promote various aspects of the infection cycle including viral RNA, CP and particle accumulation. HSP70 homologs were also found to assist CNV CP in chloroplast targeting possibly to attenuate chloroplast-mediated plant defence and thereby allow further spread of the virus. It was also determined that the HSP70 homologue, Hsc70-2 is bound to CNV virions and that this association appears to facilitate the uncoating efficiency of CNV particles likely via triggering a conformational change in particles. This is the first report that a plant virus utilizes HSP70 homologs for disassembly. A highly basic “KGRKPR” sequence in the ε-region of the CNV CP arm was also examined for its role in virion assembly and encapsidation of viral RNA. Through mutational analysis, it was found that the basic residues promote T=3 versus T=1 virion formation and encapsidation of full-length viral RNA in vivo. Moreover, mutants lacking 2-4 of the basic residues encapsidated proportionately greater amounts of host RNA suggesting the role of these basic residues in selection of viral RNA during assembly. It was also shown that heat shock enhances transcription of heat-inducible ONSEN-like retrotransposons known to be induced during CNV infection. Since retrotransposons are known to play an important role in genome variation, the described studies may be helpful in understanding the importance of plant viruses in inducing genome variation and perhaps adaptation of plants to changes in the environment.