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UBC Theses and Dissertations

Studies toward the identification of the origin of assembly on cucumber necrosis virus RNA and encapsidation of host RNA Ghoshal, Kankana


Assembly is one of the major steps in the virus multiplication cycle. Recognition of viral RNA by coat protein (CP) is one means to ensure specific packaging of viral RNA over host RNA for the production of infectious virus particles. Viral RNAs possess specific sequences and/or structures [origin of assembly sequences (OASs)] which serve as high-affinity binding sites for the CP. In this thesis, I aimed to identify the OAS of Cucumber necrosis virus (CNV). Serendipitously, it was found that besides viral RNA, CNV also encapsidates host RNAs albeit to a lower level (~0.1%). Therefore, I extended my research to characterize the host RNAs present in CNV virions and in virus-like particles (VLPs) formed during agro-infiltration with CP. Characterization of encapsidated RNAs showed that both CNV virions and VLPs contained a variety of host RNA species, the most predominant being chloroplast encoded RNAs. Remarkably, certain retrotransposon or retrotransposon-like sequences were among the most efficiently encapsidated nuclear encoded RNAs, indicating that CNV virions may possibly serve as a vehicle for horizontal transmission of retrotransposons to new hosts and thereby significantly influence genome evolution. To my knowledge, this is the first report of a plant virus encapsidating retrotransposon-like sequences and complements the recent findings that Flock house virus, a small icosahedral insect virus, also encapsidates retrotransposons. Interestingly, analysis of the relative encapsidation efficiency of CP mRNA in VLPs was found to be high, indicating that the CNV CP ORF may contain an OAS(s). Towards the identification of an OAS in CNV RNA, a 1.2 kb segment encompassing the 3’ terminus of the replicase ORF and 800 nt of the CP ORF was found to stimulate encapsidation of heterologous chimeric viral RNA during co-infection with CNV. However, smaller portions of this region failed to facilitate encapsidation. Interestingly, two chimeric viral RNAs expressing CNV CP were efficiently encapsidated suggesting that the CP ORF may contain an OAS(s). This result raises the possibility that the microenvironment where virus replication and encapsidation occurs may play a role in the specificity of encapsidation, supporting the involvement of multiple factors in the specificity of CNV RNA assembly.

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