UBC Theses and Dissertations
Molecular and biochemical characterization of viral and vector components required for cucumber necrosis virus transmission Kishore Kakani, Naga
Natural transmission of Cucumber necrosis virus (CNV) occurs via zoospores of the chytrid fungus Olpidium bornovanus. Transmission involves specific adsorption of virus particles onto the zoospore plasmalemma prior to infestation of cucumber roots by virus bound zoospores. In order to determine if specific regions of the CNV capsid are involved in transmission, several naturally occurring CNV transmission mutants were isolated and characterized. Analysis of the mutants showed that the CNV trimer cavity at the particle quasi three-fold axis plays an important role in transmission, and, moreover that the reduction in transmission is at least partially due to the reduced ability of mutants to bind to zoospores. In vitro virus/zoospore binding studies have shown that pre-treatment of zoospores with trypsin and sodium periodate each decrease CNV binding by approximately 80%, whereas no reduction in binding was found when zoospores were treated with phospholipase C. These studies suggest an important role for zoospore proteins and/or glycoprotein(s) in virus attachment. In virus overlay assays, CNV virions bound to specific-sized zoospore proteins, but CNV transmission mutants showed little or reduced binding. Several sugars were used to study their inhibitory potential on CNV binding to zoospores in vitro. It was found that a variety of mannose-containing sugars inhibited CNV binding to zoospores whereas several others did not. These studies suggest that the putative zoospore receptor may be a mannose-containing glycoprotein. Many animal virus particles undergo conformational changes upon binding to their cellular receptors. CNV is an icosahedral virus and like many other isometric plant viruses, undergoes expansion in the presence of EDTA at an alkaline pH. In the case of CNV, we have demonstrated that during expansion, the internally located coat protein RNA binding domain (R) and arm domains translocate to the particle exterior, becoming protease sensitive. Protease digestion experiments of zoospore-bound virus have revealed that CNV undergoes conformational change upon binding to zoospores and that the conformationally altered virion resembles the swollen conformation. In addition, we have found that a CNV mutant defective in vector transmission is unable to undergo this conformational change. This is the first time that conformational change in a plant virus particle has been shown to be essential for vector transmission.
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