Graduate Thesis Or Dissertation
 

Search for protein-protein interactions underlying the cis-preferential replication of turnip yellow mosaic virus

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  • Coreplication experiments have revealed that replication of turnip yellow mosaic virus (TYMV) RNA in turnip protoplasts is cis-preferential. Genomes encoding mutant p141 or p66, proteins essential for virus replication, were inefficiently rescued by a helper genome. One model for the cis-preferential replication of TYMV is that p66 and p141 form a complex that associates with the RNA from which they are translated, limiting their availability in trans. Three types of experiments were used in this study in an attempt to obtain physical evidence for the hypothetical interaction between p66 and p141. Immunoprecipitations from in vitro translation reactions using antiserum that recognizes p66 (and its progenitor, p206) coprecipitate p141, indicating that the proteins form a complex in vitro. The results of coimmunoprecipitations of translation products with in-frame deletions did not lead to definitive information about interaction domains. p66 and the helicase domain of p141 do not detectably interact in the yeast two-hybrid system or in GST fusion interaction assays. Problems with the expression of full length p141 fusions make conclusions about the interaction of other p141 domains with p66 not possible at this time. Since the helicase domain of p141 does not appear to interact with p66, future experiments will focus on obtaining expression of smaller domains of p141, outside the helicase domain, and determining if they interact with p66. Variations to the model that do not necessitate the direct interaction between p66 and p141 are also considered.
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