Characterization of the Tmv Replicase Proteins: Localization and Interactions with Rab Gdi Proteins

Title of Document: CHARACTERIZATION OF THE TMV REPLICASE PROTEINS: LOCALIZATION AND INTERACTIONS WITH RAB GDI PROTEINS Sabrina Renée Kramer Doctor of Philosophy 2008 Directed By: Dr. James Culver, Associate Professor Center for Biosystems Research University of Maryland Biotechnology Institute Tobacco mosaic virus (TMV) is a model positive-strand RNA virus. TMV encodes two replicase proteins, both of which contain methyltransferase and helicase domains; the 183 kDa protein contains an additional RNA-dependent RNA polymerase domain. Using this virus, virus-host interactions important in the initial establishment of infection and formation of replicase complexes were investigated. Specifically, on the virus side, replicase proteins were examined for regions that may contribute to its localization to the endoplasmic reticulum (ER) during TMV infection. An ER localization domain was identified in a region between amino acids 599 and 701. Alanine substitutions were introduced into this region and examined for their effects on the virus. Several possible hypotheses are discussed as to how this domain may function during infection. Concerning the host, an interaction with a host protein, a Rab GDP Dissociation Inhibitor (Rab GDI), was examined. This interaction occurred with tomato and tobacco Rab GDIs as well as with the originally identified Arabidopsis thaliana Rab GDI (AtGDI2). Silencing of Rab GDI transcripts enhanced the number of infection sites in TMV:GFP-infected plants, but did not alter viral movement or overall accumulation, indicating a possible role in initial establishment of infection. Rab GDI-silenced Nicotiana benthamiana plants showed cellular morphologies similar to those of TMV-infected cells. Moreover, TMV infection results in Rab GDI proteins localizing to structures associated with viral replication. Taken together these data indicate a role for Rab GDI proteins in the initial establishment of infection. Two models of how Rab GDI proteins may contribute to TMV infection are discussed. These studies examine parts of the viral life cycle that are not very well understood, in particular the initiation and establishment of infection. Although vesicle trafficking has been shown to be important for several different pathogens, this is the first time that a Rab GDI protein has been identified as participating in viral replication. Understanding initiation of infection and susceptibility of a host to a pathogen are vital to elucidating pathogen-host interactions and developing disease resistance strategies. CHARACTERIZATION OF THE TMV REPLICASE PROTEINS: LOCALIZATION AND INTERACTIONS WITH RAB GDI PROTEINS