Functional analysis of the tobacco mosaic virus 126/183-kDa replicase-associated proteins

The Tobacco mosaic virus (TMV) 126- and 183-kDa proteins are multi-domain proteins involved in genome replication. In this dissertation, analysis of replicase-mediated cross-protection, identification of protein-protein interactions and biochemical and structural studies of replicase domains were carried out to dissect TMV replicase functions. Specific TMV replicase sequences were expressed in plants using a Potato virus X (PVX) expression vector and tested for their ability to confer protection against challenge TMV infection. Plants expressing different TMV replicase segments displayed either high or low levels of protection. High levels of protection were conferred by segments from the polymerase domain (POL) and required the presence of translatable POL segments. Low levels of protection were conferred by all replicase segments and found to be active even in the absence of protein expression. Results indicate that TMV replicase mediated cross-protection is based upon two mechanisms; low levels of protection dependent on RNA and high levels POL-mediated protection requiring translatable RNAs and possibly protein expression.;To investigate protein-protein interactions in the assembly of the TMV replicase complex, five segments covering different 126-/183-kDa domains were screened via the yeast two-hybrid system against a library of TMV protein segments. One specific interaction between the helicase (HEL) domain and a protein, IRnHEL, covering the C-terminal region of the intervening region (IR) and N-terminal portion of the HEL domain was identified. This interaction was found to be important in virus replication as mutations that completely disrupted the two-hybrid interaction also abolished TMV replication. To investigate the importance of this interaction in assembly of the replicase complex, in vitro expressed proteins covering the HEL/IRnHEL regions were biochemically and structurally characterized. The HEL protein was found to be active in all helicase functions including ATP hydrolysis, RNA-binding and double-stranded RNA unwinding. Gel filtration studies and electron microscopy showed that the HEL protein as well as a protein covering the entire HEL and IRnHEL domains were present in the form of large order multimers. Studying the multimeric states of these proteins would help understand the conformation of the TMV 126- and 183-kDa proteins in the functional replicase complex.