Replication of the genomes of positive-strand RNA viruses of eukaryotic organisms requires coordination of viral proteins, viral RNA and cellular factors or structures. Proteins that integrate virus and host functions, or that modulate host functions to promote infection or restrict defense responses, are of prime importance during virus infection. The functional interactions among viral and host components during replication and spread are understood relatively poorly. The tobacco etch virus (TEV) system was developed during this project to dissect the interactions between viral replication factors and the host cell using novel genetic, biochemical and cell biological approaches. The multi-functional NIa protein was found to integrate several core replication functions and to interact with the host to confer compatibility functions. The TEV systems offers a number of unique benefits to investigate viral and host components involved in the infection process, including the ability to apply classical, molecular and reverse genetics to both the virus and the host. This proposal focuses on two Specific Aims:
In Aim 1, the functional significance of the interaction between the multi-functional VPg- proteinase (NIa) and the RNA-dependent RNA polymerase (NIb) will be addressed. It is postulated that NIb polymerase is recruited to TEV RNA replication complexes through direct interaction with the proteinase domain of NIa.
This Aim will use a large collection of conditional (temperature sensitive) interaction-defective mutants and intergenic suppressor mutants we have generated. The functional consequences of this interaction in infected cells will be examined. In addition, the effects of NIb polymerase binding on the NIa proteinase activity will be tested.
In Aim 2, the interactions between NIa and the host cell will be investigated. Cellular proteins with which NIa potential interacts have been identified, and the significance of the interacting proteins will be determined using a systemic series of functional assays. We will also use a highly effective positive-selection strategy to isolate host (Arabidopsis thaliana) mutants that exhibit NIa-allele dependent infection phenotypes. Identification of these host mutants will provide the starting point for isolation of genes involved in NIa-interacting reactions and pathways.
