The complex interplay between positive-strand RNA viruses and eukaryotic host cells is understood poorly. Although considerable information is available about the expression and function of proteins encoded by several model positive-strand RNA viruses, significant gaps exist in our understanding of the cellular structures and factors involved in virus replication, assembly and transport. In addition, relatively little is known about the critical virus-host interactions that govern the alteration or sequestration of cellular functions (such as transcription, translation, protein transport) and internal cellular structures (such as membranes) by positive-strand RNA viruses. In large part, this knowledge deficit is due to a lack of model virus-host systems in which rigorous host genetics can be applied. Thus, invaluable mutants with altered susceptibilities or responses to viruses have been isolated only rarely. This proposal centers on exploitation of the tobacco etch virus (TEV)/Arabidopsis thaliana model system to gain insight into intracellular host factors and functions involved in infection by positive-strand RNA viruses. The genome of TEV has proven to be highly amenable to modification by insertion of numerous foreign genes, including those encoding reporter proteins and positive and negative selectable markers. Arabidopsis offers an unbounding resource for identification, cloning and analysis of host genes through versatile genetic approaches. This system will be used to pursue three Specific Aims.
In Specific Aim 1, the Arabidopsis gene RTM1 (Restricted TEV Movement 1), which limits TEV infection to inoculated organs and which we mapped recently to position 16cM on chromosome 1, will be isolated through map-based cloning methods. A naturally occurring rtm1 allele, which permits systemic infection by TEV, will also be isolated.
Specific Aim 2 focuses on RTM1 function and the basis for recognition and limitation of TEV infection.
In Specific Aim 3, a series of novel genetic screens to isolate Arabidopsis mutants with altered susceptibilities or responses to TEV will be pursued and the affected genes will be subjected to map-based cloning. The TEV/Arabidopsis system provides a unique opportunity to identify, using a full complement of genetic resources, critical host factors affecting invasion and spread by positive-strand RNA viruses.
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