Regulatory Mechanisms of Cucumber Mosaic Virus Replication

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The subject of this proposal is cucumber mosaic virus (CMV), a wide host-range plant virus for which there is very little natural resistance available. The goals of this project are: (1) to localize sequences in the viral genome involved in the regulation of the rate of replication, and the inhibition of replication by high temperatures; (2) to determine the underlying mechanisms of differential replication in plant cells (protoplast) affected by various genetic alterations; and (3) to determine the regulatory effects of various genetic alterations on the viral replicase in vitro. These goals will be achieved by using biologically-active DNA clones of strains of CMV, that show differences in the regulation various aspects of CMV (and satellite) RNA replication in several cucurbit hosts. The sequences controlling the various replicate-related phenotypes will be mapped by recombination and site-directed mutagenesis of the DNA clones. The specific functions altered by these sequence substitutions will be determined by analyses of the replication products both in in vitro infected protoplasts, as well as of the purified viral replicase provided with the appropriate templates and incubated under the experimental conditions. These analyses will relate differences in viral protein structure to specific functions.***//