Mapping functional domains in cucumber mosaic virus and its satellite rnas

Various strains of cucumber mosaic virus (CMV) show differences in host range, pathogenicity, replication efficiency, replication of satellite RNAs, and/or efficiency of transmission by aphid vectors. A number of these properties have been delimited to specific RNAs by re-assorting the genomic RNAs between strains (i.e. pseudorecombination): however, until recently, delimitation of the above properties to specific nucieotides was not possible. We have been using a combination of pseudorecombination and recombination of biologically active cDNA clones of viral RNAs to delimit and/or localize various biological properties associated with particular strains of CMV. Thus, we have determined that 1) rapid and severe symptom induction in zucchini squash by the Fny-strain of CMV map to the 5' half of RNA 1. as does the ability to replicate in some melon cultivars at 37°C; 2) the poor replication of satellite RNAs by the Sny-strain of CMV maps to RNA 1; 3) satellite RNAmediated chlorosis-induction in tobacco is strain-specific and maps to RNA 2 of subgroup II CMV strains: and 4) the induction of systemic chlorosis in tobacco, the cultivar-specific resistance in zucchini squash, and the loss of aphid-transmissibility associated with the M-strain of CMV all map to the coat protein gene of RNA 3. By a combination of sequence analysis, recombination, and site-directed mutagenesis of cDNA clones of satellite RNAs, we have determined that 1) the induction of necrosis in tomato by some satellite RNAs is associated with the conservation of particular sequences in a domain of approximately 25 nucieotides within the 3'-half of the satellite RNAs; 2) the induction of chlorosis in either tobacco or tomato by some satellite RNAs is associated with a domain delimited to 45 nucieotides within the 5'-half of the satellite RNAs; 3) the hosl specificity for chlorosis, i.e. either tobacco or tomato, is determined by a single nucleotide change within this chlorosis-induction domain; and 4) the level of replication of satellite RNAs in cucurbit hosts is influenced by specific sequences within the 3'-half of satellite RNAs. These studies are the first step in determining the role of host components in various aspects of viral replication, nonhost resistance, and viral-induced pathogenicity.