RNA viruses are among the most important pathogens of humans, animals and plants. In several, replication involves a tRNA (e.g., retroviruses) or a complex folded 3' tRNA-like structure (e.g., brome mosaic virus, BMV). The maintenance in the RNA genome of properties such as aminoacylation implies the operation of a positive selection pressure, e.g., a controlling role in infection. Our overall objective is to identify how this function contributes to infective processes. A 3' 128 nucleotide (nt) fragment, common to all four BMV RNAs functions in vitro as a substrate for aminoacylation. Recombinant DNA techniques will be used to construct novel BMV RNA sequences with specified nucleotide substitutions, deletions or insertions. Their functionality will be tested in vitro using aminoacyl-tRNA synthetase and our unique BMV RNA template-specific and -dependent replicase. Such experiments, impossible by classical genetics, also permit evaluation of models proposed for secondary and tertiary structures of 3' sequences of viral RNAs. We have placed a cDNA clone of the 3'-terminal 200 nt of BMV RNAs under the control of an SP6 phage promoter and used SP6 RNA polymerase to transcribe microgram quantities of RNA from this DNA template. The cDNA clones was engineered to contain a Tth111 I site, and transcription of cDNA templates cleaved at this site yielded RNAs accurately terminating in -CCA. These transcripts are functional in aminoacylation and replication. Altered RNAs will be constructed by oligodeoxynucleotide-directed site-specific mutations in the cDNA clone and by other cloning techniques. Subsequently, full-length BMV RNA transcripts containing native and altered 3' sequences will be tested for infectivity in protoplasts. We have devised a hybrid-arrested replication assay to localize regions within BMV RNA intrinsic to replication events. We propose to further define regions of native and modified viral RNAs involved in initiation of replication and in binding of replicase (and synthetase) by photocrosslinking and other techniques. We also intend to expand our characterization of replicase products to provide sensitive detection of de novo (+) strand synthesis during replication in vitro. Together with proposed approaches for improvements in the purity and activity of our replicase, the results of these studies are expected to yield an understanding of replication processes.
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