Rubella virus is a human pathogen that can cause birth defects and a fatal neurodegenerative disorder. Little is known about the molecular biology of rubella virus, largely because it replicates poorly in cell culture. The goal of the proposed research is to use recombinant DNA methodology to overcome the obstacle of poor growth and characterized the molecular biology of rubella virus. The genome nuclei acid of rubella virus is a single-stranded RNA approximately 8,000 nucleotides in length, which is of positive polarity (it functions as a messenger RNA upon entry into the infected cell). The central experimental objective is to use reverse transcriptase to synthesize a DNA complementary (cDNA) to the genome RNA, to insert this cDNA into a bacterial plasmid vector and then to determine the nucleotide sequence of the cDNA. This cDNA sequence in conjunction with partial amino acid sequences to be determined from the virion structural proteins will be used to locate the exact coding sequences for these structural proteins. The complete amino acid sequences of these proteins can then be deduced from the coding sequences. Subsequently, open reading frames within the cDNA sequence known not to code for structural proteins will be used as a guide for the synthesis of short peptides against which antibodies will be raised and used to immunoprecipitate virus nonstructural proteins that contain these peptides from extracts of infected cells. With completion of this analysis, most or all of the proteins synthesized by rubella virus will have been identified, and the location of these proteins on the rubella virus genetic map will have been determined. Finally, the plasmid-linked rubella cDNA will be used as a hybridization probe to study rubella virus intracellular RNA synthesis. Medically, the research to be accomplished in this grant application will provide information essential for development of a synthetic rubella vaccine and methods for rapid diagnosis of rubella infection. Since it can naturally establish persistent infections in humans, rubella virus is an excellent virus with which to study persistent infection. However, before such studies can be undertaken, much more needs to be known about the virus's genetic structure and replication cycle, and the proposed research will fulfill this need. Virologically, rubella virus is classed in the Togaviridae family with a number of arthropod-borne viruses to which it is probably only distantly related. However, comparison between rubella virus and these other togaviruses of both replication strategy and nucleotide sequence could reveal similarities which would provide insight into virus evolution and identify regions of viral protein and RNA which have been conserved evolutionarily and therefore must be very important to virus replication.
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