Phenotypic mapping of the rubella virus genome

by Lund, Karen Diane

Abstract (Summary)
An infectious clone of the Cendehill vaccine strain of rubella virus (RV) has been constructed, as well as two chimeric clones containing cDNAs from Cendehill and Therien (wt+) strains. These clones were used to map the regions of the genome responsible for restriction of the Cendehill strain in human synovial cells (SC). Attenuating mutations have been mapped to two of the nonstructural gene regions. Substitution of Cendehill cDNA representing nts 2803 - 5355 into the Therien infectious clone (pROB0302) resulted in a decreased yield of progeny following electroporation of RNA transcripts into SC. This region contained five mutations, at nts 2829, 3060, 3164, 3528 and 4530, in the COOH-third of P150. Substitution of the Cendehill sequence representing nts 1 - 2803 contributed to a further restriction of the progeny in SC. This region contained two mutations at nts 37 and 55, within the 5' stem-loop structure. The further observation that Cendehill bound equally well to SC and the permissive Vero cell Iine indicated that restriction was not at the level of receptor binding, a function of the envelope proteins. Moreover, substitution of the Cendehill structural gene region into pROB0302 had no effect on replication of the progeny of transfection suggesting that the structurai genes were not involved in restriction. In addition it was found that transfection of RV RNA resulted in progeny with an altered phenotype relative ta the parental strain. Phenotypic alteration rernained stable after six consecutive passages in Vero cells and occurred regardless of the cell type being transfected, the transfection reagent or whether replicative intermediates or purified genomic RNAs were used. Phenotypic properties that were altered were: plaque morphology, growth rate, temperature sensitivity and cell tropism. iii
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Source Type:Master's Thesis



Date of Publication:01/01/1998

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