Genetic analyses of the terminal protein (VPg) and of spacer II in the 5'-nontranslated region of poliovirus RNA
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VPg is a small protein (20-30 amino acids long), which is present in every virus of the Picornaviridae family. A hydroxyl group of tyrosine at position 3 of VPg is covalently linked to the 5'-terminal UMP of genomic Poliovirus RNA via a phosphodiester bond and is crucial for virus viability. Poliovirus mutants in whom this tyrosine is substituted with a phenylalanine are quasi-infectious if threonine is present at position 4, which also has a hydroxyl group. The aim of the first part of this research project was to determine whether serine can also serve as an acceptor of UMP. An F3S4 mutant was constructed, which has a double mutation at this crucial position. This F3S4 mutant was found to be quasi-infectious and gave revertants to Y3T4 (WT) and Y3S4 after the first passage. Furthermore, mutants S3A4, S3T4 were also made and tested to determine whether serine at position 3 could also act as a substrate for uridylylation. The results of these experiments showed that both S3A4 and S3T4 are mutants with a severe replication defect, leading to a lethal growth phenotype, which suggests that serine at position 3 could not be uridylylated.The second project refers to the spacer II region of the 5'-nontranslated region of the Poliovirus whose function is not yet known. Interestingly, this spacer contains a region (Box C), which is 12 nucleotides long and conserved among all members of Enteroviruses. These 12 nucleotides were deleted in order to test the viability and growth phenotype of this mutant. Furthermore, the growth kinetics of the spacer II mutant and the poliovirus wild-type were compared in different cell lines at different temperatures. The results showed no differences in either the plaque phenotype or the growth kinetics between the spacer II mutant and poliovirus wild-type, an observation suggesting that the highly conserved region is dispensable under the conditions of the experiments.