Histidine at position 1042 of the p150 region of a KRT live attenuated rubella vaccine strain is responsible for the temperature sensitivity

Abstract

The Japanese live attenuated KRT rubella vaccine strain has a temperature sensitivity ( ts) phenotype. The objective of this study is to identify the region responsible for this phenotype. Genomic sequences of the KRT strain and the wild-type strain (RVi/Matsue.JPN/68) with the non- ts phenotype were investigated and reverse genetic systems (RG) for these strains were developed. The ts phenotype of KRT varied drastically on replacement of the p150 gene (encoding a methyltransferase and a nonstructural protease). Analysis of four chimeric viruses showed the region responsible for the ts phenotype to be located between Bsm I and Nhe I sites (genome position 2803–3243). There were two amino acid differences at positions 1007 and 1042. Mutations were introduced into the KRT cDNA clone, designated G1007D, H1042Y and G1007D-H1042Y. H1042Y and G1007D-H1042Y grew well at a restrictive temperature with a 100-fold higher titer than G1007D and the KRT strain, but a 10-fold lower titer than RVi/Matsue.JPN/68. Since the growth of H1042Y was not completely the same as that of the wild-type strain at the restrictive temperature, we also assessed whether other genomic regions have an additive effect with H1042Y on the ts phenotype. H1042Y-RViM SP having structural proteins of RVi/Matsue.JPN/68 grew better than H1042Y, similar to RVi/Matsue.JPN/68. Thus, we concluded that one mutation, of the histidine at position 1042 of p150, was essential for the ts phenotype of the KRT strain, and structural proteins of KRT had an additive effect with H1042Y on the ts phenotype.