Characterization of immediate early gene ORF62 of live attenuated varicella vaccine Oka strains pro-duced in China

Abstract

Objective To investigate mutations in immediate early （ IE） gene ORF62 of three varicella vaccine Oka strains （ vOka ） including two strains produced in China and their parental Oka strain （pOka）, and then to further elucidate its possible roles in attenuation mechanism by comparing their ORF 62 promoter sequences and its activities , ORF62 coding regions and its transactivities .Methods ORF62 promoter-reporter plasmids and ORF62-expressing plasmids of pOka and three vOka strains （ vOka-BK from Changchun BCHT Biotechnology Co ., vOka-SH from Shanghai Institute of Biological Product Co .Ltd., and vOka-GSK from GlaxoSmithKline plc, as control） were constructed, respectively.ORF62 promoter regions and coding regions of the four strains were sequenced and then compared with each other .Differences of activities of the ORF62 promoter, and transactivities of the ORF62-encoded IE62 upon immediate early （ORF4）, early （ORF28） and late （ORF67） gene promoters between pOka and vOka strains were assayed with transient transfection technique .Results Compared with pOka strain , three vOka strains had a consistent T deletion mutation at site 110 050 in ORF62 promoters, which did not result in any change of tran-scription factor binding motif .However , activities of ORF62 promoters from three vOka strains were significantly lower than those of pOka strain .Three consistent substitution mutations were observed in ORF 62 coding regions of three vOka strains and three new enzyme restriction sites including SmaⅠ, NaeⅠand BssHⅡwere generated, respectively.Transactivities of IE62 from three vOka strains upon ORF4, ORF28 and ORF67 promoters were significantly higher than those of pOka both in CV-1 and MeWo cells , except that vOka-SH IE62 showed significantly lower transactivities upon ORF 4 promoter than those of pOka strain in CV-1 cells.Conclusion Consistent T deletion mutation at site 110 050 in ORF62 promoters of three vOka strains might be responsible for the reduced promoter activities and the changes of IE 62 transactivities .However , it seemed that cell types have no significant effect on ORF 62 promoter activity or IE 62 transactivity between pOka and vOka strains . Key words: Varicella-zoster virus; Promoter activity; Transactivity