ABSTRACT
Objective To investigate mutations in immediate early ( IE) gene ORF62 of three var-icella 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 pro-moter-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 ac-tivities 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 con-sistent 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 signifi-cantly lower than those of pOka strain .Three consistent substitution mutations were observed in ORF 62 cod-ing 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 .How-ever , it seemed that cell types have no significant effect on ORF 62 promoter activity or IE 62 transactivity be-tween pOka and vOka strains .
ABSTRACT
Objective To further investigate inhibitory mechanism(s) of resveratrol on varicellazoster virus (VZV) in vitro with our previously generated reporter cell line MV9G.Methods Cell-free VZVs were directly inoculated onto MV9G cells (CFVs direct-infection) or cell-associated VZVs wereco-cultured with MV9G cells (CAVs co-culture) to activate expression of reporter gene firefly luciferase in MV9G cells.Resveratrol was added before or after virus infection,roles of resveratrolon direct inactivation,on viral attachment to and penetration into MV9G cells,on intracellular viral replication and its IC50,inhibitorytime points and reversibility were assayed by comparing the luciferase activities reduction by resveratrol.Thereductions of VZV IE62 mRNA copies and IE62-antibody positive cells by resveratrol were further assayed.Results ATPs contents of MV9G cells in the presence of resveratrol over 30.0 μg/ml were concentrationdependently reduced,the CD50 of which was around 60.3 μg/ml.CFVs were premixed with 25.0 μg/ml resveratrol andincubated at 37℃ waterbath for two hours and then directly inoculated onto MV9G cells,luciferases activated by resveratrol-treated CFVs were reduced to around half of the untreated controls.MV9G cells were pre-incubated with resveratrol at 37℃ for 2 h and then directly infected with CFVs at 37℃ for another 2 h,the CFVs-activated luciferase was concentration-dependently reduced,but no big change was observed in those pre-incubated at 4℃.MV9G cells were co-cultured with CAVs in the presence of resvertrolfor 72 h,the CAVs-activated luciferases were markedly reduced in a concentration-dependent manner,the IC50 of which was around 8.7 μgml.Resveratrol was added in CAVs co-culture at 1,3,6,9,12,24,30,and 36 h post infection,the CAVs-activated luciferase in those resveratrol was added at 3,6,9,12,and 24 h post infection were significantly higher than those of controls.Resveratrol was withdrawn from CAVs coculture media,the CAVs-activated luciferases after withdrawal were significantly higher than those before,especially in those withdrswn at 24 and 72 h post infection.The IE62 mRNA levels shown by cDNA copiesdetected with SYBR Green RT-PCR and IE62 positive cells shown by monoclonal anti-IE62 antibody of thevirus-infected cells treated with resveratrol were significantly reduced with increase of incubation time withresveratrol.Conclusion Resveratrol was cytotoxic to MV9G cells,and the maximum resistant concentrationon MV9G cells was around 30.0 μg/ml,the CD50 of which was around 60.3 μg/ml.Non-cytotoxic resveratrol partly inactivated CFVs,inhibited viral penetration into rather than attachment to MV9G cells.Resveratrol inhibited CAVs' intmcellular replication strongly but reversibly in a concentration-dependent manner,the IC50 of which was around 8.7 μ/ml.The inhibition of resveratrol on VZV in vitro might be through suppression of IE62 gene transcription and expression in the early stage of infection.