Generation of a Cell Culture-Adapted Hepatitis C Virus with Longer Half Life at Physiological Temperature 英文参考文献.docVIP

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Generation of a Cell Culture-Adapted Hepatitis C Virus with Longer Half Life at Physiological Temperature 英文参考文献.doc

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Generation of a Cell Culture-Adapted Hepatitis C Virus with Longer Half Life at Physiological Temperature 英文参考文献

GenerationofaCellCulture-AdaptedHepatitisCVirus withLongerHalfLifeatPhysiologicalTemperature ChonSaengKim1.,SunJuKeum1.,SungKeyJang1,2,3 * 1MolecularVirologyLaboratory,POSTECHBiotechCenter,DepartmentofLifeScience,PohangUniversityofScienceandTechnology,Pohang,Kyungbuk,Republicof Korea, 2DivisionofIntegrativeBiosciencesandBiotechnology, PohangUniversityofScienceandTechnology,Pohang,Kyungbuk,RepublicofKorea, 3Biotechnology ResearchCenter,PohangUniversityofScienceandTechnology,Pohang,Kyungbuk,RepublicofKorea Abstract Background:WepreviouslyreportedinfectiousHCVclonesthatcontaintheconvenientreporters,greenfluorescentprotein (GFP) and Renilla luciferase (Rluc), in the NS5a-coding sequence. Although these viruses were useful in monitoring viral proliferationandscreeningofanti-HCVdrugs,theinfectivityandyieldoftheviruseswerelow. Methodology/PrincipalFindings:InordertoobtainahighlyefficientHCVcultivationsystem,wetransfectedHuh7.5.1cells [1]withJFH5a-GFPRNAandthencultivatedcellsfor20days.WefoundahighlyinfectiousHCVclonecontainingtwocell culture-adapted mutations.Twocellculture-adapted mutations whichwereresponsiblefortheincreased viralinfectivity were located in E2 and p7 protein coding regions. The viral titer of the variant was ,100-fold higher than that of the parentalvirus.ThemutationintheE2proteinincreasedtheviabilityofvirusat37uCbyacquiringprolongedinteraction capability with a HCV receptor CD81. The wild-type and p7-mutated virus had a half-life of ,2.5 to 3hours at 37uC. In contrast, the half-life of viruses, which contained E2 mutation singly and combination with the p7 mutation, was 5 to 6hoursat37uC.Themutationinthep7protein,eithersinglyorincombinationwiththeE2mutation,enhancedinfectious virusproductionabout10–50-foldbyfacilitatinganearlystepofvirionproduction. Conclusion/Significance:ThemutationintheE2proteingeneratedbytheculturesystemincreasesvirionviabilityat37uC. The adaptive mutation in the p7 protein facilitates an earlier stage of virus productio