title: Analysis of adaptive mutations in Kunjin virus replicon RNA reveals a novel role for the flavivirus nonstructural protein NS2A in inhibition of beta interferon promoter-driven transcription.
abstract: The establishment of persistent noncytopathic replication by replicon RNAs of a number of positive-strand RNA viruses usually leads to generation of adaptive mutations in nonstructural genes. Some of these adaptive mutations (e.g., in hepatitis C virus) increase the ability of RNA replication to resist the antiviral action of alpha/beta interferon (IFN-alpha/beta); others (e.g., in Sindbis virus) may also lead to more efficient IFN production. Using puromycin-selectable Kunjin virus (KUN) replicon RNA, we identified two adaptive mutations in the NS2A gene (producing Ala30-to-Pro and Asn101-to-Asp mutations in the gene product; for simplicity, these will be referred to hereafter as Ala30-to-Pro and Asn101-to-Asp mutations) that, when introduced individually or together into the original wild-type (wt) replicon RNA, resulted in approximately 15- to 50-fold more efficient establishment of persistent replication in hamster (BHK21) and human (HEK293 and HEp-2) cell lines. Transfection with a reporter plasmid carrying the luciferase gene under the control of the IFN-beta promoter resulted in approximately 6- to 7-fold-higher luciferase expression in HEp-2 cells stably expressing KUN replicon RNA with an Ala30-to-Pro mutation in the NS2A gene compared to that observed in HEp-2 cells stably expressing KUN replicon RNA with the wt NS2A gene. Moreover, cotransfection of plasmids expressing individual wt or Ala30-to-Pro-mutated NS2A genes with the IFN-beta promoter reporter plasmid, followed by infection with Semliki Forest virus to activate IFN-beta promoter-driven transcription, showed approximately 7-fold inhibition of luciferase expression by the wt but not by the Ala30-to-Pro-mutated NS2A protein. The results show for the first time a role for the flavivirus nonstructural protein NS2A in inhibition of IFN-beta promoter-driven transcription and identify a single-amino-acid mutation in NS2A that dramatically reduces this inhibitory activity. The findings determine a new function for NS2A in virus-host interactions, extend the range of KUN replicon vectors for noncytopathic gene expression, and identify NS2A as a new target for attenuation in the development of live flavivirus vaccines.
authors.affiliation: Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Queensland 4029, Australia.
authors.forename: Wen Jun
authors.initials: WJ
authors.lastname: Liu
authors.forename: Hua Bo
authors.initials: HB
authors.lastname: Chen
authors.forename: Xiang Ju
authors.initials: XJ
authors.lastname: Wang
authors.forename: Hester
authors.initials: H
authors.lastname: Huang
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Khromykh
mesh_terms: D000818:Animals; D002460:Cell Line; D006801:Humans; D016899:Interferon-beta / Q000235:genetics*; D009154:Mutation; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000096:biosynthesis*; D012093:Replicon; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000502:physiology*; D019065:Virus Assembly; D014779:Virus Replication; D014902:West Nile virus / Q000235:genetics* / Q000502:physiology
chemical_list: D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D016899:Interferon-beta