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.
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.
Liu
Wen Jun
WJ
Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Queensland 4029, Australia.
Chen
Hua Bo
HB
Wang
Xiang Ju
XJ
Huang
Hester
H
Khromykh
Alexander A
AA
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
D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D016899:Interferon-beta