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Journal of Virology, November 2004, p. 12225-12235, Vol. 78, No. 22
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.22.12225-12235.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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{dagger}

Wen Jun Liu,1,2 Hua Bo Chen,1,2 Xiang Ju Wang,1,2 Hester Huang,1,2 and Alexander A. Khromykh1,2*

Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital,1 Clinical Medical Virology Centre, University of Queensland, Brisbane, Queensland, Australia2

Received 1 April 2004/ Accepted 20 July 2004

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}/ß); 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 ~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-ß promoter resulted in ~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-ß promoter reporter plasmid, followed by infection with Semliki Forest virus to activate IFN-ß promoter-driven transcription, showed ~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-ß 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.

* Corresponding author. Mailing address: Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Brisbane, Queensland 4029, Australia. Phone: 617 36361568. Fax: 617 36361401. E-mail: a.khromykh{at}uq.edu.au.

{dagger} This is publication number 206 from the Clinical Medical Virology Centre and the Sir Albert Sakzewski Virus Research Centre.

Journal of Virology, November 2004, p. 12225-12235, Vol. 78, No. 22
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.22.12225-12235.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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