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

Liu, Wen Jun, Chen, Hua Bo, Xiang, Ju Wang, Huang, Hester and Khromykh, Alexander A. (2004) 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. Journal of Virology, 78 22: 12225-12235.


Author Liu, Wen Jun
Chen, Hua Bo
Xiang, Ju Wang
Huang, Hester
Khromykh, Alexander A.
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
Journal name Journal of Virology  (ERA 2012 Listed)    (ERA 2010 Rank A*)   Check publisher's open access policy
Publication date 2004-11
Sub-type Article
DOI 10.1128/JVI.78.22.12225-12235.2004
Volume number 78
Issue number 22
ISSN 0022-538X
Start page 12225
End page 12235
Total pages 11
Place of publication Washington
Publisher American Society of Microbiology
Language eng
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 similar to15- 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 similar to6- 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 similar to7-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.
Keyword Virology
West-nile-virus
Sindbis Virus
Cell-culture
Subgenomic Replicons
Regulatory Factor-3
Heterologous Genes
Ns1-ns2a Cleavage
Mammalian-cells
Dengue Virus
Replication
Q-Index Code C1

Document type: Journal Article
Sub-type: Article
Collections: Excellence in Research Australia (ERA) - Collection
School of Chemistry and Molecular Biosciences
 
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