Role of Nonstructural Protein NS2A in Flavivirus Assembly

Leung, Jason Y., Pijlman, Gorben P., Kondratieva, Natasha, Hyde, Jennifer, Mackenzie, Jason M. and Khromykh, Alexander A. (2008) Role of Nonstructural Protein NS2A in Flavivirus Assembly. Journal of Virology, 82 10: 4731-4741. doi:10.1128/JVI.00002-08

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Author Leung, Jason Y.
Pijlman, Gorben P.
Kondratieva, Natasha
Hyde, Jennifer
Mackenzie, Jason M.
Khromykh, Alexander A.
Title Role of Nonstructural Protein NS2A in Flavivirus Assembly
Journal name Journal of Virology   Check publisher's open access policy
ISSN 0022-538X
Publication date 2008-05-12
Year available 2008
Sub-type Article (original research)
DOI 10.1128/JVI.00002-08
Open Access Status File (Publisher version)
Volume 82
Issue 10
Start page 4731
End page 4741
Total pages 11
Place of publication Washington, DC, United States
Publisher American Society for Microbiology
Language eng
Subject C1
060502 Infectious Agents
060506 Virology
920109 Infectious Diseases
970111 Expanding Knowledge in the Medical and Health Sciences
970106 Expanding Knowledge in the Biological Sciences
Abstract Flavivirus nonstructural (NS) proteins are involved in RNA replication and modulation of the host antiviral response; however, evidence is mounting that some NS proteins also have essential roles in virus assembly. Kunjin virus (KUN) NS2A is a small, hydrophobic, transmembrane protein that is part of the replication complex and inhibits interferon induction. Previously, we have shown that an isoleucine (I)-to-asparagine (N) substitution at position 59 of the NS2A protein blocked the production of secreted virus particles in cells electroporated with viral RNA carrying this mutation. We now show that prolonged incubation of mutant KUN NS2A-I59N replicon RNA, in an inducible BHK-derived packaging cell line (expressing KUN structural proteins C, prM, and E), generated escape mutants that rescued the secretion of infectious virus-like particles. Sequencing identified three groups of revertants that included (i) reversions to wild-type, hydrophobic Ile, (ii) pseudorevertants to more hydrophobic residues (Ser, Thr, and Tyr) at codon 59, and (iii) pseudorevertants retaining Asn at NS2A codon 59 but containing a compensatory mutation (Thr-to-Pro) at NS2A codon 149. Engineering hydrophobic residues at NS2A position 59 or the compensatory T149P mutation into NS2A-I59N replicon RNA restored the assembly of secreted virus-like particles in packaging cells. T149P mutation also rescued virus production when introduced into the full-length KUN RNA containing an NS2A-I59N mutation. Immunofluorescence and electron microscopy analyses of NS2A-I59N replicon-expressing cells showed a distinct lack of virus-induced membranes normally present in cells expressing wild-type replicon RNA. The compensatory mutation NS2A-T149P restored the induction of membrane structures to a level similar to those observed during wild-type replication. The results further confirm the role of NS2A in virus assembly, demonstrate the importance of hydrophobic residues at codon 59 in this process, implicate the involvement of NS2A in the biogenesis of virus-induced membranes, and suggest a vital role for the virus-induced membranes in virus assembly.
Keyword Virology
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: 2009 Higher Education Research Data Collection
School of Chemistry and Molecular Biosciences
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Citation counts: TR Web of Science Citation Count  Cited 87 times in Thomson Reuters Web of Science Article | Citations
Scopus Citation Count Cited 92 times in Scopus Article | Citations
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Created: Wed, 01 Apr 2009, 20:36:13 EST by Glenda Chown on behalf of School of Chemistry & Molecular Biosciences