Translation of the flavivirus Kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging

Pijlman, Gorben P., Kondratieva, Natasha and Khromykh, Alexander A. (2006) Translation of the flavivirus Kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging. Journal of Virology, 80 22: 11255-11264. doi:10.1128/JVI.01559-06


Author Pijlman, Gorben P.
Kondratieva, Natasha
Khromykh, Alexander A.
Title Translation of the flavivirus Kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging
Journal name Journal of Virology   Check publisher's open access policy
ISSN 0022-538X
1098-5514
Publication date 2006-11
Year available 2006
Sub-type Article (original research)
DOI 10.1128/JVI.01559-06
Open Access Status DOI
Volume 80
Issue 22
Start page 11255
End page 11264
Total pages 10
Place of publication Washington, DC, United States
Publisher American Society for Microbiology
Collection year 2006
Language eng
Subject C1
270303 Virology
730101 Infectious diseases
Abstract Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.
Keyword West-nile-virus
Yellow-fever Virus
Hepatitis-c Virus
Acting Replication Element
Viral Diarrhea Virus
Full-length Cdna
Coding Region
Nonstructural Proteins
Stranded-rna
Replicon Rna
Q-Index Code C1
Institutional Status UQ

 
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Created: Wed, 15 Aug 2007, 09:26:06 EST