Coupling between replication and packaging of flavivirus RNA: Evidence derived from the use of DNA-based full-length cDNA clones of kunjin virus

Khromykh, A. A., Varnavski, A. N., Sedlak, P. L. and Westaway, E. G. (2001) Coupling between replication and packaging of flavivirus RNA: Evidence derived from the use of DNA-based full-length cDNA clones of kunjin virus. Journal of Virology, 75 10: 4633-4640. doi:10.1128/JVI.75.10.4633-4640.2001


Author Khromykh, A. A.
Varnavski, A. N.
Sedlak, P. L.
Westaway, E. G.
Title Coupling between replication and packaging of flavivirus RNA: Evidence derived from the use of DNA-based full-length cDNA clones of kunjin virus
Journal name Journal of Virology   Check publisher's open access policy
ISSN 0022-538X
Publication date 2001-05
Sub-type Article (original research)
DOI 10.1128/JVI.75.10.4633-4640.2001
Volume 75
Issue 10
Start page 4633
End page 4640
Total pages 8
Editor T. E. Shenk
Place of publication Washington USA
Publisher American Society for Microbiology
Collection year 2001
Language eng
Subject C1
730101 Infectious diseases
270303 Virology
Abstract In order to study whether flavivirus RNA packaging is dependent on RNA replication, we generated two DNA-based Kunjin virus constructs, pKUN1 and pKUN1dGDD, allowing continuous production of replicating (wild-type) and nonreplicating (with a deletion of the NS5 gene RNA-polymerase motif GDD) full-length Kunjin virus RNAs, respectively, via nuclear transcription by cellular RNA polymerase II. As expected, transfection of pKUN1 plasmid DNA into BHK cells resulted in the recovery of secreted infectious Kunjin virions. Transfection of pKUN1dGDD DNA into BHK cells, however, did not result in the recovery of any secreted virus particles containing encapsidated dGDD RNA, despite an apparent accumulation of this RNA in cells demonstrated by Northern blot analysis and its efficient translation demonstrated by detection of correctly processed labeled structural proteins (at least prM and E) both in cells and in the culture fluid using coimmunoprecipitation analysis with anti-E antibodies. In contrast, when dGDD RNA was produced even in much smaller amounts in PKUN1dGDD DNA-transfected repBHK cells (where it was replicated via complementation), it was packaged into secreted virus particles, Thus, packaging of defective Kunjin virus RNA could occur only when it was replicated. Our results with genome-length Kunjin virus RNA and the results with poliovirus replicon RNA (C, I. Nugent et al,, J, Virol, 73:427-435, 1999), both demonstrating the necessity for the RNA to be replicated before it can be packaged, strongly suggest the existence of a common mechanism for minimizing amplification and transmission of defective RNAs among the quasispecies in positive-strand RNA viruses, This mechanism may thus help alleviate the high-copy error rate of RNA-dependent RNA polymerases.
Keyword Virology
Double-stranded-rna
Replicon Rna
Heterologous Genes
Infected Cells
Transcomplementation
Proteins
Ns1
Trans
Components
Expression
Q-Index Code C1

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Chemistry and Molecular Biosciences
 
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