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Journal of Virology, May 2001, p. 4633-4640, Vol. 75, No. 10
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.10.4633-4640.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Coupling between Replication and Packaging of Flavivirus RNA: Evidence Derived from the Use of DNA-Based Full-Length cDNA Clones of Kunjin Virus†

Alexander A. Khromykh,1,2,* Andrei N. Varnavski,1,2,Dagger Petra L. Sedlak,1,2 and Edwin G. Westaway1,2

Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital,1 and Clinical Medical Virology Centre, University of Queensland,2 Brisbane, Australia

Received 27 November 2000/Accepted 13 February 2001

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.

* Corresponding author. Mailing address: Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, Herston Rd., Herston, Brisbane, Queensland 4029, Australia. Phone: (617) 3636-1568. Fax: (617) 3636-1401. E-mail: a.khromykh{at}uq.edu.au.

dagger Publication no. 127 from the Sir Albert Sakzewski Virus Research Centre.

Dagger Present address: Institute for Human Gene Therapy, Philadelphia, Pa.

Journal of Virology, May 2001, p. 4633-4640, Vol. 75, No. 10
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.10.4633-4640.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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