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Journal of Virology, July 2001, p. 6719-6728, Vol. 75, No. 14
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.14.6719-6728.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Essential Role of Cyclization Sequences in Flavivirus RNA Replication†

Alexander A. Khromykh,* Hedije Meka, Kimberley J. Guyatt,Dagger and Edwin G. Westaway

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

Received 16 February 2001/Accepted 25 April 2001

A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5'- and 3'-terminal regions of Flavivirus RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5' genomic region (first ~160 nucleotides) and the 3' untranslated region (last ~115 nucleotides) for a range of mosquito-borne Flavivirus species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5' or 3') in the replicon RNA of the mosquito-borne Kunjin virus were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5' or only the 3' cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5'- and 3'-terminal regions of three tick-borne virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito-borne species. They base-paired with Delta G values of approximately -20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5' and 3' complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne Flavivirus species.

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

dagger Publication no. 132 from the Sir Albert Sakzewski Virus Research Centre.

Dagger Present address: Queensland Agricultural Biotechnology Centre, University of Queensland, Brisbane, Australia.

Journal of Virology, July 2001, p. 6719-6728, Vol. 75, No. 14
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.14.6719-6728.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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