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Essential role of cyclization sequences in flavivirus RNA replication
A A Khromykh1, H Meka, K J Guyatt
1Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital, University of Queensland, Herston Rd., Herston, Brisbane, QLD 4029, Australia. a.khromykh@mailbox.uq.edu.au
Journal of Virology
|June 20, 2001
Summary
Flavivirus RNA replication requires base-pairing interactions between conserved 5' and 3' terminal sequences. Disrupting these interactions prevents RNA replication, while restoring complementarity allows it, confirming their essential role.
Area of Science:
- Virology
- Molecular Biology
- RNA Biology
Background:
- Flavivirus RNA replication involves interactions between conserved complementary sequences at the 5' and 3' ends.
- Previous studies suggested a role for these cyclization sequences but lacked in vivo experimental validation.
Purpose of the Study:
- To investigate the in vivo role of base-pairing interactions between conserved 5' and 3' terminal sequences in Flavivirus RNA replication.
- To experimentally validate predicted RNA secondary structures and base-pairing interactions.
Main Methods:
- Utilized M-fold program for RNA secondary structure prediction of viral genomic regions.
- Employed site-directed mutagenesis in Kunjin virus replicon RNA to disrupt and restore complementary cyclization sequences.
- Assessed RNA replication competence through immunofluorescence for NS3 protein and Northern blot analysis.
Main Results:
- M-fold predicted base-pairing between conserved 5' and 3' terminal regions in mosquito-borne Flaviviruses.
- Mutations disrupting base-pairing in Kunjin virus replicon RNA abolished replication.
- Compensatory mutations restoring complementarity rescued RNA replication.
- Identified novel conserved complementary sequences in tick-borne Flaviviruses with significant base-pairing stability.
Conclusions:
- Base-pairing between 5' and 3' terminal sequences, not the specific nucleotide sequence, is critical for Flavivirus RNA replication.
- Multiple pairs of cyclization sequences may be involved in the replication of tick-borne Flaviviruses.