A complex RNA motif defined by three discontinuous 5-nucleotide-long strands is essential for Flavivirus RNA replication

  1. Byung-Hak Song1,
  2. Sang-Im Yun1,
  3. Yu-Jeong Choi1,
  4. Jeong-Min Kim1,
  5. Chan-Hee Lee2, and
  6. Young-Min Lee1
  1. 1Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, South Korea
  2. 2Division of Life Sciences, College of Natural Sciences and Research Institute for Genetic Engineering, Chungbuk National University, Cheongju, South Korea

Abstract

Tertiary or higher-order RNA motifs that regulate replication of positive-strand RNA viruses are as yet poorly understood. Using Japanese encephalitis virus (JEV), we now show that a key element in JEV RNA replication is a complex RNA motif that includes a string of three discontinuous complementary sequences (TDCS). The TDCS consists of three 5-nt-long strands, the left (L) strand upstream of the translation initiator AUG adjacent to the 5′-end of the genome, and the middle (M) and right (R) strands corresponding to the base of the Flavivirus-conserved 3′ stem–loop structure near the 3′-end of the RNA. The three strands are arranged in an antiparallel configuration, with two sets of base-pairing interactions creating L-M and M-R duplexes. Disrupting either or both of these duplex regions of TDCS completely abolished RNA replication, whereas reconstructing both duplex regions, albeit with mutated sequences, fully restored RNA replication. Modeling of replication-competent genomes recovered from a large pool of pseudorevertants originating from six replication-incompetent TDCS mutants suggests that both duplex base-pairing potentials of TDCS are required for RNA replication. In all cases, acquisition of novel sequences within the 3′M-R duplex facilitated a long-range RNA–RNA interaction of its 3′M strand with either the authentic 5′L strand or its alternative (invariably located upstream of the 5′ initiator), thereby restoring replicability. We also found that a TDCS homolog is conserved in other flaviviruses. These data suggest that two duplex base-pairings defined by the TDCS play an essential regulatory role in a key step(s) of Flavivirus RNA replication.

Keywords

Footnotes

  • Reprint requests to: Young-Min Lee, Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University, 12 Gaeshin-Dong, Heungduk-Ku, Cheongju-Si, South Korea; e-mail: ymlee{at}chungbuk.ac.kr; fax: 82-43-272-1603.

  • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.993608.

    • Received January 7, 2008.
    • Accepted May 23, 2008.

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  1. Published in Advance July 30, 2008, doi: 10.1261/rna.993608 RNA 14: 1791-1813 Copyright © 2008 RNA Society

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