A complex RNA motif defined by three discontinuous 5-nucleotide-long strands is essential for Flavivirus RNA replication.
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.
Song
Byung-Hak
BH
Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, South Korea.
Yun
Sang-Im
SI
Choi
Yu-Jeong
YJ
Kim
Jeong-Min
JM
Lee
Chan-Hee
CH
Lee
Young-Min
YM
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D004664:Encephalitis Virus, Japanese / Q000235:genetics / Q000502:physiology*; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014779:Virus Replication / Q000235:genetics
D012367:RNA, Viral