The 5'-terminal region of the Aichi virus genome encodes cis-acting replication elements required for positive- and negative-strand RNA synthesis.
Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5' end) formed at the 5' end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5'-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5'-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5'-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5'-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5' end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3' end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5' end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis.
Nagashima
Shigeo
S
Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.
Sasaki
Jun
J
Taniguchi
Koki
K
D000818:Animals; D001483:Base Sequence; D002474:Cell-Free System; D002678:Chimera / Q000235:genetics; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000235:genetics; D004686:Encephalomyocarditis virus / Q000235:genetics; D016679:Genome, Viral; D019791:Guanidine / Q000494:pharmacology; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010849:Picornaviridae / Q000187:drug effects / Q000235:genetics* / Q000502:physiology*; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012093:Replicon / Q000235:genetics; D014709:Vero Cells; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics
D004279:DNA, Viral; D012367:RNA, Viral; D019791:Guanidine