Construction of an infectious cDNA clone of aichi virus (a new member of the family Picornaviridae) and mutational analysis of a stem-loop structure at the 5 ' end of the genome

Aichi virus is the type species of a new genus, Kobuvirus, of the family Pi cornaviridae. In this study, we constructed a full-length cDNA clone of Aic hi virus whose in vitro transcripts were infectious to Vero cells. During c onstruction of the infectious eDNA clone, a novel sequence of 32 nucleotide s was identified at the 5' end of the genome. Computer-assisted prediction of the secondary structure of the 5' end of the genome, including the novel sequence, suggested the formation of a stable stem-loop structure consisti ng of 42 nucleotides. The function of this stem-loop in virus replication w as investigated using various site-directed mutants derived from the infect ious cDNA clone. Our data indicated that correct folding of the stem-loop a t the 5' end of the positive strand, but not at the 3' end of the negative strand, is critical for viral RNA replication. The primary sequence in the lower part of the stem was also suggested to be crucial for RNA replication . In contrast, nucleotide changes in the loop segment did not so severely r educe the efficiency of virus replication. A double mutant, in which both n ucleotide stretches of the middle part of the stem were replaced by their c omplementary nucleotides, had efficient RNA replication and translation abi lities but was unable to produce viruses. These results indicate that the s tem-loop at the 5' end of the Aichi virus genome is an element involved in both viral RNA replication and production of infectious virus particles.