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Journal of Virology, September 2001, p. 8021-8030, Vol. 75, No. 17
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.17.8021-8030.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Jun Sasaki,1,* Yasuhiro Kusuhara,1 Yoshimasa Maeno,1 Nobumichi Kobayashi,2 Teruo Yamashita,3 Kenji Sakae,3 Naokazu Takeda,4 and Koki Taniguchi1

Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192,1 Department of Hygiene, School of Medicine, Sapporo Medical University, Sapporo 060-0061,2 Department of Virology, Aichi Prefectural Institute of Public Health, Nagoya, Aichi 462-8576,3 and Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640,4 Japan

Received 9 March 2001/Accepted 5 June 2001

Aichi virus is the type species of a new genus, Kobuvirus, of the family Picornaviridae. In this study, we constructed a full-length cDNA clone of Aichi virus whose in vitro transcripts were infectious to Vero cells. During construction of the infectious cDNA clone, a novel sequence of 32 nucleotides 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 consisting of 42 nucleotides. The function of this stem-loop in virus replication was investigated using various site-directed mutants derived from the infectious cDNA clone. Our data indicated that correct folding of the stem-loop at 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 reduce the efficiency of virus replication. A double mutant, in which both nucleotide stretches of the middle part of the stem were replaced by their complementary nucleotides, had efficient RNA replication and translation abilities but was unable to produce viruses. These results indicate that the stem-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.

* Corresponding author. Mailing address: Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan. Phone: 81-562-93-2486. Fax: 81-562-93-4008. E-mail: jsasaki{at}fujita-hu.ac.jp.

Journal of Virology, September 2001, p. 8021-8030, Vol. 75, No. 17
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.17.8021-8030.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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