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

被引:63
作者
Sasaki, J [1 ]
Kusuhara, Y
Maeno, Y
Kobayashi, N
Yamashita, T
Sakae, K
Takeda, N
Taniguchi, K
机构
[1] Fujita Hlth Univ, Sch Med, Dept Virol & Prasitol, Toyoake, Aichi 4701192, Japan
[2] Sapporo Med Univ, Sch Med, Dept Hyg, Sapporo, Hokkaido 0600061, Japan
[3] Aichi Prefectural Inst Publ Hlth, Dept Virol, Nagoya, Aichi 4628576, Japan
[4] Natl Inst Infect Dis, Dept Virol 2, Tokyo 1628640, Japan
关键词
D O I
10.1128/JVI.75.17.8021-8030.2001
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 eDNA 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.
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页码:8021 / 8030
页数:10
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