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
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
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DOI：
10.1128/jvi.75.17.8021-8030.2001
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发表时间：
2001-09-01
影响因子：
5.4
通讯作者：
Taniguchi, K
中科院分区：
文献类型：
--
作者：
Sasaki, J;Kusuhara, Y;Taniguchi, K
爱知病毒是小核糖核酸病毒科科的一个新属--科布病毒属的模式种。在这项研究中，我们构建了一个全长的爱知病毒的cDNA克隆，其体外转录本感染Vero细胞。在构建感染性eDNA克隆的过程中，在基因组的5'端鉴定了一个新的32个核苷酸的序列。计算机辅助预测基因组5'端的二级结构，包括新序列，表明形成了由42个核苷酸组成的稳定茎环结构。使用来自感染性cDNA克隆的各种定点突变体研究了该茎环在病毒复制中的功能。我们的数据表明，在正链的5'端而不是在负链的3'端的茎环的正确折叠对于病毒RNA复制是至关重要的。茎下部的一级序列也被认为是RNA复制的关键。相比之下，环片段中的核苷酸变化并没有如此严重地降低病毒复制的效率。一个双突变体，其中茎中间部分的两个核苷酸片段被它们的互补核苷酸取代，具有有效的RNA复制和翻译能力，但不能产生病毒。这些结果表明，在爱知病毒基因组的5'端的茎环是参与病毒RNA复制和感染性病毒颗粒的产生的元件。
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.