Ishii T, Shiroki K, Hong D H, Aoki T, Ohta Y, Abe S, Hashizume S, Nomoto A
Department of Microbiology, Institute of Medical Science, The University of Tokyo, Japan.
J Virol. 1998 Mar;72(3):2398-405. doi: 10.1128/JVI.72.3.2398-2405.1998.
Four mutants of the virulent Mahoney strain of poliovirus were generated by introducing mutations in nucleotides (nt) 128 to 134 of the genome, a region that contains a part of the stem-loop II (SLII) structure located within the internal ribosomal entry site (IRES; nt 120 to 590) (K. Shiroki, T. Ishii, T. Aoki, Y. Ota, W.-X. Yang, T. Komatsu, Y. Ami, M. Arita, S. Abe, S. Hashizume, and A. Nomoto, J. Virol. 71:1-8, 1997). These mutants (SLII mutants) replicated well in human HeLa cells but not in mouse TgSVA cells that had been established from the kidney of a poliovirus-sensitive transgenic mouse. Their neurovirulence in mice was also greatly attenuated compared to that of the parental virus. The poor replication activity of the SLII mutants in TgSVA cells appeared to be attributable to reduced activity of the IRES. Two and three naturally occurring revertants that replicated well in TgSVA cells were isolated from mutants SLII-1 and SLII-5, respectively. The revertants recovered IRES activity in a cell-free translation system from TgSVA cells and returned to a neurovirulent phenotype like that of the Mahoney strain in mice. Two of the revertant sites that affected the phenotype were identified as being at nt 107 and within a region from nt 120 to 161. A mutation at nt 107, specifically a change from uridine to adenine, was observed in all the revertant genomes and exerted a significant effect on the revertant phenotype. Exhibition of the full revertant phenotype required mutations in both regions. These results suggested that nt 107 of poliovirus RNA is involved in structures required for the IRES activity in mouse cells.
通过在脊髓灰质炎病毒强毒株Mahoney株基因组的核苷酸（nt）128至134处引入突变，产生了四个突变体，该区域包含位于内部核糖体进入位点（IRES；nt 120至590）内的茎环II（SLII）结构的一部分（K. Shiroki、T. Ishii、T. Aoki、Y. Ota、W.-X. Yang、T. Komatsu、Y. Ami、M. Arita、S. Abe、S. Hashizume和A. Nomoto，《病毒学杂志》71:1 - 8，1997年）。这些突变体（SLII突变体）在人HeLa细胞中复制良好，但在从对脊髓灰质炎病毒敏感的转基因小鼠肾脏建立的小鼠TgSVA细胞中不能复制。与亲本病毒相比，它们在小鼠中的神经毒力也大大减弱。SLII突变体在TgSVA细胞中复制活性差似乎归因于IRES活性降低。分别从突变体SLII - 1和SLII - 5中分离出两个和三个在TgSVA细胞中复制良好的自然回复突变体。这些回复突变体在来自TgSVA细胞的无细胞翻译系统中恢复了IRES活性，并在小鼠中恢复到类似Mahoney株的神经毒力表型。确定了两个影响表型的回复突变位点，分别位于nt 107以及nt 120至161区域内。在所有回复突变体基因组中均观察到nt 107处的突变，具体为尿苷变为腺嘌呤，并且对回复突变体表型有显著影响。完整回复突变体表型的展现需要两个区域都发生突变。这些结果表明脊髓灰质炎病毒RNA的nt 107参与了小鼠细胞中IRES活性所需的结构。
