Ribavirin-Resistant Mutants of Human Enterovirus 71 Express a High Replication Fidelity Phenotype during Growth in Cell Culture
Ribavirin-Resistant Mutants of Human Enterovirus 71 Express a High Replication Fidelity Phenotype during Growth in Cell Culture
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DOI：
10.1128/jvi.02139-12
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发表时间：
2013-02-01
影响因子：
5.4
通讯作者：
McMinn, Peter C.
中科院分区：
文献类型：
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作者：
Sadeghipour, Sara;Bek, Emily J.;McMinn, Peter C.
在动物模型中已经显示，在其3D聚合酶中具有G64 S突变的利巴韦林耐药脊髓灰质炎病毒具有高复制保真度，同时具有减弱的毒力。在这里，我们描述了在人肠道病毒71（HEV 71）的三维聚合酶的利巴韦林耐药性和复制保真度的诱变的影响。在HEV 71全长感染性cDNA克隆（26 M）的氨基酸位置3D-G64处引入七个取代。从G64 N、G64 R和G64 T突变体cDNA克隆中拯救活克隆衍生的病毒群体。克隆衍生的G64 R和G64 T突变病毒群体在1，600 μ M利巴韦林存在下对生长抑制具有抗性，而亲本26 M和G64 N突变病毒的生长在800 μ M利巴韦林存在下受到抑制。2C和3D编码区的核苷酸测序显示，在400 μ M利巴韦林存在下13次传代后，26 M基因组中的随机诱变率比突变型G64 R病毒基因组中的随机诱变率高近10倍。此外，在26 M和G64 N的2C编码区中获得的随机突变赋予了在0.5 mM胍存在下对生长抑制的抗性，而G64 R和G64 T突变病毒群体仍然对0.5 mM胍的生长抑制敏感。有趣的是，在利巴韦林选择后在26 M的3D编码区中鉴定的S264 L突变也与利巴韦林耐药和高复制保真度表型相关。这些发现与以下假设一致：3D-G64 R、3D-G64 T和3D-S264 L突变赋予HEV 71对抗病毒诱变剂利巴韦林的抗性，以及在细胞培养物中生长期间的高复制保真度表型。
It has been shown in animal models that ribavirin-resistant poliovirus with a G64S mutation in its 3D polymerase has high replication fidelity coupled with attenuated virulence. Here, we describe the effects of mutagenesis in the human enterovirus 71 (HEV71) 3D polymerase on ribavirin resistance and replication fidelity. Seven substitutions were introduced at amino acid position 3D-G64 of a HEV71 full-length infectious cDNA clone (26M). Viable clone-derived virus populations were rescued from the G64N, G64R, and G64T mutant cDNA clones. The clone-derived G64R and G64T mutant virus populations were resistant to growth inhibition in the presence of 1,600 mu M ribavirin, whereas the growth of parental 26M and the G64N mutant viruses were inhibited in the presence of 800 mu M ribavirin. Nucleotide sequencing of the 2C and 3D coding regions revealed that the rate of random mutagenesis after 13 passages in the presence of 400 mu M ribavirin was nearly 10 times higher in the 26M genome than in the mutant G64R virus genome. Furthermore, random mutations acquired in the 2C coding regions of 26M and G64N conferred resistance to growth inhibition in the presence of 0.5 mM guanidine, whereas the G64R and G64T mutant virus populations remained susceptible to growth inhibition by 0.5 mM guanidine. Interestingly, a S264L mutation identified in the 3D coding region of 26M after ribavirin selection was also associated with both ribavirin-resistant and high replication fidelity phenotypes. These findings are consistent with the hypothesis that the 3D-G64R, 3D-G64T, and 3D-S264L mutations confer resistance upon HEV71 to the antiviral mutagen ribavirin, coupled with a high replication fidelity phenotype during growth in cell culture.