Infectious Diseases and Immunology, Sydney Medical School, University of Sydney, New South Wales, Australia.
J Virol. 2013 Feb;87(3):1759-69. doi: 10.1128/JVI.02139-12. Epub 2012 Nov 21.
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 μM ribavirin, whereas the growth of parental 26M and the G64N mutant viruses were inhibited in the presence of 800 μM 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 μM 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.
在动物模型中已经表明，具有 3D 聚合酶 G64S 突变的耐利巴韦林脊髓灰质炎病毒具有高复制保真度和减弱的毒力。在这里，我们描述了人肠道病毒 71（HEV71）3D 聚合酶中的突变对利巴韦林耐药性和复制保真度的影响。在 HEV71 全长感染性 cDNA 克隆（26M）的 3D-G64 氨基酸位置引入了七个取代。从 G64N、G64R 和 G64T 突变 cDNA 克隆中拯救出可存活的克隆衍生病毒群体。衍生的 G64R 和 G64T 突变病毒群体对 1600 μM 利巴韦林存在时的生长抑制具有抗性，而亲本 26M 和 G64N 突变病毒的生长在 800 μM 利巴韦林存在时受到抑制。2C 和 3D 编码区的核苷酸测序显示，在存在 400 μM 利巴韦林的情况下，经过 13 次传代后，26M 基因组中的随机突变率几乎是突变 G64R 病毒基因组的 10 倍。此外，在 26M 和 G64N 的 2C 编码区获得的随机突变赋予了对 0.5 mM 胍抑制生长的抗性，而 G64R 和 G64T 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 26M 的 3D 编码区中鉴定的 S264L 突变也与利巴韦林耐药性和高复制保真度表型相关。这些发现与以下假设一致，即 3D-G64R、3D-G64T 和 3D-S264L 突变赋予 HEV71 对抗病毒诱变剂利巴韦林的抗性，并且在细胞培养物中生长时具有高复制保真度表型。
