Department of Microbiology & Molecular Genetics and Center for Virus Research, University of California, Irvine, CA 92697, USA.
Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, UK.
Viruses. 2020 Jan 31;12(2):166. doi: 10.3390/v12020166.
In this study, we characterized the role of host cell protein tyrosyl-DNA phosphodiesterase 2 (TDP2) activity, also known as VPg unlinkase, in picornavirus infections in a human cell model of infection. TDP2/VPg unlinkase is used by picornaviruses to remove the small polypeptide, VPg (Virus Protein genome-linked, the primer for viral RNA synthesis), from virus genomic RNA. We utilized a CRISPR/Cas-9-generated TDP2 knock out (KO) human retinal pigment epithelial-1 (hRPE-1) cell line, in addition to the wild type (WT) counterpart for our studies. We determined that in the absence of TDP2, virus growth kinetics for two enteroviruses (poliovirus and coxsackievirus B3) were delayed by about 2 h. Virus titers were reduced by ~2 log10 units for poliovirus and 0.5 log10 units for coxsackievirus at 4 hours post-infection (hpi), and by ~1 log10 unit at 6 hpi for poliovirus. However, virus titers were nearly indistinguishable from those of control cells by the end of the infectious cycle. We determined that this was not the result of an alternative source of VPg unlinkase activity being activated in the absence of TPD2 at late times of infection. Viral protein production in TDP2 KO cells was also substantially reduced at 4 hpi for poliovirus infection, consistent with the observed growth kinetics delay, but reached normal levels by 6 hpi. Interestingly, this result differs somewhat from what has been reported previously for the TDP2 KO mouse cell model, suggesting that either cell type or species-specific differences might be playing a role in the observed phenotype. We also determined that catalytically inactive TDP2 does not rescue the growth defect, confirming that TDP2 5' phosphodiesterase activity is required for efficient virus replication. Importantly, we show for the first time that polysomes can assemble efficiently on VPg-linked RNA after the initial round of translation in a cell culture model, but both positive and negative strand RNA production is impaired in the absence of TDP2 at mid-times of infection, indicating that the presence of VPg on the viral RNA affects a step in the replication cycle downstream of translation (e.g., RNA synthesis). In agreement with this conclusion, we found that double-stranded RNA production (a marker of viral RNA synthesis) is delayed in TDP2 KO RPE-1 cells. Moreover, we show that premature encapsidation of nascent, VPg-linked RNA is not responsible for the observed virus growth defect. Our studies provide the first lines of evidence to suggest that either negative- or positive-strand RNA synthesis (or both) is a likely candidate for the step that requires the removal of VPg from the RNA for an enterovirus infection to proceed efficiently.
在这项研究中，我们在感染人细胞模型中描述了宿主细胞蛋白酪氨酸-DNA 磷酸二酯酶 2（TDP2）活性（也称为 VPg 解连酶）在小核糖核酸病毒感染中的作用。TDP2/VPg 解连酶被小核糖核酸病毒用于从病毒基因组 RNA 上除去小多肽 VPg（病毒蛋白基因组连接，病毒 RNA 合成的引物）。我们利用 CRISPR/Cas-9 生成的 TDP2 敲除（KO）人视网膜色素上皮-1（hRPE-1）细胞系，以及野生型（WT）对照进行了研究。我们发现，在没有 TDP2 的情况下，两种肠病毒（脊髓灰质炎病毒和柯萨奇病毒 B3）的病毒生长动力学延迟了约 2 小时。在感染后 4 小时（hpi）时，脊髓灰质炎病毒和柯萨奇病毒的病毒滴度分别降低了约 2 个对数单位和 0.5 个对数单位，在 6 hpi 时，脊髓灰质炎病毒的病毒滴度降低了约 1 个对数单位。然而，在感染周期结束时，病毒滴度与对照细胞几乎没有区别。我们发现，这并不是由于在感染后期缺乏 TPD2 时激活了替代的 VPg 解连酶活性所致。在脊髓灰质炎病毒感染的 4 hpi 时，TDP2 KO 细胞中的病毒蛋白产量也大幅降低，与观察到的生长动力学延迟一致，但在 6 hpi 时达到正常水平。有趣的是，这一结果与之前在 TDP2 KO 鼠细胞模型中报道的结果略有不同，表明细胞类型或物种特异性差异可能在观察到的表型中发挥作用。我们还确定，无催化活性的 TDP2 不能挽救生长缺陷，这证实了 TDP2 5' 磷酸二酯酶活性是病毒复制所必需的。重要的是，我们首次表明，在细胞培养模型中，在第一轮翻译后，多核糖体可以有效地组装在 VPg 连接的 RNA 上，但在感染中期缺乏 TDP2 时，正链和负链 RNA 的产生都受到损害，这表明病毒 RNA 上的 VPg 会影响翻译后的复制周期中的一个步骤（例如，RNA 合成）。这一结论与我们的发现一致，即在 TDP2 KO RPE-1 细胞中，双链 RNA 的产生（病毒 RNA 合成的标志物）延迟。此外，我们表明，新生 VPg 连接的 RNA 的过早封装不是导致观察到的病毒生长缺陷的原因。我们的研究首次提供了证据，表明无论是负链还是正链 RNA 合成（或两者）都可能是肠病毒感染中需要从 RNA 上去除 VPg 以有效进行的步骤的候选者。
