On the Recognition of Natural Substrate CTP and Endogenous Inhibitor ddhCTP of SARS-CoV‑2 RNA-Dependent RNA Polymerase: A Molecular Dynamics Study

The novel coronavirus SARS-CoV-2 is the causative agent of the COVID-19 outbreak that is affecting the entire planet. As the pandemic is still spreading worldwide, with multiple mutations of the virus, it is of interest and of help to employ computational methods for identifying potential inhibitors of the enzymes responsible for viral replication. Attractive antiviral nucleotide analogue RNA-dependent RNA polymerase (RdRp) chain terminator inhibitors are investigated with this purpose. This study, based on molecular dynamics (MD) simulations, addresses the important aspects of the incorporation of an endogenously synthesized nucleoside triphosphate, ddhCTP, in comparison with the natural nucleobase cytidine triphosphate (CTP) in RdRp. The ddhCTP species is the product of the viperin antiviral protein as part of the innate immune response. The absence of the ribose 3′-OH in ddhCTP could have important implications in its inhibitory mechanism of RdRp. We built an in silico model of the RNA strand embedded in RdRp using experimental methods, starting from the cryo-electron microscopy structure and exploiting the information obtained by spectrometry on the RNA sequence. We determined that the model was stable during the MD simulation time. The obtained results provide deeper insights into the incorporation of nucleoside triphosphates, whose molecular mechanism by the RdRp active site still remains elusive.严重急性呼吸综合征冠状病毒2（SARS-CoV-2，即新型冠状病毒）是引发当前全球肆虐的新冠肺炎（COVID-19）疫情的病原体。随着这场大流行仍在全球范围内蔓延，且病毒出现多种突变，借助计算方法识别负责病毒复制的酶的潜在抑制剂，兼具重要研究价值与应用意义。针对此研究目标，本研究对具有开发潜力的抗病毒核苷酸类似物RNA依赖的RNA聚合酶（RNA-dependent RNA polymerase, RdRp）链终止抑制剂展开了相关研究。本研究基于分子动力学（molecular dynamics, MD）模拟，对比天然核苷碱基胞苷三磷酸（cytidine triphosphate, CTP），探讨了内源性合成的三磷酸二脱氧胞苷（ddhCTP）在RdRp中的掺入过程这一关键科学问题。ddhCTP是干扰素诱导的内质网相关病毒抑制蛋白（viperin）作为先天免疫应答组分所产生的产物。该物质缺失核糖3'-羟基（3'-OH），这一结构特征可能对其RdRp抑制机制产生重要影响。本研究依托冷冻电子显微镜（cryo-electron microscopy）结构，并利用光谱法获得的RNA序列信息，通过实验手段构建了嵌入RdRp的RNA链的计算机模拟模型。经验证，该模型在分子动力学模拟时长内保持结构稳定。本研究所得结果为三磷酸核苷的掺入过程提供了更为深入的认知，而RdRp活性位点介导该过程的分子机制至今仍未完全阐明。