利用转录组动态测序和分子对接揭示冠状 病毒与宿主的相互作用

随着新型冠状病毒的大规模爆发，感染人类的冠状病毒达到7种，其中3种曾经对人类生命健康构成严重威胁，包括SARS-CoV、MERS-CoV和SARS-CoV-2。在新冠发生后，新的感染人的冠状病毒的发现，提示着自然界中存在未知的病毒对人类的健康造成威胁。并且，长新冠的发生也提示人类，除了感染过程中引起的急性期症状，冠状病毒感染后遗症同样值得科学家的关注。针对严重的冠状病毒感染，目前仍未有效果明显的特效药。为了更好地制定有效的防护措施，更加彻底地了解冠状病毒的致病机制是必然的先决条件。但是，病毒感染是一个复杂的过程，在宿主细胞中触发不同的调节反应，病毒感染的进程涉及许多细胞内的转录调控，随着感染进展到不同阶段，细胞内的转录事件也发生改变。并且抗病毒药物作用病毒的同时也干扰宿主细胞转录组的变化。掌握不同感染阶段和治疗阶段宿主细胞的转录动态将有助于更加精确地对症下药。然而，常规RNA测序（RNA-seq）的结果不仅包括了新合成的mRNA，还包括已合成的mRNA。已合成的mRNA可能会掩盖了新合成mRNA的信息。所以，病毒感染时细胞的瞬时转录的mRNA在常规RNA测序技术下无法被检测到，RNA-seq分析无法检测细胞内的所有转录事件。因此，对新合成转录本的监控有助于深入了解病毒的复制机制和致病机理。

在本研究中，我们采用了巯基(SH)-链接烷基化代谢测序（SLAM-seq），通过检测掺入到新合成转录本中的修饰核苷酸4-硫尿嘧啶（S4U）来区分新合成和已合成的RNA转录本的方法，以不同角度揭示冠状病毒HCoV-OC43感染初期稳定状态下总的RNA和新合成RNA的水平变化。结果表明，在HCoV-OC43感染初期，Wnt/β-catenin信号通路中新合成的转录本显著富集。此外，利用常规的生物学技术，我们发现在感染的早期阶段抑制Wnt/β-catenin信号通路会促进病毒的复制，但在感染的后期阶段却抑制了病毒复制。通过利用瑞德西韦进行的药物测试，我们发现其抑制HCoV-OC43的复制可能依赖于感染中对Wnt/β-catenin信号通路的激活。综合而言，我们的研究揭示了Wnt/β-catenin信号通路在HCoV-OC43感染不同阶段的多样角色，为抗病毒治疗提供了潜在的靶点。另外，尽管HCoV-OC43感染导致BHK21细胞发生细胞病变，但抑制细胞凋亡并不影响病毒在细胞内的复制。这一发现强调了抗病毒策略需要综合考虑细胞生存和病毒复制之间的平衡。

在本研究中，我们采用了标记核苷酸的方法监测新合成RNA，能够在不同时间点追踪RNA的合成，为揭示病毒感染的动态过程提供了有力的工具，也为病毒感染不同阶段的治疗提供分子调节机制的基础研究。总体而言，本研究不仅深入探讨了HCoV-OC43感染的分子机制，而且发现了通路调节在不同感染阶段的重要作用。这不仅为理解冠状病毒感染的基础生物学提供了新的视角，而且为未来的抗病毒治疗研究提供了潜在的靶点。在病毒感染的不同阶段，针对相应的转录组变化，设计合理的抗病毒药物，提供更加精确的治疗方式与手段。我们的研究成果揭示了细胞内信号通路与病毒复制之间的复杂交互关系，为制定更有效的抗病毒治疗策略奠定了基础。这些发现不仅对理解和治疗冠状病毒感染有重要意义，也为其他病毒感染的研究提供了可借鉴的理论和启示。

在论文的第二部分，我们主要利用全原子分子动力学模拟表征了新型纳米抗体IBT-CoV144与新型冠状病毒SARS-CoV-2刺突蛋白的受体结合域的结合性质和相互作用位点。由于SARS-CoV-2的持续突变，现有抗病毒药物的有效性面临严峻的挑战，因此探索具有功效的新的治疗方法至关重要。在此之前我们报道的一种新型纳米抗体IBT-CoV144，它表现出对SARS-CoV-2突变株具有出色和广泛的中和能力，我们利用冷冻电镜技术解析了其与SARS-CoV-2刺突蛋白的结构。然而，冷冻电镜未能阐明IBT-CoV144中和的冠状病毒刺突蛋白上的氨基酸表位。于是我们深入探讨了IBT-CoV144与冠状病毒刺突蛋白受体结合域在原子水平上的结合机制。利用全原子分子动力学模拟，表征了IBT-CoV144与刺突蛋白RBD的结合性质和相互作用位点并利用分子对接系统地探索IBT-CoV144与野生型和BQ1.1突变株的结合位点，从而揭示IBT-CoV144对突变株中和能力下降的原因，全面了解了IBT-CoV144与新冠病毒RBD相互作用的具体位点，为IBT-CoV144中和能力提供了理论支持。

综上所述，本研究主要利用SLAM-seq技术和分子动力学模拟技术，弥补了常规RNA-seq技术和冷冻电镜技术，探索并优化了对冠状病毒相关研究，为理解冠状病毒以及探索冠状病毒疾病发生、药物研究提供理论基础。

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