Abstract:

RNA-dependent RNA polymerase （RdRp） is the main antiviral target due to its crucial role in severe acute respiratory syndrome coronavirus 2（SARS-CoV-2） replication. The function of RdRp is strictly dependent on its spatial structure. Since mutations in key amino acid sites may lead to changes in spatial structure and function， it is necessary to investigate whether the RdRp inhibitor effective on SARS-CoV-2 prototype strain still has inhibitory effect on Omicron variants. Then 913 RdRp high-quality sequences of Omicron variants prevalent in China in the past two years were analyzed by multiple sequence alignment. Compared with the prototype strain， these Omicron variants had both synonym and missense mutations. Among the missense mutations， with the exception of a few low-frequency mutation sites， 44% had P323L single mutation， 34% had P323L and G671S dual mutation， and 12% had P323L， G671S and D63N triple mutation. The Omicron RdRp sequences containing these three types of mutations were respectively grouped as 1_RdRp， 2_RdRp and 3_RdRp. AlphaFold2 was used to construct the protein structure model of these three types of RdRp， and PyMOL was used to superposition the RdRp structure of Omicron and the prototype strain. The results showed that all the three protein structure models could be highly superimposed with the RdRp of the prototype （6M71_RdRp）， suggesting that these mutations did not alter the spatial structure of RdRp. Further， three Omicron RdRp protein models were respectively docked with four reported RdRp inhibitors， and the results showed that these four inhibitors could still stably bind to the active center of each Omicron RdRp model， suggesting that RdRp is a long-term effective target. Based on this， an extracellular RdRp enzymatic reaction and detection system was constructed， which would be helpful for the evaluation or screening of antiviral drugs targeting RdRp.

Key words: SARS-CoV-2, Omicron, RdRp, multiple sequence alignment, molecular docking

摘要：

RNA依赖的RNA聚合酶（RNA-dependent RNA polymerase, RdRp）在新型冠状病毒——严重急性呼吸综合征冠状病毒2型（severe acute respiratory syndrome coronavirus 2, SARS-CoV-2）复制中发挥重要作用，是主要的抗病毒靶点。RdRp的功能严格依赖其空间结构，而关键氨基酸位点的突变可能导致空间结构和功能的改变。为了探究对SARS-CoV-2原型株有效的RdRp抑制剂是否对Omicron变异株仍存在抑制效果，对我国近两年流行的Omicron变异株的913个RdRp优质序列进行多序列比对。与原型株相比，这些Omicron变异株的RdRp序列既存在同义突变又存在错义突变。在错义突变中，除少数低频突变位点外，44%为P323L，34%为P323L和G671S双重突变，12%为P323L、G671S和D63N三重突变，分别将包含这3类突变的Omicron RdRp归为1_RdRp、2_RdRp和3_RdRp。利用AlphaFold2构建这3类RdRp的蛋白质结构模型，并通过PyMOL将Omicron与原型株的RdRp结构进行叠合比对，结果显示3类蛋白质3D结构均与原型株的RdRp（6M71_RdRp）高度重叠，提示突变未对RdRp的空间结构产生显著影响。在此基础上，将3种Omicron RdRp蛋白模型分别与4种已报道的RdRp抑制剂对接，结果显示这4种抑制剂依然能稳定结合在各Omicron RdRp模型的活性中心，提示RdRp是一个长期有效的靶标。基于此，构建的细胞外RdRp酶活反应和检测体系将有助于靶向RdRp的抗病毒药物的评价或筛选。

关键词: SARS-CoV-2, Omicron, RdRp, 多序列比对, 分子对接