The nucleotide analog Remdesivir (RDV) is the only FDA-approved antiviral therapy to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The physical basis for efficient utilization of RDV by SARS-CoV-2 polymerase is unknown. Here, we characterize the impact of RDV and other nucleotide analogs on RNA synthesis by the polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. The location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We reveal that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into deep backtrack, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this nucleotide analog well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.

中文翻译：

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核苷酸类似物对 SARS-CoV-2 聚合酶的抑制作用：单分子观点

核苷酸类似物 Remdesivir (RDV) 是 FDA 批准的唯一一种用于治疗严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 感染的抗病毒疗法。SARS-CoV-2 聚合酶有效利用 RDV 的物理基础尚不清楚。在这里，我们使用高通量、单分子、磁性镊子平台描述了 RDV 和其他核苷酸类似物对聚合酶合成 RNA 的影响。修饰在核糖或碱基中的位置决定了用于其掺入的催化途径。我们发现 RDV 掺入不会终止病毒 RNA 合成，但会导致聚合酶进入深度回溯，这可能在传统的整体分析中表现为终止。SARS-CoV-2 能够逃避毒蛇抗病毒蛋白 ddhCTP 的内源性合成产物，尽管聚合酶很好地结合了这种核苷酸类似物。这种实验范式对于发现和开发针对病毒聚合酶的疗法至关重要。