Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective.
Inhibition of SARS-CoV-2 polymerase by nucleotide analogs from a single-molecule perspective.
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核苷酸类似物对SARS-CoV-2聚合酶的单分子抑制作用
DOI：
10.7554/elife.70968
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发表时间：
2021-10-07
期刊：
影响因子：
7.7
通讯作者：
Dulin D
中科院分区：
文献类型：
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作者：
Seifert M;Bera SC;van Nies P;Kirchdoerfer RN;Shannon A;Le TT;Meng X;Xia H;Wood JM;Harris LD;Papini FS;Arnold JJ;Almo S;Grove TL;Shi PY;Xiang Y;Canard B;Depken M;Cameron CE;Dulin D
在疫苗开发过程中缺乏“现成的”抗冠状病毒药物，使SARS-CoV-2大流行严重恶化。此外，新的疫苗耐药性变种和冠状病毒疫情可能在不久的将来发生，我们必须准备好面对这种可能性。然而，由于我们对靶向冠状病毒聚合酶以损害其基本活性的核苷酸类似物的掺入模式和作用机制的理解不足，至今仍然缺乏有效的抗病毒药物。在这里，我们使用高通量，单分子，磁镊平台表征了remdesivir（RDV，唯一FDA批准的抗冠状病毒药物）和其他核苷酸类似物（NAs）对冠状病毒聚合酶RNA合成的影响。我们揭示了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。我们表明，RDV掺入并不终止病毒RNA的合成，但导致聚合酶回溯到30 nt，这可能会出现在传统的合奏测定终止。SARS-CoV-2能够逃避蝰蛇蛋白抗病毒蛋白ddhCTP的内源性合成产物，尽管聚合酶很好地结合了该NA。这种实验范式对于发现和开发靶向病毒聚合酶的治疗剂是必不可少的。为了在细胞间繁殖和传播，COVID-19（也称为SARS-CoV-2）病毒必须首先复制其遗传信息。这个过程涉及一种“聚合酶”蛋白质复合物，通过组装一个精确的构建模块或核苷酸序列来进行忠实的复制。Remdesivir是美国食品和药物管理局（FDA）批准的唯一一种针对SARS-CoV-2的药物，由核苷酸类似物组成，这种分子的结构与复制所需的实际构建模块相似。如果聚合酶识别这些类似物并将其整合到不断生长的基因序列中，复制机制就会被破坏，病毒就无法繁殖。大多数研究这一过程的方法似乎表明，remdesivir通过阻止聚合酶并完全终止复制来起作用。然而，Remdesivir和其他类似物究竟如何损害病毒新拷贝的合成仍然不确定。为了探索这个问题，Seifert，Bera等人采用了一种称为磁镊子的方法，该方法使用磁场以非常精确的方式操纵微粒。与其他方法不同，这种技术允许类似物在类似于细胞中发现的条件下整合，并在单个分子水平上进行检查。结果表明，与之前的假设相反，remdesivir不会终止复制;相反，它会导致聚合酶暂停和回溯（这可能在其他技术中表现为终止）。然后将相同的方法应用于其他核苷酸类似物，其中一些也被发现靶向SARS-CoV-2聚合酶。然而，这些类似物与remdesivir不同地掺入，并且效率较低。它们还以不同的方式阻碍聚合酶。综上所述，塞弗特，贝拉等人的结果表明，磁镊可以是一种强有力的方法来揭示类似物如何干扰复制。这些信息可用于改进目前可用的类似物，以及开发对SARS-CoV-2更有效的新的抗病毒药物。在仍然缺乏COVID-19治疗方法的时候，这些知识将是关键，并且可能需要防止新的变种和未来的爆发。
The absence of ‘shovel-ready’ anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day, due to our poor understanding of the mode of incorporation and mechanism of action of nucleotides analogs that target the coronavirus polymerase to impair its essential activity. Here, we characterize the impact of remdesivir (RDV, the only FDA-approved anti-coronavirus drug) and other nucleotide analogs (NAs) on RNA synthesis by the coronavirus polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. We reveal that the location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We show that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into backtrack as far as 30 nt, 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 NA well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases. To multiply and spread from cell to cell, the virus responsible for COVID-19 (also known as SARS-CoV-2) must first replicate its genetic information. This process involves a ‘polymerase’ protein complex making a faithful copy by assembling a precise sequence of building blocks, or nucleotides. The only drug approved against SARS-CoV-2 by the US Food and Drug Administration (FDA), remdesivir, consists of a nucleotide analog, a molecule whose structure is similar to the actual building blocks needed for replication. If the polymerase recognizes and integrates these analogs into the growing genetic sequence, the replication mechanism is disrupted, and the virus cannot multiply. Most approaches to study this process seem to indicate that remdesivir works by stopping the polymerase and terminating replication altogether. Yet, exactly how remdesivir and other analogs impair the synthesis of new copies of the virus remains uncertain. To explore this question, Seifert, Bera et al. employed an approach called magnetic tweezers which uses a magnetic field to manipulate micro-particles with great precision. Unlike other methods, this technique allows analogs to be integrated under conditions similar to those found in cells, and to be examined at the level of a single molecule. The results show that contrary to previous assumptions, remdesivir does not terminate replication; instead, it causes the polymerase to pause and backtrack (which may appear as termination in other techniques). The same approach was then applied to other nucleotide analogs, some of which were also found to target the SARS-CoV-2 polymerase. However, these analogs are incorporated differently to remdesivir and with less efficiency. They also obstruct the polymerase in distinct ways. Taken together, the results by Seifert, Bera et al. suggest that magnetic tweezers can be a powerful approach to reveal how analogs interfere with replication. This information could be used to improve currently available analogs as well as develop new antiviral drugs that are more effective against SARS-CoV-2. This knowledge will be key at a time when treatments against COVID-19 are still lacking, and may be needed to protect against new variants and future outbreaks.