Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease.
Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) Is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease.
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DOI：
10.1128/mbio.00221-18
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发表时间：
2018-03-06
期刊：
影响因子：
6.4
通讯作者：
Denison MR
中科院分区：
文献类型：
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作者：
Agostini ML;Andres EL;Sims AC;Graham RL;Sheahan TP;Lu X;Smith EC;Case JB;Feng JY;Jordan R;Ray AS;Cihlar T;Siegel D;Mackman RL;Clarke MO;Baric RS;Denison MR
新出现的冠状病毒（CoV）在人类中引起严重疾病，但没有获得批准的治疗方法。CoV nsp 14核糖核酸外切酶（ExoN）由于其校对活性而具有复杂的抗病毒核苷开发。我们最近报道了核苷类似物GS-5734（remdesivir）在体外和严重急性呼吸道综合征冠状病毒（SARS-CoV）小鼠模型中有效抑制人类和人畜共患冠状病毒。然而，关于GS-5734的研究尚未报道与GS-5734相关的耐药性，我们也不了解GS-5734在野生型（WT）校对CoV中的作用。在这里，我们发现GS-5734抑制鼠肝炎病毒（MHV）的50%有效浓度值（EC 50）与SARS-CoV和中东呼吸综合征冠状病毒（MERS-CoV）相似。在存在GS-5734亲本核苷的情况下，WT MHV的传代在所有CoV中保守的残基处选择了nsp 12聚合酶中的两个突变，其赋予对GS-5734高达5.6倍的耐药性，如通过EC 50所确定的。在不存在GS-5734的情况下，耐药病毒无法在直接共感染传代中与WT竞争。将MHV抗性突变引入SARS-CoV导致相同的体外抗性表型和小鼠模型中的SARS-CoV致病性减弱。最后，我们证明了缺乏ExoN校正的MHV突变体对GS-5734显著更敏感。结合起来，结果表明，即使在完整的ExoN校正活性的情况下，GS-5734也会干扰nsp 12聚合酶，并且可以通过增加无毒浓度的GS-5734来克服耐药性，进一步支持GS-5734作为广谱治疗药物的开发，以防止当代和新兴的CoV。冠状病毒（CoV）引起严重的人类感染，但没有批准的抗病毒药物来治疗这些感染。用于CoV感染的基于核苷的治疗剂的开发受到校对核糖核酸外切酶的存在的阻碍。在这里，我们扩展了核苷酸前药remdesivir（GS-5734）的已知功效，以包括β-2a CoV组。此外，GS-5734有效抑制具有完整校对的CoV。在用GS-5734亲本核苷选择后，nsp 12聚合酶中在跨CoV相同的残基处的2个氨基酸取代提供了对GS-5734的低水平抗性。耐药突变降低了体外MHV的病毒适应性，并减弱了SARS-CoV感染动物模型的发病机制。总之，这些研究确定了GS-5734活性的靶点，并证明耐药性难以选择，仅部分耐药，并损害MHV和SARS-CoV的适应性和毒力，支持GS-5734作为潜在有效的泛CoV抗病毒剂的进一步开发。
Emerging coronaviruses (CoVs) cause severe disease in humans, but no approved therapeutics are available. The CoV nsp14 exoribonuclease (ExoN) has complicated development of antiviral nucleosides due to its proofreading activity. We recently reported that the nucleoside analogue GS-5734 (remdesivir) potently inhibits human and zoonotic CoVs in vitro and in a severe acute respiratory syndrome coronavirus (SARS-CoV) mouse model. However, studies with GS-5734 have not reported resistance associated with GS-5734, nor do we understand the action of GS-5734 in wild-type (WT) proofreading CoVs. Here, we show that GS-5734 inhibits murine hepatitis virus (MHV) with similar 50% effective concentration values (EC50) as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Passage of WT MHV in the presence of the GS-5734 parent nucleoside selected two mutations in the nsp12 polymerase at residues conserved across all CoVs that conferred up to 5.6-fold resistance to GS-5734, as determined by EC50. The resistant viruses were unable to compete with WT in direct coinfection passage in the absence of GS-5734. Introduction of the MHV resistance mutations into SARS-CoV resulted in the same in vitro resistance phenotype and attenuated SARS-CoV pathogenesis in a mouse model. Finally, we demonstrate that an MHV mutant lacking ExoN proofreading was significantly more sensitive to GS-5734. Combined, the results indicate that GS-5734 interferes with the nsp12 polymerase even in the setting of intact ExoN proofreading activity and that resistance can be overcome with increased, nontoxic concentrations of GS-5734, further supporting the development of GS-5734 as a broad-spectrum therapeutic to protect against contemporary and emerging CoVs. Coronaviruses (CoVs) cause severe human infections, but there are no approved antivirals to treat these infections. Development of nucleoside-based therapeutics for CoV infections has been hampered by the presence of a proofreading exoribonuclease. Here, we expand the known efficacy of the nucleotide prodrug remdesivir (GS-5734) to include a group β-2a CoV. Further, GS-5734 potently inhibits CoVs with intact proofreading. Following selection with the GS-5734 parent nucleoside, 2 amino acid substitutions in the nsp12 polymerase at residues that are identical across CoVs provide low-level resistance to GS-5734. The resistance mutations decrease viral fitness of MHV in vitro and attenuate pathogenesis in a SARS-CoV animal model of infection. Together, these studies define the target of GS-5734 activity and demonstrate that resistance is difficult to select, only partial, and impairs fitness and virulence of MHV and SARS-CoV, supporting further development of GS-5734 as a potential effective pan-CoV antiviral.