Coronavirus nsp10/nsp16 Methyltransferase Can Be Targeted by nsp10-Derived Peptide In Vitro and In Vivo To Reduce Replication and Pathogenesis
Coronavirus nsp10/nsp16 Methyltransferase Can Be Targeted by nsp10-Derived Peptide In Vitro and In Vivo To Reduce Replication and Pathogenesis
复制标题
冠状病毒 nsp10/nsp16 甲基转移酶可以在体外和体内被 nsp10 衍生肽靶向，以减少复制和发病机制。
DOI：
10.1128/jvi.00948-15
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发表时间：
2015-08-01
影响因子：
5.4
通讯作者：
Guo, Deyin
中科院分区：
文献类型：
--
作者：
Wang, Yi;Sun, Ying;Guo, Deyin
真核生物mRNA的5 '帽结构对于RNA稳定性和蛋白质翻译是重要的。许多在真核生物细胞质中复制的病毒已经进化出2'-O-甲基转移酶（2'-O-MTase）以自主修饰其mRNA并在5 '端携带cap-1结构（m7GpppNm），从而促进病毒复制并逃避宿主细胞中的先天免疫识别。以往的研究表明，严重急性呼吸综合征冠状病毒（SARS CoV）非结构蛋白16（nsp 16）的2'-O-MTase活性需要被nsp 10激活，而猫冠状病毒（FCoV）的nsp 16单独具有2'-O-MTase活性（E. Decroly等人，J Virol 82：8071 - 8084，2008，http：//dx·doi·org/10·1128/JVI·00407 - 08; M. Bouvet等人，PLoS Pathog 6：e1000863，2010，http：//dx.doi.org/10.1371/journal.ppat. Decroly等人，PLoS Pathog 7：e1002059，2011，http：//dx.doi.org/10.1371/journal.ppat. Chen等，PLoS Pathog 7：e1002294，2011，http：//dx.doi.org/10.1371/journal.ppat.1002294）。在这项研究中，我们证明了nsp10刺激nsp16 2'-O-MTase活性是冠状病毒（包括FCoV）中的一种普遍和保守的机制，并且nsp10在刺激不同冠状病毒的nsp16中具有功能互换性。基于我们目前和以前的研究，我们从小鼠肝炎病毒（MHV）nsp 10的相互作用界面序列中设计了一个肽（TP 29），并在生化测定和MHV感染和SARS-CoV复制子模型中证明了该肽抑制不同冠状病毒的2'-O-MTase活性。有趣的是，肽TP 29在MHV感染的小鼠中通过在感染的早期阶段抑制病毒复制和增强I型干扰素的产生来削弱MHV毒力和发病机制，从而在体内发挥强大的抑制作用。因此，作为原理证明，目前的结果表明冠状病毒2'-O-MTase活性可以在体外和体内靶向。重要性冠状病毒是动物和人类的重要病原体，具有高度的人畜共患病潜力。SARS-CoV编码由催化亚基nsp16和刺激亚基nsp10组成的2'-O-MTase，在病毒基因组复制和逃避先天免疫中起重要作用。我们目前的研究结果表明，nsp10刺激nsp16 2'-O-MTase活性是冠状病毒的常见机制，并且nsp10在不同冠状病毒中刺激nsp16的功能是可互换的，这是开发抑制肽的基本原理。我们证明了来自MHV nsp10的nsp16相互作用结构域的肽可以在体外抑制不同冠状病毒的2'-O-MTase活性以及在细胞培养中抑制MHV和SARS-CoV复制子的病毒复制，并且它可以强烈抑制MHV感染小鼠中的病毒复制和致病性。这项工作使得通过靶向冠状病毒的nsp16/nsp10 2'-O-MTase开发广谱肽抑制剂成为可能。
The 5' cap structures of eukaryotic mRNAs are important for RNA stability and protein translation. Many viruses that replicate in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases (2'-O-MTase) to autonomously modify their mRNAs and carry a cap-1 structure (m7GpppNm) at the 5' end, thereby facilitating viral replication and escaping innate immune recognition in host cells. Previous studies showed that the 2'-O-MTase activity of severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 16 (nsp16) needs to be activated by nsp10, whereas nsp16 of feline coronavirus (FCoV) alone possesses 2'-O-MTase activity (E. Decroly et al., J Virol 82:8071-8084, 2008, http://dx.doi.org/10.1128/JVI.00407-08; M. Bouvet et al., PLoS Pathog 6:e1000863, 2010, http://dx.doi.org/10.1371/journal.ppat.1000863; E. Decroly et al., PLoS Pathog 7:e1002059, 2011, http://dx.doi.org/10.1371/journal.ppat.1002059; Y. Chen et al., PLoS Pathog 7:e1002294, 2011, http://dx.doi.org/10.1371/journal.ppat.1002294). In this study, we demonstrate that stimulation of nsp16 2'-O-MTase activity by nsp10 is a universal and conserved mechanism in coronaviruses, including FCoV, and that nsp10 is functionally interchangeable in the stimulation of nsp16 of different coronaviruses. Based on our current and previous studies, we designed a peptide (TP29) from the sequence of the interaction interface of mouse hepatitis virus (MHV) nsp10 and demonstrated that the peptide inhibits the 2'-O-MTase activity of different coronaviruses in biochemical assays and the viral replication in MHV infection and SARS-CoV replicon models. Interestingly, the peptide TP29 exerted robust inhibitory effects in vivo in MHV-infected mice by impairing MHV virulence and pathogenesis through suppressing virus replication and enhancing type I interferon production at an early stage of infection. Therefore, as a proof of principle, the current results indicate that coronavirus 2'-O-MTase activity can be targeted in vitro and in vivo.IMPORTANCE Coronaviruses are important pathogens of animals and human with high zoonotic potential. SARS-CoV encodes the 2'-O-MTase that is composed of the catalytic subunit nsp16 and the stimulatory subunit nsp10 and plays an important role in virus genome replication and evasion from innate immunity. Our current results demonstrate that stimulation of nsp16 2'-O-MTase activity by nsp10 is a common mechanism for coronaviruses, and nsp10 is functionally interchangeable in the stimulation of nsp16 among different coronaviruses, which underlies the rationale for developing inhibitory peptides. We demonstrate that a peptide derived from the nsp16-interacting domain of MHV nsp10 could inhibit 2'-O-MTase activity of different coronaviruses in vitro and viral replication of MHV and SARS-CoV replicon in cell culture, and it could strongly inhibit virus replication and pathogenesis in MHV-infected mice. This work makes it possible to develop broad-spectrum peptide inhibitors by targeting the nsp16/nsp10 2'-O-MTase of coronaviruses.