The N-Terminal Region of Middle East Respiratory Syndrome Coronavirus Accessory Protein 8b Is Essential for Enhanced Virulence of an Attenuated Murine Coronavirus.
The N-Terminal Region of Middle East Respiratory Syndrome Coronavirus Accessory Protein 8b Is Essential for Enhanced Virulence of an Attenuated Murine Coronavirus.
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DOI：
10.1128/jvi.01842-21
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发表时间：
2022-02-09
影响因子：
5.4
通讯作者：
Zhao J
中科院分区：
文献类型：
--
作者：
Li Y;Jin Y;Kuang L;Luo Z;Li F;Sun J;Zhu A;Zhuang Z;Wang Y;Wen L;Liu D;Chen C;Gan M;Zhao J;Zhao J
中东呼吸综合征冠状病毒（MERS-CoV）是2012年出现的一种β冠状病毒，可引起严重的肺炎和肾衰竭。MERS-CoV编码5个辅助蛋白。其中一些已被证明干扰宿主的抗病毒免疫反应。然而，蛋白8b在天然免疫和病毒毒力中的作用很少被研究。我们将MERS-CoV辅助蛋白基因分别导入减毒鼠冠状病毒（Mouse hepatitis virus，MHV）基因组中，发现辅助蛋白8b能增强病毒在体内和体外的复制，提高感染小鼠的致死率。RNA-seq分析显示，蛋白8b可显著抑制表达蛋白8b的MHV感染小鼠的I型干扰素（IFN-I）产生和天然免疫应答。我们还发现MERS-CoV蛋白8b可以从多个内部甲硫氨酸位点起始，并且鉴定出至少三种蛋白变体。蛋白质8b的残基1-23被证明是体内毒力增加的原因。此外，蛋白8b对IFN-I的抑制作用可能不会有助于其毒力增强，因为aa 1 -23缺失不影响IFN-I在体外和体内的产生。接下来，我们还发现蛋白8b定位于感染细胞的内质网（ER）/高尔基体膜，其被C-末端区域aa 88-112缺失破坏。该研究将为MERS-CoV感染的发病机制提供新的见解。重要性多种冠状病毒（CoV）会导致严重的呼吸道感染，并成为全球公共卫生威胁，例如SARS-CoV、MERS-CoV和SARS-CoV-2。每种冠状病毒都含有不同数量的辅助蛋白，这些蛋白在不同的冠状病毒之间表现出高度的变异性。辅助蛋白在冠状病毒的发病机制中起重要作用。MERS-CoV含有5个辅助蛋白（蛋白3、4a、4 b、5、8b），并且所有4个辅助蛋白（蛋白3、4a、4 b、5）的缺失显著影响MERS-CoV的复制和致病性。然而，ORF 8b是否也调节MERS-CoV感染尚不清楚。本研究构建了表达MERS-CoV蛋白8b的小鼠肝炎病毒（MHV）重组病毒，并证明了蛋白8b能够显著增强MHV的毒力，这种增强作用是由蛋白8b的N端结构域介导的。该研究将有助于了解MERS-CoV感染的发病机制。
Middle East respiratory syndrome coronavirus (MERS-CoV) is a beta coronavirus that emerged in 2012, causing severe pneumonia and renal failure. MERS-CoV encodes five accessory proteins. Some of them have been shown to interfere with host antiviral immune response. However, the roles of protein 8b in innate immunity and viral virulence was rarely studied. Here, we introduced individual MERS-CoV accessory protein genes into the genome of an attenuated murine coronavirus (Mouse hepatitis virus, MHV), respectively, and found accessory protein 8b could enhance viral replication in vivo and in vitro and increase the lethality of infected mice. RNA-seq analysis revealed that protein 8b could significantly inhibit type I interferon production (IFN-I) and innate immune response in mice infected with MHV expressing protein 8b. We also found that MERS-CoV protein 8b could initiate from multiple internal methionine sites and at least three protein variants were identified. Residues 1-23 of protein 8b was demonstrated to be responsible for increased virulence in vivo. In addition, the inhibitory effect on IFN-I of protein 8b might not contribute to its virulence enhancement as aa1-23 deletion did not affect IFN-I production in vitro and in vivo. Next, we also found that protein 8b was localized to the endoplasmic reticulum (ER)/Golgi membrane in infected cells, which was disrupted by C-terminal region aa 88-112 deletion. This study will provide new insight into the pathogenesis of MERS-CoV infection. IMPORTANCE Multiple coronaviruses (CoV) cause severe respiratory infections and become global public health threats such as SARS-CoV, MERS-CoV, and SARS-CoV-2. Each coronavirus contains different numbers of accessory proteins which show high variability among different CoVs. Accessory proteins are demonstrated to play essential roles in pathogenesis of CoVs. MERS-CoV contains 5 accessory proteins (protein 3, 4a, 4b, 5, 8b), and deletion of all four accessory proteins (protein 3, 4a, 4b, 5), significantly affects MERS-CoV replication and pathogenesis. However, whether ORF8b also regulates MERS-CoV infection is unknown. Here, we constructed mouse hepatitis virus (MHV) recombinant virus expressing MERS-CoV protein 8b and demonstrated protein 8b could significantly enhance the virulence of MHV, which is mediated by N-terminal domain of protein 8b. This study will shed light on the understanding of pathogenesis of MERS-CoV infection.