Identifying host and viral correlates for coronavirus pathogenesis

摘要 冠状病毒(CoV)刺突蛋白是一种关键的病毒决定簇，负责与受体结合和 融合/进入。尖峰蛋白也被预测为驱动跨物种的主要因素。 传播，允许出现像SARS和MERS冠状病毒这样的流行毒株。在之后的第一个十年里 SARS-CoV的出现，允许与新宿主受体结合的流行高峰的变化是 被认为是这种人畜共患病出现的基础。然而，我们的研究表明，蝙蝠物种已经 具有能够感染人类细胞的尖峰蛋白的类SARS冠状病毒。这些结果表明，对于一个子集， 蝙蝠冠状病毒、受体结合和人类细胞感染并不是病毒出现的主要障碍。 我们发现，尽管在体外复制相当，但含有蝙蝠冠状病毒尖峰的嵌合病毒已经减少 体内毒力。感染了表达BAT来源SHC014-CoV尖峰的嵌合SARS-CoV的小鼠 与SARS冠状病毒对照组相比，体重减轻和致命性降低。重要的是，这种衰减会发生在 相当于SARS-CoV在肺部的复制。结果表明，毒力由多个因素决定 仅仅是在体外感染宿主细胞的能力。值得注意的是，我们还发现SHC014尖峰嵌合体减少了 肺部大呼吸道的感染。这些初步数据形成了我们的核心假设，即SARS- 冠状病毒的毒力取决于宿主与冠状病毒刺突蛋白的相互作用和病毒基序。 了解导致呼吸道感染减少的宿主和病毒机制可能会在体内预测 发病机制，并对人畜共患病的出现具有重要意义。 在这项建议中，我们探索宿主因素和冠状病毒尖峰的变化，以减弱人畜共患病的SHC014尖峰。 在活体内。在第一部分中，我们检查了趋向性的变化，发现人畜共患病的SHC014棘突已经损害了上端 呼吸道感染。我们预测这种不亲和性与宿主蛋白酶活性的差异有关。我们 随后利用体外和体内实验确定了介导这种衰减的特定宿主蛋白水解酶 接近了。在第二部分中，我们使用小鼠适应和结构分析来预测脉冲变化 负责SHC014尖峰的衰减。我们随后产生突变病毒并恢复 SHC014在体内刺激性或减弱SARS刺激性刺激物。最后，我们评价了衰减的机理。 重点研究了与宿主蛋白水解酶的相互作用。总之，该提案确定了宿主蛋白水解酶和Spike 在冠状病毒感染后改变呼吸道感染和指示毒力的相互作用。这些发现 为理解毒力提供关键见解，并对出现和 冠状病毒的传播。

Abstract The coronavirus (CoV) spike protein is a key viral determinant responsible for receptor binding and fusion/entry. The spike protein has also been predicted to be the major factor driving cross-species transmission, allowing the emergence of epidemic strains like SARS- and MERS-CoV. In the first decade after SARS-CoV emergence, changes to the epidemic spike that allowed binding to a new host receptor were thought to underlie this zoonotic emergence. However, our work has shown that bat species already harbor SARS-like CoVs with spike proteins capable of infecting human cells. These results argue that for a subset of bat CoVs, receptor binding and infection of human cells is not the major barrier for emergence. We found that despite equivalent replication in vitro, chimeric viruses containing bat CoV spikes have reduced virulence in vivo. Mice infected with a chimeric SARS-CoV expressing the bat derived SHC014-CoV spike had reduced weight loss and lethality compared to SARS-CoV controls. Importantly, this attenuation occurs despite equivalent replication to SARS-CoV in the lung. The results indicate that virulence is dictated by more than just the ability to infect host cells in vitro. Notably, we also found that the SHC014 spike chimera has reduced infection of the large airways of the lung. These preliminary data shaped our central hypothesis that SARS- CoV virulence is predicated on both host interactions with and viral motifs in the CoV spike protein. Understanding the host and viral mechanisms that drive reduced airway infection may predict in vivo pathogenesis and have critical implications for zoonotic emergence. In this proposal, we explore the host factors and CoV spike changes that attenuate the zoonotic SHC014 spike in vivo. In part one, we examine tropism changes finding that the zoonotic SHC014 spike has impaired upper airway infection. We predict that this incompatibility relates to differences in host protease activity. We subsequently define the specific host proteases that mediate this attenuation using both in vitro and in vivo approaches. In part two, we use mouse-adaptation and structural analysis to predict spike changes responsible for attenuation of the SHC014 spike. We subsequently generate mutant viruses and restore the SHC014 spike or attenuate the SARS spike in vivo. Finally, we evaluate the mechanism of attenuation focusing on spike interactions with host proteases. Together, the proposal identifies host proteases and spike interactions that alter airway infection and dictate virulence following coronavirus infection. These findings provide critical insights for understanding virulence as well as have important implications for emergence and transmission of coronaviruses.