Abstract：
Viruses are obligate parasites that depend on the cellular machinery for their propagation. Several viruses also incorporate cellular proteins that facilitate viral spread. Defining these cellular proteins is critical to decipher viral life cycles and delineate novel therapeutic strategies. While numerous studies have explored the importance of host proteins in coronavirus spread, information about their presence in mature virions is limited. In this study, we developed a protocol to highly enrich mature HCoV-OC43 virions and characterize them by proteomics. Recognizing that cells release extracellular vesicles whose content is modulated by viruses, and given our ability to separate virions from these vesicles, we also analyzed their protein content in both uninfected and infected cells. We uncovered 69 unique cellular proteins associated with virions including 31 high-confidence hits. These proteins primarily regulate RNA metabolism, enzymatic activities, vesicular transport, cell adhesion, metabolite interconversion, and translation. We further discovered that the virus had a profound impact on exosome composition, incorporating 47 novel cellular proteins (11 high confidence) and excluding 92 others (61 high confidence) in virus-associated extracellular vesicles compared to uninfected cells. Moreover, a dsiRNA screen revealed that 11 of 18 select targets significantly impacted viral yields, including proteins found in virions or extracellular vesicles. Overall, this study provides new and important insights into the incorporation of numerous host proteins into HCoV-OC43 virions, their biological significance, and the ability of the virus to modulate extracellular vesicles.
OBJECTIVE: In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles.
OBJECTIVE: In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles.
摘要：
病毒是专性寄生虫，依赖于细胞机制进行繁殖。几种病毒还掺入促进病毒传播的细胞蛋白。定义这些细胞蛋白对于破译病毒生命周期和描绘新的治疗策略至关重要。虽然许多研究探索了宿主蛋白在冠状病毒传播中的重要性，关于它们在成熟病毒体中存在的信息是有限的。在这项研究中,我们开发了一种方案来高度富集成熟的HCoV-OC43病毒体，并通过蛋白质组学对其进行表征。认识到细胞释放细胞外囊泡，其内容受病毒调节，考虑到我们能够从这些囊泡中分离病毒体，我们还分析了它们在未感染和感染细胞中的蛋白质含量。我们发现了与病毒体相关的69种独特的细胞蛋白，包括31种高置信度命中。这些蛋白质主要调节RNA代谢，酶活性，囊泡运输，细胞粘附，代谢物相互转化，和翻译。我们进一步发现该病毒对外泌体成分有深远的影响，与未感染细胞相比，在病毒相关的细胞外囊泡中掺入了47种新型细胞蛋白（11种高置信度），并排除了92种其他蛋白（61种高置信度）。此外，一个dsiRNA屏幕显示，11的18个目标选择显著影响病毒产量，包括病毒体或细胞外囊泡中的蛋白质。总的来说,这项研究为将许多宿主蛋白掺入HCoV-OC43病毒体提供了新的重要见解，它们的生物学意义，以及病毒调节细胞外囊泡的能力。
目标：近年来，冠状病毒已经引起了全球的关注，这使得开发控制它们和防止未来大流行的方法变得至关重要。除了病毒蛋白,宿主蛋白在病毒繁殖中起着重要作用，并提供了潜在的治疗靶点。靶向宿主蛋白是有利的，因为与病毒蛋白相比，它们不太可能突变和产生抗性，许多抗病毒治疗的常见问题。在这项研究中,我们检查了毒性较低的生物安全级别2HCoV-OC43病毒的蛋白质含量，以代替毒性较大的SARS-CoV-2。我们的发现表明，整合到病毒体中的几种细胞蛋白调节病毒传播。此外,我们报告说，该病毒广泛调节细胞外囊泡的含量，增强病毒传播。这强调了病毒之间的关键相互作用，宿主蛋白，和细胞外囊泡。
目标：近年来，冠状病毒已经引起了全球的关注，这使得开发控制它们和防止未来大流行的方法变得至关重要。除了病毒蛋白,宿主蛋白在病毒繁殖中起着重要作用，并提供了潜在的治疗靶点。靶向宿主蛋白是有利的，因为与病毒蛋白相比，它们不太可能突变和产生抗性，许多抗病毒治疗的常见问题。在这项研究中,我们检查了毒性较低的生物安全级别2HCoV-OC43病毒的蛋白质含量，以代替毒性较大的SARS-CoV-2。我们的发现表明，整合到病毒体中的几种细胞蛋白调节病毒传播。此外,我们报告说，该病毒广泛调节细胞外囊泡的含量，增强病毒传播。这强调了病毒之间的关键相互作用，宿主蛋白，和细胞外囊泡。
