Biomechanics Study of SARS-CoV-2 Virus-Like Particles

SARS-CoV-2是COVID-19的病原体，COVID-19在2019年成为一种强有力的人类病原体。分泌后的病毒颗粒与环境处于稳定状态，可以被视为组装良好的纳米机器，准备感染下一个宿主。SARS-CoV-2的病毒基因组和大部分病毒蛋白隐藏在病毒的脂质包膜内，形成直径为130 ± 30 nm的球体。病毒包膜也是刺突蛋白S的多个拷贝的家园，刺突蛋白S具有约160 kD的分子量，并且插入具有单个跨膜螺旋结构域的膜中。包膜也由SARS-CoV-2的M蛋白的多个拷贝支撑，其约为25 kD，具有三重跨膜螺旋结构域。SARS-CoV-2的S、M和包膜一起为病毒颗粒提供了结构完整性，因为颗粒在宿主之间的各种环境中传播。利用RAPID NSF奖，在过去的6个月里，PI已经建立了最小的系统来收获SARS-CoV-2病毒样颗粒（VLP），并确定了S和M蛋白中可以容忍遗传标签而不影响VLP结构的区域。这些VLP具有与完全感染性病毒体相似的形态（以及S和M蛋白含量），但不包装基因组，因此不具有感染性。PI将研究这种病毒病原体的机制。他们将增加代表性不足的群体在犹他州大学追求基础科学的参与，并为大学的跨学科培训创造机会。PI还将培训学生并与更广泛的公众进行科学交流。PI的初步数据显示，S蛋白可以脱落并重新插入病毒包膜，这与其他包膜病毒的其他跨膜糖蛋白的已知行为形成强烈对比。SARS-CoV-2 S蛋白是免疫系统识别的主要抗原。因此，S蛋白的脱落与COVID-19疾病的进展相关;然而，需要详细了解S蛋白脱落/重新插入的基本物理学和随机热力学观点，因为在其他包膜病毒中没有类似的行为模型。为了阐明S蛋白脱落/重新插入的机制及其对病毒感染的意义，PI将：（1）测量单个固定化SARS-CoV-2 VLP中S蛋白脱落和重新插入的动力学。S蛋白的生物活性形式是蛋白质三聚体。他们将利用单分子荧光以及力谱技术来建立S蛋白脱落/重新插入动力学。此外，PI将研究S蛋白脱落-重新插入对三聚体形成的影响，并建立促进和抑制S蛋白脱落的化学和物理因素。(2)模拟S蛋白平衡对病毒生命周期的影响。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

SARS-CoV-2 is the causative agent of COVID-19, which has emerged as a potent human pathogen in 2019. The virus particles after secretion are in steady state with their environment and can be viewed as well assembled nano-machines ready to infect their next host. The viral genome and most of the viral proteins of SARS-CoV-2 are hidden within the lipid envelope of the virus, which forms a sphere of diameter 130 ± 30 nm. The viral envelope is also home to multiple copies of the spike protein S, which has a molecular weight of ~160kD and is inserted in the membrane with a single membrane spanning helical domain. The envelope is also underpinned by multiple copies of the M protein of SARS-CoV-2, which is approximately 25kD with triple membrane spanning helical domains. Together the S, M and the envelope of SARS-CoV-2 provide structural integrity for the virus particles as the particles travel through various environments between hosts. Utilizing a RAPID NSF award, over the past 6 months the PIs have established the minimal system to harvest SARS-CoV-2 virus like particles (VLPs) and identified regions in S and M proteins where genetic tags can be tolerated without effecting VLP structures. These VLPs have similar morphology (as well as S and M protein content) as fully infectious virions, but do not package the genome and therefore are not infectious. The PIs will study the mechanics of this viral pathogen. They will increase the participation of underrepresented groups in the pursuit of basic science at University of Utah and create opportunities for cross-disciplinary training at the university. The PIs will also train students and communicate science with the broader public.The PIs preliminary data show that the S protein can shed and re-insert back into the viral envelope, in strong contrast with known behavior of other envelope-spanning glycoproteins from other enveloped viruses. The SARS-CoV-2 S protein is a major antigen recognized by the immune system. Shedding of the S protein is therefore relevant for progression of COVID-19 disease; however the basic physics and stochastic thermodynamics view of the S protein shedding/reinsertion needs to be understood in detail since there are no parallel models of such behavior among other enveloped viruses. To elucidate the mechanism of S protein shedding/reinsertion and its implication for viral infection the PIs will: (1) Measure the S protein shedding and re-insertion dynamics in single immobilized SARS-CoV-2 VLPs. The biologically active form of the S protein is a protein trimer. They will utilize single molecule fluorescence as well as force spectroscopy techniques to establish the S protein shedding/re-insertion dynamics. In addition the PIs will investigate the effects of S protein shedding-re-insertion on formation of trimers and also establish chemical and physical factors which promote and inhibit S protein shedding. (2) Model the effect of the S protein equilibrium on the viral life cycle.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.