Discerning the mechanisms of cell membrane budding by coronavirus non-structural proteins nsp3, nsp4, and nsp6

人类群体中病毒感染的发生可以追溯到世纪早期，并且从那时起出现了大量的病毒病原体。具体而言，冠状病毒科引起严重急性呼吸综合征（SARS）流行、中东呼吸综合征（MERS）和SARS-CoV-2流行。在宿主细胞中的冠状病毒感染过程中，一组非结构蛋白（nsps）对于促进病毒复制至关重要--nsp 3、nsp 4和nsp 6。这些跨膜蛋白被确定为在与其他宿主蛋白结合以形成双膜囊泡（DMV）的内质网（ER）膜变形中发挥积极作用。DMV是重要的复制结构，其通过提供细胞和病毒成分的理想环境同时提供对宿主细胞防御机制的保护而有助于病毒RNA合成。尽管由nsp蛋白诱导的DMV结构的形成在冠状病毒和SARS流行病中是保守的，但可追溯到十年前，控制nsp诱导的DMV形成的基本生化和生物物理机制仍然知之甚少。因此，我们建议使用重建的仿生膜系统和SARS-CoV-2衍生的nsp蛋白和肽来研究DMV形成的生物化学和生物力学方面。这可以通过确定生物物理特性和相互作用动力学的脂质膜结合到nsps时如何改变，通过广泛的生物物理和成像方法来实现。该项目的结果将大大加深我们对nsp驱动的冠状病毒复制的分子机制的理解。

The occurrence of viral infections in human populations dates to the early 20th century and a large variety of viral pathogens have emerged since. Specifically, Coronaviridae have caused the severe acute respiratory syndrome (SARS) epidemic, the Middle-East respiratory syndrome (MERS), and the SARS-CoV-2 pandemic. Throughout coronaviral infections in host cells, a set of non-structural proteins (nsps) is crucial for facilitating viral replication - nsp3, nsp4, and nsp6. These membrane-spanning proteins were determined to play an active role in deforming the endoplasmic reticulum (ER) membrane in conjunction with other host proteins to form double-membrane vesicles (DMV). DMVs are important replicative structures, which contribute to viral RNA synthesis by providing an ideal environment of cellular and viral constituents while offering protection from host cell defence mechanisms. Despite the formation of DMV structures induced by nsp proteins being conserved in coronaviruses and the SARS epidemic dating back a decade, essential biochemical and biophysical mechanisms governing nsp-induced DMV formation remain poorly understood. Thus, we propose the use of reconstituted biomimetic membrane systems and SARS-CoV-2-derived nsp proteins and peptides to study the biochemical and biomechanical aspects underlying DMV formation. This can be achieved by determining how biophysical properties and interaction dynamics of lipid membranes are altered when bound to nsps, by means of a wide range of biophysical and imaging methods. The results of this project will greatly further our understanding of the molecular mechanisms in nsp-driven coronaviral replication.