Investigating the molecular mechanisms of membrane remodeling by coronaviruses

研究冠状病毒膜重塑的分子机制

• 批准号: 10724399
• 负责人: Michael Joseph Ragusa
• 金额: $ 20.44万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-16 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10724399
• 关键词: 2019-nCoV; Amino Acids; Antiviral Agents; Appearance; Binding; Biochemical; Biological Assay; Cell membrane; Cells; Cessation of life; Complex; Confocal Microscopy; Coronavirus; Coronavirus Infections; Cryo-electron tomography; Cryoelectron Microscopy; Detergents; Development; Drug Targeting; Endoplasmic Reticulum; Fluorescence Spectroscopy; Freezing; Future; Genetic Transcription; Healthcare Systems; Human; In Vitro; Infection; Integral Membrane Protein; Investigation; Length; Liposomes; Membrane; Membrane Structure and Function; Middle East Respiratory Syndrome Coronavirus; Molecular; Nonstructural Protein; Population; Positioning Attribute; Process; Production; Protein Region; Proteins; RNA Transport; RNA Viruses; Resolution; Respiratory Tract Infections; Role; SARS coronavirus; Shapes; Shelter facility; Site; Social Distance; Structure; Surface; Testing; Transmembrane Domain; Vaccines; Vesicle; Viral Nonstructural Proteins; Virus; Virus Replication; Work; experimental study; fighting; human pathogen; insight; membrane model; membrane reconstitution; mortality; novel; novel coronavirus; pandemic disease; particle; protein purification; protein structure; reconstitution; screening; success; tool; transmission process; unilamellar vesicle; viral RNA

Coronaviruses are enveloped positive-sense RNA viruses. Over the last two decades, coronaviruses have led to severe respiratory infections in humans. Most recently, SARS-CoV-2 led to a global pandemic and resulted in more than 6.5 million deaths globally since December 2019. We currently lack a sufficiently broad set of antiviral drugs targeting different aspects of coronavirus replication. Therefore, developing new antiviral drugs targeting currently untargeted aspects of coronavirus replication may help reduce the mortality of future coronavirus infections. As such, it is critical to understand the molecular mechanisms of many different aspects of coronavirus replication as this will help to determine which aspects of viral replication may be useful targets for the development of new antiviral drugs. One aspect of coronavirus replication that is not well understood is the mechanisms by which coronaviruses remodel host cell membranes. Once coronaviruses infect host cells, a set of nonstructural proteins (nsps) are produced from the viral RNA. Three of these nsps, nsp3, nsp4 and nsp6, are integral membrane proteins that remodel host cell membranes to generate double-membrane vesicles (DMVs) from the endoplasmic reticulum (ER). These DMVs serve as the assembly sites for the replication and transcription complexes that are critical to producing viral RNA. In addition, DMVs have been shown to contain viral RNA further highlighting the critical role of DMVs in viral RNA production. While it is clear that membrane remodeling by coronaviruses is essential for their replication, we currently lack an understanding of the molecular mechanisms by which coronaviruses remodel host cell membranes to generate DMVs. One major reason for our limited understanding of this process, is that no studies have investigated the structure and function of the membrane-spanning regions of nsp3, nsp4 and nsp6 using purified proteins. As such, we will purify nsp3, nsp4 and nsp6 for structural studies using cryo-EM and for biochemical investigations using model membranes including liposomes and giant unilamellar vesicles. Importantly, this will work will not only provide new insight into the mechanisms of coronavirus replication, but it will also help reveal if membrane remodeling by coronaviruses may be a useful target for the development of future antiviral drugs.

冠状病毒是有包膜的正义RNA病毒。在过去的二十年里，冠状病毒已经导致了人类严重的呼吸道感染。最近，SARS-CoV-2导致了一场全球大流行，自2019年12月以来，全球已有超过650万人死亡。我们目前缺乏针对冠状病毒复制不同方面的足够广泛的抗病毒药物。因此，开发针对目前冠状病毒复制的非靶向方面的新的抗病毒药物可能有助于降低未来冠状病毒感染的死亡率。因此，了解冠状病毒复制的许多不同方面的分子机制至关重要，因为这将有助于确定病毒复制的哪些方面可能是开发新的抗病毒药物的有用靶点。冠状病毒复制的一个不太清楚的方面是冠状病毒重塑宿主细胞膜的机制。冠状病毒一旦感染宿主细胞，病毒RNA就会产生一系列非结构蛋白(NSP)。其中三个NSP，NSP3，NSP4和NSP6，是完整的膜蛋白，可以重塑宿主细胞膜，从内质网(ER)产生双膜小泡(DMV)。这些DMV是复制和转录复合体的组装部位，而复制和转录复合体是产生病毒RNA的关键。此外，DMV已被证明含有病毒RNA，进一步强调了DMV在病毒RNA生产中的关键作用。虽然冠状病毒的膜重塑对于它们的复制是至关重要的，但目前我们对冠状病毒重塑宿主细胞膜以产生DMV的分子机制缺乏了解。我们对这一过程了解有限的一个主要原因是，还没有研究利用纯化的蛋白来研究nsp3、nsp4和nsp6的跨膜区域的结构和功能。因此，我们将提纯NSP3、NSP4和NSP6，以便使用冷冻EM进行结构研究，并使用包括脂质体和巨大单层囊泡在内的模型膜进行生化研究。重要的是，这项工作不仅将为冠状病毒复制的机制提供新的见解，而且还将有助于揭示冠状病毒的膜重构是否可能是未来抗病毒药物开发的有用靶点。