The Role of the Host Ubiquitin System in Promoting SARS-CoV-2 Replication and Pathogenesis

宿主泛素系统在促进 SARS-CoV-2 复制和发病机制中的作用

• 批准号: 10345011
• 负责人: Ricardo Rajsbaum
• 金额: $ 25.68万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-21 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10345011
• 关键词: 2019-nCoV; ACE2; Affect; Affinity; Antibodies; Antiviral Agents; Appearance; Binding; Biochemical; Bioinformatics; Biological Assay; Blocking Antibodies; COVID-19; Cells; Cessation of life; Clinical Trials; Co-Immunoprecipitations; Coronavirus; Data; Development; Disease; Epidemic; Family; Flaviviridae; Flavivirus; Future; Goals; Human; Immune; Immune Evasion; Immunity; Impairment; In Vitro; Infection; Integration Host Factors; Interferons; Knowledge; Lead; Link; Lung diseases; Lysine; Mass Spectrum Analysis; Mediating; Membrane Proteins; Molecular; Mouse Cell Line; Mutagenesis; Mutation; Names; Nature; Outcome; Outcome Study; Pathogenesis; Pathogenicity; Pharmacology; Play; Process; Protein Family; Proteins; Publishing; RNA Viruses; Receptor Cell; Recombinants; Reporting; Role; Severe Acute Respiratory Syndrome; Site; Structural Protein; Structure; System; TRIM Motif; Testing; Therapeutic Intervention; Time; Ubiquitin; Ubiquitination; Vaccines; Variant; Viral; Viral Matrix Proteins; Viral Packaging; Viral Structural Proteins; Virion; Virus; Virus Receptors; Virus Replication; biosafety level 3 facility; env Gene Products; health economics; in vivo; in vivo Model; infection rate; member; multiple myeloma M Protein; mutant; neutralizing antibody; novel; novel coronavirus; pandemic disease; pathogenic virus; public health emergency; receptor; targeted treatment; transmission process; ubiquitin-protein ligase; virus envelope

The Severe Acute Respiratory Syndrome Coronavirus 2 (CoV-2) belongs to a family of pathogenic enveloped RNA viruses of the family Coronaviridae. The ongoing pandemic has caused a public health emergency worldwide, accompanied by dire health and economic consequences. There is evidence suggesting that CoV-2 may have relatively higher infection rates compared to previous epidemic strains of SARS and higher affinity to the receptor ACE2 than SARS-1. In addition, new CoV-2 variants have appeared recently with mutations that correlate with higher infection rates and the ability to escape specific immunity, causing major concerns. A major gap in knowledge remains as to how CoV-2 may have acquired the ability to spread more efficiently, and how new mutations may affect virus infectivity. The overarching goal of this proposal is to better understand the molecular mechanisms that regulate CoV-2 cell entry and replication, and how the appearance of new variants could lead to immune escape. We will focus on the role of the host Ubiquitin (Ub) system in promoting CoV-2 infection. This information could help predict appearance of more transmissible variants of coronaviruses, and to develop antiviral approaches by targeting specific steps of the ubiquitination process. Our data recently published in Nature, show that the envelope protein of flaviviruses is K63-linked polyubiquitinated, which enhances virus attachment to host cell receptors. Therefore, we asked whether a similar mechanism applies to SARS-CoV-2. Our preliminary data indicate that CoV-2 structural proteins are ubiquitinated on multiple lysine residue, some of which are not conserved in the original epidemic CoV strain. In addition, new variants of CoV-2 have appeared with mutations on these ubiquitination sites. Our data also suggest that ubiquitination of Spike (S) protein may play a role in stabilizing the CoV-2 S-ACE2 interaction, potentially leading to enhanced entry and pathogenesis. It is currently unknown whether any member of the Coronaviridae family, including SARS-CoV-2, utilize ubiquitination of viral structural proteins as a mechanism of virus attachment and entry. We have also identified E3-Ub ligases of the Tripartite Motif (TRIM) family of proteins, which ubiquitinates viral structural proteins. Our general hypothesis is that the variants of CoV-2 that have gained specific lysine residues provide new Ub acceptor sites on structural proteins, which can enhance virus replication and immune escape. By using in vitro biochemical approaches, novel recombinant mutant viruses, and in vivo models, we will assess how ubiquitination of structural CoV-2 proteins contribute to CoV-2 infectivity. In Aim 1 we will determine the mechanistic role of ubiquitination of the CoV-2 S protein in virus replication and antibody escape, and in Aim 2 we will determine the mechanistic role of ubiquitination of the CoV-2 Membrane protein in virus replication and IFN antagonism. The outcome of these studies may help explain how new more infectious viruses may appear by gaining ubiquitination sites and will provide the basis for the development of an antiviral approach that could be applied to a broad range of enveloped viruses.

严重的急性呼吸道综合征冠状病毒2（COV-2）属于家族冠状病毒的致病性RNA病毒家族。正在进行的大流行在全球范围内引起了公共卫生紧急情况，并伴随着严重的健康和经济后果。有证据表明，与先前的SARS流行病相比，COV-2的感染率可能比SARS-1更高，对受体ACE2的亲和力更高。此外，最近出现了新的COV-2变体，其突变与较高的感染率和逃避特定免疫力的能力相关，引起了重大关注。知识的主要差距仍然是关于COV-2如何获得更有效传播的能力以及新突变如何影响病毒感染的能力。该提案的总体目标是更好地了解调节COV-2细胞进入和复制的分子机制，以及新变体的外观如何导致免疫逃生。我们将重点关注宿主泛素（UB）系统在促进COV-2感染中的作用。该信息可以帮助预测冠状病毒的更可传播变体的外观，并通过靶向泛素化过程的特定步骤来开发抗病毒方法。我们最近在自然界中发表的数据表明，黄素的包膜蛋白是K63连接的多泛素化的，它增强了病毒附着在宿主细胞受体上。因此，我们询问是否适用于SARS-COV-2。我们的初步数据表明，COV-2结构蛋白在多种赖氨酸残基上被泛素化，其中一些在原始流行病COV菌株中并不保守。此外，在这些泛素化位点上，COV-2的新变体出现了突变。我们的数据还表明，尖峰蛋白的泛素化可能在稳定COV-2 S-ACE2相互作用方面发挥作用，可能导致进入和发病机理的增强。目前，尚不清楚包括SARS-COV-2在内的冠状科家族的任何成员是否利用病毒结构蛋白作为病毒附着和进入的机制。我们还鉴定了三方基序（Trim）蛋白质的E3-UB连接酶，该蛋白会泛素化病毒结构蛋白。我们的一般假设是，获得特定赖氨酸残基的COV-2变体在结构蛋白上提供了新的UB受体位点，从而可以增强病毒复制和免疫逃生。通过使用体外生化方法，新型的重组突变病毒和体内模型，我们将评估结构COV-2蛋白的泛素化如何有助于COV-2感染性。在AIM 1中，我们将确定COV-2 S蛋白在病毒复制和抗体逃逸中泛素化的机械作用，并且在AIM 2中，我们将确定COV-2膜蛋白在病毒重复和IFN拮抗作用中泛素化的机械作用。这些研究的结果可能有助于解释如何通过获得泛素化地点来出现新的更具传染性病毒，并将为开发一种可以应用于广泛包围病毒的抗病毒药方法的发展提供基础。