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

严重急性呼吸综合征冠状病毒2型(CoV-2)属于冠状病毒科的病原性囊膜RNA病毒家族。持续的大流行在世界范围内造成了公共卫生紧急状态，并伴随着可怕的健康和经济后果。有证据表明，与以往的SARS流行株相比，CoV-2的感染率可能相对更高，与受体ACE2的亲和力可能比SARS-1更高。此外，最近出现了新的CoV-2变异株，其变异与更高的感染率和逃避特定免疫的能力有关，引起了重大关注。关于CoV-2可能如何获得了更有效传播的能力，以及新的突变可能如何影响病毒的传染性，知识中的一个主要空白仍然存在。这项提议的首要目标是更好地了解调控CoV-2细胞进入和复制的分子机制，以及新变异的出现如何导致免疫逃逸。我们将重点介绍宿主泛素(Ub)系统在促进CoV-2感染中的作用。这些信息可以帮助预测更易传播的冠状病毒变种的出现，并通过针对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》杂志上发表的数据表明，黄病毒的包膜蛋白是K63连接的多泛素化的，它增强了病毒对宿主细胞受体的附着。因此，我们询问类似的机制是否适用于SARS-CoV-2。我们的初步数据表明，CoV-2结构蛋白在多个赖氨酸残基上泛素化，其中一些在最初的流行CoV株中不保守。此外，CoV-2的新变体已经出现，在这些泛素化位点上发生了突变。我们的数据还表明，SPEKE(S)蛋白的泛素化可能在稳定冠状病毒-2 S-ACE2相互作用中发挥作用，可能导致增强进入和致病。目前尚不清楚冠状病毒家族中是否有任何成员，包括SARS-CoV-2，利用病毒结构蛋白的泛素化作为病毒附着和进入的机制。我们还鉴定了三方基序(TRIM)家族的E3-Ub连接酶，它泛化病毒结构蛋白。我们的一般假设是，获得特定赖氨酸残基的CoV-2变异株在结构蛋白上提供了新的Ub受体位点，这可以促进病毒复制和免疫逃逸。通过使用体外生化方法、新型重组突变病毒和体内模型，我们将评估结构CoV-2蛋白的泛素化如何促进CoV-2的感染性。在目标1中，我们将确定CoV-2 S蛋白泛素化在病毒复制和抗体逃避中的机制作用，在目标2中，我们将确定CoV-2膜蛋白泛素化在病毒复制和干扰素拮抗中的机制作用。这些研究的结果可能有助于解释新的、更具传染性的病毒如何通过获得泛素化位点而出现，并将为开发可应用于广泛包膜病毒的抗病毒方法提供基础。

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.