Small GTP Binding Proteins in Gastrointestinal Mucosa

项目总结 所有冠状病毒，包括小鼠肝炎病毒、SARS、MERS SARS-CoV-2和猪疫情 腹泻病毒(PEDV)是由多种蛋白质组分组成。所有这些冠状病毒都利用 糖基化的M蛋白作为病毒在感染细胞内组装的核心。虽然许多冠状病毒 感染与严重的肺部疾病有关，它们还会导致一系列胃肠道异常， 尤其是拉肚子。通过酵母双杂交筛选，我们已经鉴定了细胞质的相互作用 MHV M蛋白与肌球蛋白VB(MYO5B)的尾部。在过去的20年里，我们研究了监管 Rab11a及其相互作用蛋白MYO5B的顶膜循环系统 Rab11家族相互作用蛋白(Rab11-FIP)。我们之前已经证明了MYO5B可以相互作用 具有多个Rab小GTP酶，包括Rab11a、Rab11b、Rab25、Rab8a和Rab10。MHV M蛋白 与MYO5B(外显子D)中的一个选择性剪接外显子相互作用，该外显子编码我们拥有的序列 先前显示与Rab10结合。MYO5B D与MHV M蛋白Rab10和Rab11a共定位 在极化的上皮细胞中表达。MYO5B D还与PEDV共表达的M蛋白共定位， MERS和SARS-CoV-2。缺失外显子D的MYO5B(MYO5B-D)不与冠状病毒M蛋白结合。一个 MHV M细胞质尾部的随机突变确定了与MHV M相互作用的点突变 MYO5B外显子D其中一个突变，MHV M(E121K)，以前曾被报道阻止病毒组装。 所有这些发现都使我们推测，冠状病毒M蛋白与MYO5B D的关联 是M蛋白通过心尖循环系统和病毒组装进行运输的关键一步。评估 这一假设，我们将追求3个具体目标：第一，我们将定义MYO5B外显子D的结构基础 与M蛋白和Rab10的相关性。我们将利用酵母双杂交筛选来确定外显子D的残基 负责MHV M与Rab10的结合。此外，我们将使用裂解泛素酵母两个- 杂交筛选评估其他冠状病毒M蛋白与MYO5B外显子D的相关性 确定冠状病毒M蛋白与循环系统成分的关联。我们将利用 研究MHV M和其他病毒利用的转运途径的分化的人肠道肠样 冠状病毒M蛋白通过质膜循环系统。我们将检查MHV和 冠状病毒样颗粒在极化的上皮细胞中聚集。第三，我们将针对M蛋白相互作用 通过检测MHV M蛋白最小结合MYO5B外显子ABCDE结构域的表达能力 为了破坏M蛋白和全长MYO5B D之间的相互作用。此外，我们将执行一个小的 分子筛查可破坏MHV M胞浆尾巴与MHV M相互作用的化合物 MYO5B(ABCDE)这些研究将建立破坏冠状病毒组装的共同途径 极化的上皮细胞。

PROJECT SUMMARY All coronaviruses including Mouse hepatitis virus (MHV), SARS, MERS SARS-CoV-2 and Porcine Epidemic Diarrhea Virus (PEDV), are assembled from multiple protein components. All of these coronaviruses utilize a glycosylated M protein as the nidus for assembly of viruses within infected cells. While many coronavirus infections have been linked to severe lung disease, they also cause a range of gastrointestinal abnormalities, especially diarrhea. Through a yeast two-hybrid screen, we have identified the interaction of the cytoplasmic tail of the MHV M protein with Myosin Vb (MYO5B). Over the past 20 years, we have studied the regulation of the apical membrane recycling system by Rab11a and its associated interacting proteins MYO5B and the Rab11 Family Interacting Proteins (Rab11-FIPs). We have previously demonstrated that MYO5B can interact with multiple Rab small GTPases, including Rab11a, Rab11b, Rab25, Rab8a and Rab10. MHV M protein interacts with an alternatively spliced exon in MYO5B (Exon D), which codes for a sequence that we have previously shown to bind Rab10. MYO5B+D co-localizes with MHV M protein, Rab10 and Rab11a, when co- expressed in polarized epithelial cells. MYO5B+D also co-localized with co-expressed M proteins from PEDV, MERS and SARS-CoV-2. MYO5B lacking Exon D (MYO5B-D) did not localize with coronavirus M proteins. A random mutagenesis of the MHV M cytoplasmic tail identified point mutations that abrogate interactions with MYO5B Exon D. One these mutations, MHV M(E121K), has previously been reported to block viral assembly. All of these findings have led us to hypothesize that the association of coronavirus M proteins with MYO5B+D is a critical step in M protein trafficking through the apical recycling system and virus assembly. To evaluate this hypothesis, we will pursue 3 specific aims: First, we will define the structural basis of MYO5B exon D association with M proteins and Rab10. We will utilize yeast 2-hybrid screening to define residues in Exon D that are responsible for binding of MHV M versus Rab10. Additionally, we will use split-ubiquitin yeast two- hybrid screening to evaluate association of other coronavirus M proteins with MYO5B Exon D. Second, we will determine the association of coronavirus M proteins with elements of the recycling system. We will utilize differentiated human intestinal enteroids to examine the trafficking pathway utilized by MHV M and other coronavirus M proteins through the plasma membrane recycling system. We will examine MHV and coronavirus virus like particle assembly in polarized epithelial cells. Third, we will target M protein interaction with MHV M proteins by testing the ability of expression of the minimal binding MYO5B Exons ABCDE domain to disrupt interactions between M proteins and full length MYO5B+D. Additionally, we will perform a small molecule screen to identify compounds that can disrupt the interaction of MHV M cytosolic tail and MYO5B(ABCDE). These studies will establish common pathways for disrupting coronavirus assembly in polarized epithelial cells.