Small GTP Binding Proteins in Gastrointestinal Mucosa

项目摘要 所有冠状病毒，包括小鼠肝炎病毒（MHV）、SARS、MERS SARS-CoV-2和猪流行病 腹泻病毒（PEDV）由多种蛋白质组分组装而成。所有这些冠状病毒都利用 糖基化M蛋白作为病毒在感染细胞内组装的巢。虽然许多冠状病毒 感染与严重的肺部疾病有关，它们还导致一系列胃肠道异常， 尤其是腹泻。通过酵母双杂交筛选，我们已经确定了细胞质的相互作用， MHV M蛋白的尾部与肌球蛋白Vb（MYO 5 B）。在过去20年，我们研究了 Rab 11 a及其相关的相互作用蛋白MYO 5 B的顶端膜再循环系统， Rab 11家族相互作用蛋白（Rab 11-FIPs）。我们之前已经证明MYO 5 B可以与 具有多种Rab小GTP酶，包括Rab 11 a、Rab 11b、Rab 25、Rab 8a和Rab 10。MHV M蛋白 与MYO 5 B中的可变剪接外显子（外显子D）相互作用，该外显子编码一个序列， 以前显示结合Rab 10。MYO 5 B +D与MHV M蛋白Rab 10和Rab 11 a共定位， 在极化上皮细胞中表达。MYO 5 B +D还与来自PEDV的共表达的M蛋白共定位， MERS和SARS-CoV-2。MYO 5 B缺乏外显子D（MYO 5 B-D）不定位于冠状病毒M蛋白。一 MHV M胞质尾区的随机诱变鉴定了消除与 MYO 5 B外显子D。这些突变之一，MHV M（E121 K），以前曾报道过阻止病毒组装。 所有这些发现使我们假设冠状病毒M蛋白与MYO 5 B +D的关联 是M蛋白通过顶端再循环系统和病毒装配运输的关键步骤。评价 基于这一假设，我们将追求3个具体目标：首先，我们将确定MYO 5 B外显子D的结构基础 与M蛋白和Rab 10的关联。我们将利用酵母双杂交筛选来确定外显子D中的残基 负责MHV M与Rab 10的结合。此外，我们将使用分裂泛素酵母二- 杂交筛选以评估其他冠状病毒M蛋白与MYO 5 B外显子D的关联。二是 确定冠状病毒M蛋白与再循环系统元素的关联。我们将利用 分化的人肠类肠，以检查MHV M和其他 冠状病毒M蛋白通过质膜回收系统。我们将检查MHV， 极化上皮细胞中的冠状病毒样颗粒组装。第三，我们将靶向M蛋白相互作用 通过检测MYO 5 B外显子ABCDE结构域的最小结合表达能力， 破坏M蛋白和全长MYO 5 B +D之间的相互作用。此外，我们还将举办一个小型的 分子筛选以鉴定可以破坏MHV M胞质尾和 MYO5B（ABCDE）。这些研究将建立破坏冠状病毒组装的共同途径， 极化上皮细胞

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.