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 wil...