# Small GTP Binding Proteins in Gastrointestinal Mucosa

> **NIH NIH R01** · VANDERBILT UNIVERSITY MEDICAL CENTER · 2022 · $615,274

## Abstract

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...

## Key facts

- **NIH application ID:** 10450147
- **Project number:** 5R01DK048370-27
- **Recipient organization:** VANDERBILT UNIVERSITY MEDICAL CENTER
- **Principal Investigator:** James Richard Goldenring
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $615,274
- **Award type:** 5
- **Project period:** 1997-08-01 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10450147

## Citation

> US National Institutes of Health, RePORTER application 10450147, Small GTP Binding Proteins in Gastrointestinal Mucosa (5R01DK048370-27). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10450147. Licensed CC0.

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