(519.2) A Myosin Vb Splice Variant Regulates Coronavirus M Protein Trafficking in Polarized Epithelial Cells

Poster Board Number: A437

James Goldenring (Vanderbilt University Medical Center, Nashville VA Medical Center), Lynne Lapierre (Vanderbilt University Medical Center), Joseph Roland (Vanderbilt University Medical Center), Elizabeth Manning (Vanderbilt University Medical Center), Cathy Caldwell (Vanderbilt University Medical Center), Honor Glenn (Arizona State University), Pierre-Olivier Vidalain (University of Lyon), Frederic Tangy (Institut Pasteur), Brenda Hogue (Arizona State University), C.A.M. de Haan (University of Utrecht)

The membrane (M) glycoprotein of coronaviruses (CoVs) serves as the nidus for virion assembly.  Using a yeast two-hybrid screen, we identified the interaction of the cytosolic tail of Murine Hepatitis Virus (MHV-CoV) M protein with Myosin Vb (MYO5B) (Figure 1A).  MHV-CoV M interacts specifically with the alternative splice variant of cellular MYO5B including Exon D (MYO5B+D), that also mediates interaction with cellular Rab10.  When co-expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co-localized with MHV-CoV M protein, as well as with M proteins from porcine epidemic diarrhea Virus (PEDV-CoV), Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome 2 (SARS-CoV-2) (Figure 2).  M-GFP chimeric proteins co-expressed with mCherry-MYO5B+D also co-localized with endogenous Rab10 and Rab11a (Figure 1B,C).  We identified point mutations in MHV-CoV M that blocked the interaction with MYO5B+D in yeast 2-hybrid assays (Figure 1B).  One of these point mutations (E121K) was previously shown to block MHV-CoV virion assembly, and it blocked interaction with MYO5B+D.  The E to K mutation at homologous positions in PEDV-CoV, MERS-CoV and SARS-CoV-2 M proteins also blocked colocalization with MYO5B+D.  Knockdown of Rab10 blocked the co-localization of the M proteins with MYO5B+D (Figure 2).  Re-expression of Cerulein-Rab10 in Rab10 KD cells re-established colocalization between M proteins and MYO5B+D (Figure 2).  Our results suggest that the interaction of CoV M proteins with MYO5B+D may play a role in regulating their trafficking through Rab10-containing membrane systems in epithelial cells.

National Science Foundation RAPID grant NSF2032016 and R01 DK48370 to JRG.
NSF RAPID:IIBR Award 2032199, NSF Science Technology Center Award 1231306; Mercatus Center Emergent Ventures Fast Grant to B.G.H.





Figure 1: The MHV M cytoplasmic tail interacts with MYO5B+D. A. Yeast 2-hybrid interactions between pAD-Myosin Vb targets and various interacting proteins.  B. Truncation and random mutagenesis reveals requisite binding regions in MHV M tail.  C. Co-expression of MHV M-GFP with Cherry-MYO5B+D or Cherry MYO5-D andwith immunostaining for Rab11a (cyan) and F-actin (blue) in MDCK cells.  D. Co-expression of MHV M-GFP with Cherry MYO5B+D and Rab10 immunostaining and  staining of F-actin . Bar = 5 µm.; Figure 2: Knockdown of Rab10 expression in A549 cells significantly decreases co-localization of Coronavirus M proteins with Cherry-MYO5B+D tail.  A549 lung cells were transfected with MHV M-GFP, PEDV M-GFP, MERS M-GFP or SARS-CoV-2 M-GFP with Cherry-MYO5B+D 902 tail (902T) in non-targeting (NT) wild type cells, Rab10 knockdown (KD) cells and Rab10 knockdown cells with rescue with Cer-Rab10.  3-dimensional Manders’ coefficients were calculated for Z-stack images.   *p < 0.01.  **p < 0.001.