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

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.