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), specifically with the alternative splice variant of cellular MYO5B including exon D (MYO5B+D), which mediates interaction with Rab10. When co-expressed in human lung epithelial A549 and canine kidney epithelial MDCK cells, MYO5B+D co-localized with the MHV-CoV M protein, as well as with the M proteins from Porcine Epidemic Diarrhea Virus (PEDV-CoV), Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome 2 (SARS-CoV-2). Co-expressed M proteins and MYO5B+D co-localized with endogenous Rab10 and Rab11a. We identified point mutations in MHV-CoV M that blocked the interaction with MYO5B+D in yeast 2-hybrid assays. One of these point mutations (E121K) was previously shown to block MHV-CoV virion assembly and its 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. The knockdown of Rab10 blocked the co-localization of M proteins with MYO5B+D and was rescued by re-expression of CFP-Rab10. Our results suggest that CoV M proteins traffic through Rab10-containing systems, in association with MYO5B+D.
Keywords: M protein; MHV; MYO5B; Myosin Vb; Rab10; Rab11a; coronavirus; membrane recycling.