Coronavirus M Protein Trafficking in Epithelial Cells Utilizes a Myosin Vb Splice Variant and Rab10.

Area of Science:

• Virology
• Cell Biology
• Molecular Biology

Background:

• The membrane (M) glycoprotein is essential for coronavirus (CoV) virion assembly.
• Understanding M protein interactions is key to deciphering viral replication mechanisms.

Purpose of the Study:

• To investigate the interaction between the M protein of Murine Hepatitis Virus (MHV-CoV) and cellular proteins.
• To determine the role of Myosin Vb (MYO5B) and Rab GTPases in CoV M protein trafficking.

Main Methods:

• Yeast two-hybrid screening to identify interacting partners of the MHV-CoV M protein cytosolic tail.
• Co-expression of viral M proteins and MYO5B+D in human and canine epithelial cell lines.
• Confocal microscopy to assess co-localization of M proteins, MYO5B+D, Rab10, and Rab11a.
• Site-directed mutagenesis to identify critical residues for M protein-MYO5B+D interaction.
• Rab10 knockdown and rescue experiments.

Main Results:

• The cytosolic tail of MHV-CoV M protein interacts with Myosin Vb (MYO5B), specifically the MYO5B+D splice variant.
• MYO5B+D co-localizes with M proteins from various CoVs (MHV, PEDV, MERS-CoV, SARS-CoV-2) and with endogenous Rab10 and Rab11a.
• Specific point mutations in the M protein, including E121K in MHV-CoV and homologous mutations in other CoVs, abolish MYO5B+D interaction and co-localization.
• Rab10 knockdown disrupts M protein-MYO5B+D co-localization, which can be rescued by Rab10 re-expression.

Conclusions:

• Coronaviruses M proteins utilize the MYO5B+D and Rab10 machinery for their intracellular trafficking.
• The identified interaction and trafficking pathway are conserved across different CoVs, including SARS-CoV-2.
• Targeting this M protein-host interaction pathway could offer a strategy for antiviral development.