RAPID: Interaction of the Coronavirus M protein with a Myosin V motor protein

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The COVID-19 coronavirus represents one of the most acute and dangerous threat to global health in a century. The present COVID-19 outbreak is caused by the SARS-CoV-2 virus and is exacerbated by our present inability to inhibit the assembly of virus inside cells, which affects the spread of coronavirus within human patients. SARS-Cov-2 is a member of a family of related coronaviruses that includes SARS, MERS, and Mouse Hepatitis Virus (MHV). These are RNA viruses wrapped in a lipid membrane, which has a number of different proteins embedded in it. In general however, relatively little is known about how viral proteins are packaged together inside human cells to assemble coronavirus particles. This lack of knowledge impedes the development of strategies that could target the assembly of virus particles inside human cells. However, one membrane protein encoded by the virus, designated the M protein because of its membrane association, serves as the starting seed for the assembly of all the other coronavirus proteins. It has recently been discovered that the tail of the MHV version of the M protein can interact with a motor protein used for trafficking inside human cells. Because the sequence of the M-protein tails in this class of coronaviruses is very conserved, this project seeks to determine if this interaction is generalizable among SARS-CoV-2, and other related coronaviruses. If this interaction is present in SARS-CoV-2, then this project will identify the parts of the M protein that interact with the myosin motor. This would allow future research to evaluate drugs that can interrupt this interaction, which may alter the ability of the SARS-CoV-2 and other coronaviruses to assemble and spread. As a Broader Impact, the Project will provide critically needed information for fight the Covid-19 pandemic. Relatively little is known about the processes required for the assembly of coronavirus virions in human cells. The family of beta-coronaviruses, including SARS-CoV-2, SARS, MERS and MHV all utilize a similar compendium of proteins, including a membrane glycoprotein protein, termed the M protein. M protein serves as the nidus for assembly of other coronavirus proteins into the mature virus particle. This project seeks to expand on the recent finding that the M-protein of MHV interacts with a specific 26 amino acid alternatively spliced exon of Myosin Vb (MYO5B), termed exon D. This observation is the first to identify a key intracellular trafficking protein that interacts with the M protein. This project seeks to identify whether this interaction is preserved in the M proteins of other beta-coronavirus family members, including the SARS-CoV-2 virus. The Project will also determine the molecular basis of the interaction between M protein and Exon D of MYO5B. The Project will first utilize yeast 2-hybrid assays to evaluate the binding of the internal tails of SARS-CoV-2 (COVID-19), SARS and MERS and other related coronaviruses. Second, random mutagenesis of the coronavirus tails will be utilized to determine the common amino acid motifs used for interaction with MYO5B Exon D. Third, wild type and mutant coronavirus proteins will be expressed in HeLa cells, and mutations that block interactions with MYO5B on M protein will be evaluated for their effects on trafficking through the endoplasmic reticulum, the Golgi apparatus and to the plasma membrane. If verified with the SARS-CoV-2 M protein, the site of interaction with MYO5b Exon D could be utilized as a target for disruption of the assembly of coronavirus virions. This RAPID Project is expected to provide insights for the future development of molecular strategies to disrupt virion assembly in host cells, which could lead to new therapeutics for the treatment of Covid-19.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.