Coronavirus M Proteins Inhibit Anterograde Protein Trafficking and Secretion

Abstract

Coronaviruses (CoVs) encode transmembrane proteins, such as Spike, Membrane (M) and Envelope (E), which are translated at the endoplasmic reticulum (ER) and traverse the secretory pathway to converge at sites of virus assembly. Three ER resident proteins, activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and PKR-like ER kinase (PERK), sense accumulation of unfolded proteins in the ER and initiate transcriptional responses, a process known as the unfolded protein response (UPR). I observed UPR modulation by numerous SARS-CoV-2 proteins including Spike, which activated all three arms of the UPR. By contrast, M inhibited ATF6 but was unable to inhibit activation of IRE1 or PERK. ATF6 has a unique activation mechanism whereby ER stress triggers translocation to the Golgi where ATF6 is processed by proteases to release the transcription factor ATF6-N. M did not inhibit ATF6-N activity; therefore, I reasoned M may inhibit ATF6 trafficking at a step that precedes protease cleavage and release of ATF6-N. Indeed, I observed that ectopically expressed M inhibited several proteins requiring ER-to-Golgi transport, including sterol regulatory element binding protein-2 (SREBP2) and stimulator of interferon genes (STING). M also inhibited the secretion of a soluble Gaussia luciferase protein. Using a cargo sorting assay, I observed M accumulated in the cis-Golgi and inhibited anterograde transport of a transmembrane reporter protein beyond this compartment, while dispersing the trans-Golgi network (TGN). Further investigation determined M co-localizes with cholesterol, suggesting M overexpression alters cholesterol levels at the cis-Golgi, which is known to inhibit protein trafficking from the Golgi. Together, these observations suggest CoV M proteins disrupt anterograde trafficking in the canonical secretory pathway, potentially by directly binding cholesterol causing an imbalance in Golgi membranes. This work identifies a novel role for CoV M proteins, whereby inhibition of protein trafficking inhibits several intracellular signalling pathways and cellular secretion, implicating a larger role for M during CoV replication beyond virion assembly and structure.