Coronavirus M proteins disperse the trans-Golgi network and inhibit anterograde protein trafficking in the secretory pathway

Coronaviruses (CoVs) encode Spike, Membrane (M), and Envelope (E) transmembrane proteins that are translated and processed at the endoplasmic reticulum (ER) and traverse the secretory pathway to converge at sites of virus assembly. Three transmembrane ER resident proteins, activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1), and PKR-like endoplasmic reticulum kinase (PERK), sense the accumulation of unfolded proteins in the ER and initiate the unfolded protein response (UPR) to increase ER protein folding capacity. We observed UPR modulation by numerous CoV proteins, including Spike, which broadly activated all three arms of the UPR. By contrast, M selectively inhibited the ATF6 arm of the UPR, either when it was activated by CoV proteins like Spike, or when it was activated by chemical stimuli of ER stress; however, M was unable to inhibit Spike-mediated activation of IRE1 or PERK. ATF6 inhibition was conserved amongst all human CoV M proteins. Amongst the UPR sensors, ATF6 has a unique activation mechanism whereby ER stress triggers translocation to the Golgi where ATF6 is processed by resident proteases to release the ATF6-N bZIP transcription factor. Because M had no effect on the function of the ATF6-N transcription factor, we reasoned that it may act upstream by inhibiting ATF6 trafficking in the secretory pathway. Indeed, we observed that ectopically expressed M inhibited several processes that require ER-to-Golgi transport, including sterol regulatory element binding protein-2 (SREBP2)-mediated activation of sterol responses and stimulator of interferon response cGAMP interactor 1 (STING1)-mediated activation of interferon responses. M also inhibited the secretion of a soluble Gaussia luciferase reporter protein. Using a Retention Using Selective Hooks (RUSH) cargo sorting assay, we observed that M accumulated in the cis-Golgi and inhibited further anterograde transport of a transmembrane reporter protein beyond this compartment, while dispersing the trans-Golgi network (TGN). We also observed a conserved TGN dispersal phenotype in cells infected with SARS-CoV-2, hCoV-OC43, or hCoV-229E. We determined that M is present in both detergent resistant and detergent soluble membranes and that M increased cholesterol abundance at the cis-Golgi. Together, these observations suggest that CoV M proteins disrupt the TGN and impede normal anterograde traffic in the canonical secretory pathway, potentially by increasing cholesterol levels at the cis-Golgi . Because CoV egress does not require the TGN, this mechanism could allow the virus to selectively interfere with host responses to infection without impeding egress of nascent virions. IMPORTANCEMany viruses encode proteins that limit host antiviral responses. Coronaviruses encode a remarkably diverse array of proteins that antagonize host responses at discrete steps including the detection of viral products, signal transduction, host mRNA processing and nuclear export, and protein synthesis. Here, we describe a new form of viral antagonism of host antiviral responses by remodelling the secretory pathway, dispersing a distal portion of the Golgi network and causing accumulation of proteins early in the Golgi. This is achieved by an abundant viral structural transmembrane glycoprotein M, which is best known for its role as the central player in the assembly of new viruses. This newly discovered function of M allows it to limit host antiviral responses that depend on trafficking in the late secretory pathway, while maintaining its role in virus assembly in the early secretory pathway.