Abstract
M is the most abundant structural membrane protein in coronaviruses and is essential for the formation of infectious virus particles. SARS-CoV-2 M adopts two conformations, M(short) and M(long), and regulated transition between states is hypothesized to coordinate viral assembly and budding. However, the factors that regulate M conformation and roles for each state are unknown. Here, we discover a direct M-sphingolipid interaction that controls M conformational dynamics and virus assembly. We show M binds Golgi-enriched anionic lipids including ceramide-1-phosphate (C1P). Molecular dynamics simulations show C1P interaction promotes a long to short transition and energetically stabilizes M(short). Cryo-EM structures show C1P specifically binds M(short) at a conserved site bridging transmembrane and cytoplasmic regions. Disrupting M(short)-C1P interaction alters M subcellular localization, reduces interaction with Spike and E, and impairs subsequent virus-like particle cell entry. Together, these results show endogenous signaling lipids regulate M structure and support a model in which M(short) is stabilized in the early endomembrane system to organize other structural proteins prior to viral budding.