Targeting sphingolipid metabolism: inhibition of neutral sphingomyelinase 2 impairs coronaviral replication organelle formation.

Many plus-strand RNA viruses, including coronaviruses, orchestrate the formation of specialized replication organelles in the cytoplasm of infected cells by specific membrane-associated viral nonstructural proteins (nsp's). This process involves a massive reorganization of intracellular membrane compartments and is thought to depend on a specific set of cellular enzymes that synthesize or modify cellular lipids. Here, we investigated the potential role(s) of specific sphingolipids and related enzymes in coronavirus replication. We observed in cells infected with three different coronaviruses a comparable increase in global ceramide levels, whereas sphingomyelin levels were reduced, suggesting an enhanced conversion of sphingomyelin to ceramide by sphingomyelinases. To test this hypothesis, we targeted cellular sphingomyelinases using pharmacological and genetic approaches. The data obtained in these experiments led us to conclude that neutral sphingomyelinase 2 plays a critical role in an early (but not the entry) step of coronavirus replication in Huh-7-ACE2 cells. Furthermore, neutral sphingomyelinase 2 inhibited the formation of viral replication organelles, indicating the involvement of ceramide in the formation of these membrane structures. We also confirmed colocalization of neutral sphingomyelinase 2 and ceramide, but not sphingomyelin, with replication organelles that were produced in coronavirus-infected cells or induced by co-expression of coronavirus nsp3 and nsp4. A colocalization of ceramides with coronaviral replication organelles could be consistently demonstrated for different epithelial cell systems using genetically diverse coronaviruses, describing potential cell- and virus-type independence. IMPORTANCE: Coronaviruses are enveloped plus-strand RNA viruses with a broad host range, including humans. Human coronaviruses are not only associated with endemic, mild upper respiratory tract infections but also have pandemic potential and can be associated with a severe disease burden. The recent SARS-CoV-2 pandemic especially highlighted the urgent need to identify ideal broad-spectrum and host-targeted antiviral strategies. Since lipids are involved in every step of viral replication, we compared changes in the sphingolipid metabolism of cells infected with three different coronaviruses to identify similarities and related cellular enzymes involved in facilitating viral replication. We observed increased ceramide levels while sphingomyelin levels decreased, suggesting enhanced sphingomyelin-to-ceramide conversion by cellular sphingomyelinases upon infection. Impairment of neutral sphingomyelinase 2 reduced viral replication and the formation of virus-induced membranous replication organelles. Furthermore, we found that neutral sphingomyelinase 2 and its product ceramide were associated with viral replication organelles. Ceramides consistently appeared to be integral lipid building blocks of replication organelles across different human pathogenic coronaviruses and cell types. In conclusion, our study provides new insights into novel potentially conserved druggable sphingolipid-related host factors involved in coronaviral replication, offering potential new targets for antiviral therapies against newly emerging coronaviruses.