S-acylation of SARS-CoV-2 spike protein: Mechanistic dissection, in vitro reconstitution and role in viral infectivity.

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long-chain fatty acids get attached to cysteine residues facing the cytosol. In humans, 23 members of the zDHHC family of integral membrane enzymes catalyze this modification. S-acylation is critical for the life cycle of many enveloped viruses. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among known human pathogens in the closely related family of ß-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated, and what the impact of this modification is on viral infectivity. Here we identify specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation. Interestingly, when we investigated the effect of the cysteine clusters using pseudotyped virus, mutation of the same three clusters of cysteines severely compromised viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate SARS-CoV-2 Spike protein. Finally, we reconstituted S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observe a striking heterogeneity in the S-acylation status of the different cysteines in our in cellulo experiments, which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolster our understanding of a poorly understood posttranslational modification integral to the SARS-CoV-2 Spike protein. This study opens up avenues for further mechanistic dissection and lays the groundwork toward developing future strategies that could aid in the identification of targeted small-molecule modulators.

Toward the identification of ZDHHC enzymes required for palmitoylation of viral protein as potential drug targets.

Introduction: S-acylation is the attachment of fatty acids not only to cysteines of cellular, but also of viral proteins. The modification is often crucial for the protein´s function and hence for virus replication. Transfer of fatty acids is mediated by one or several of the 23 members of the ZDHHC family of proteins. Since their genes are linked to various human diseases, they represent drug targets.Areas covered: The authors explore whether targeting acylation of viral proteins might be a strategy to combat viral diseases. Many human pathogens contain S-acylated proteins; the ZDHHCs involved in their acylation are currently identified. Based on the 3D structure of two ZDHHCs, the regulation and the biochemistry of the palmitolyation reaction and the lipid and protein substrate specificities are discussed. The authors then speculate how ZDHHCs might recognize S-acylated membrane proteins of Influenza virus.Expert opinion: Although many viral diseases can now be treated, the available drugs bind to viral proteins that rapidly mutate and become resistant. To develop inhibitors for the genetically more stable cellular ZDHHCs, their binding sites for viral substrates need to be identified. If only a few cellular proteins are recognized by the same binding site, development of specific inhibitors may have therapeutic potential.