S-acylation of SARS-CoV-2 Spike Protein: Mechanistic Dissection, In Vitro Reconstitution and Role in Viral Infectivity

Puthenveetil, Robbins; Lun, Cheng Man; Murphy, R. Elliot; Healy, Liam B.; Vilmen, Geraldine; Christenson, Eric T.; Freed, Eric O.; Banerjee, Anirban

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long chain fatty acids get attached to cytosol-facing cysteines. 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 β-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated or the impact of this modification 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 compromises viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate the 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 towards developing future strategies that could aid in the identification of targeted small-molecule modulators.