{"doi":"10.1016/j.jbc.2021.101112","title":"S-acylation of SARS-CoV-2 spike protein: Mechanistic dissection, in vitro reconstitution and role in viral infectivity","abstract":"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 β-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. 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 β-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. The Coronavirus disease 2019 (COVID-19) pandemic, the most recent epidemic caused by an outbreak of zoonotic coronaviruses in the past two decades, was preceded closely by MERS in 2012 and SARS in 2003 (1Sharif-Yakan A. Kanj S.S. Emergence of MERS-CoV in the Middle East: Origins, transmission, treatment, and perspectives.PLoS Pathog. 2014; 10e1004457Crossref PubMed Scopus (66) Google Scholar, 2Cherry J.D. The chronology of the 2002-2003 SARS mini pandemic.Paed","journal":"Journal of Biological Chemistry","year":2021,"id":155587,"datarank":2.0800548733334026,"base_score":4.07753744390572,"endowment":4.07753744390572,"self_citation_contribution":0.611630616585858,"citation_network_contribution":1.4684242567475443,"self_endowment_contribution":0.611630616585858,"citer_contribution":1.4684242567475443,"corpus_percentile":null,"corpus_rank":null,"citation_count":58,"citer_count":51,"citers_with_citation_signal":43,"citers_with_endowment":43,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9553,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":658439,"name":"Cheng Man Lun","orcid":"0000-0003-1419-3010","position":1,"is_corresponding":false},{"id":480754,"name":"R. Elliot Murphy","orcid":"0000-0002-4144-2345","position":2,"is_corresponding":false},{"id":658440,"name":"Liam B. Healy","orcid":"0000-0002-2640-1596","position":3,"is_corresponding":false},{"id":659282,"name":"Géraldine Vilmen","orcid":null,"position":4,"is_corresponding":false},{"id":251547,"name":"Eric T. Christenson","orcid":"0000-0001-7463-7694","position":5,"is_corresponding":false},{"id":124089,"name":"Eric O. Freed","orcid":"0000-0003-3345-022X","position":6,"is_corresponding":false},{"id":251551,"name":"Anirban Banerjee","orcid":"0000-0003-1494-6801","position":7,"is_corresponding":false},{"id":513371,"name":"Robbins Puthenveetil","orcid":"0000-0003-4958-6295","position":0,"is_corresponding":true}],"reference_count":47,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-18T23:44:03.750995Z","pmid":"34428449","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}