S-acylation of SARS-CoV-2 Spike Protein: Mechanistic Dissection, In Vitro Reconstitution and Role in Viral Infectivity
| Autor: | R. Elliot Murphy, Robbins Puthenveetil, Geraldine Vilmen, Liam B. Healy, Anirban Banerjee, Eric T. Christenson, Cheng Man Lun, Eric O. Freed |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
viral protein
RNA virus post-translational modification (PTM) Viral protein infectious disease Acylation Lipoylation Biology Spike protein Protein lipidation medicine.disease_cause Biochemistry protein palmitoylation Palmitoylation Viral envelope membrane reconstitution medicine Humans Protein palmitoylation membrane protein Amino Acid Sequence Cysteine Molecular Biology Research Articles Infectivity SARS-CoV-2 S-acylation COVID-19 Cell Biology Virus Internalization Recombinant Proteins Cell biology HEK293 Cells Membrane protein Spike Glycoprotein Coronavirus Mutagenesis Site-Directed Sequence Alignment membrane enzyme Acyltransferases |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|