S-acylation of SARS-CoV-2 spike protein: Mechanistic dissection,in vitro reconstitution and role in viral infectivity |
| |
Authors: | Robbins Puthenveetil Cheng Man Lun R Elliot Murphy Liam B Healy Geraldine Vilmen Eric T Christenson Eric O Freed Anirban Banerjee |
| |
Institution: | 1.Section on Structural and Chemical Biology of Membrane Proteins, Neurosciences and Cellular and Structural Biology Division, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA;2.Virus-Cell Interaction Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland, USA |
| |
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. |
| |
Keywords: | SARS-CoV-2 spike protein posttranslational modification (PTM) membrane enzyme in vitro reconstitution membrane protein infectious disease viral protein zDHHC enzyme protein palmitoylation |
|
|