A better understanding of coronavirus biology can enable development of new antivirals to help stop COVID-19 and prevent future pandemics. In recent work, Puthenveetil et al. characterized S-acylation of the Spike (S) protein of SARS-CoV-2. This post-translational modification is known to be important to the viral replication cycle of other viruses across multiple virus families but has not been studied in SARS-CoV-2.
To control for gel loading and S protein expression in the S-acylation experiments, the authors used the Azure Sapphire Biomolecular Imager to detect GAPDH by NIR fluorescence and SARS-CoV-2 S protein by chemiluminescence on Western blots (Figure 1).
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The authors expressed S protein in cultured cells and carried out metabolic labeling with a fatty acid that was detected using a rhodamine-labeled fluorescent probe. They found substantial S-acylation that was blocked by 2BP, a global inhibitor of S-acylation. The authors also found the S-acylation of the S protein was dependent on the presence of the cysteines in the C-terminal domain.
Further experiments expressing mutated versions of the S protein identified which cysteines were S-acylated, the effect of mutating these cysteines on particle infectivity, and which members of the human family of enzymes that carry out S-acylation were able to modify the S protein in cells and in an in vitro assay.
What is S-acylation?
S-acylation involves adding long-chain fatty acids to cysteine residues on the cytosolic side of transmembrane proteins. The cytoplasmic tail of the SARS-CoV-2 S protein contains 10 cysteines in 6 potential S-acylation sites. Puthenveetil et al note that all but one of these are conserved with SARS-CoV, and most are conserved with other coronaviruses that infect humans, including MERS. Still, nothing is known about what role, if any, the S-acylation of these cysteines may play in the biology of viruses, such as SARS-COV-2.