University of South Florida Study Shows That Palmitoylation Of SARS-CoV-2 Spike Protein Is Essential For Cell Entry And The Formation Of Syncytia

A new study by researchers from University of South Florida-Tampa along with experts from James A Haley Veterans Hospital-Florida and Florida International University-Port St. Lucie have found that palmitoylation of the first five cysteine residues of the C-terminal domain of the SARS-CoV-2 spike protein is essential to SARS-CoV-2, facilitating cell entry and the formation of syncytia.

 
Palmitoylation is the covalent attachment of fatty acids, such as palmitic acid, to cysteine and less frequently to serine and threonine residues of proteins, which are typically membrane proteins. The precise function of palmitoylation depends on the particular protein being considered.
 
The spike glycoproteins of almost all enveloped viruses are known to undergo post-translational attachment of palmitic acid moieties. The precise role of such palmitoylation of the spike protein in membrane fusion and infection is not completely understood.
 
The study team reported that palmitoylation of the first five cysteine residues of the c-terminal cysteine-rich domain of the SARS-CoV-2 spike are indispensable for infection and palmitoylation deficient spike mutants are defective in trimerization and subsequent membrane fusion. The DHHC9 palmitoyltransferase interacts with and palmitoylates the spike protein in the ER and Golgi, and knockdown of DHHC9 results in reduced fusion and infection of SARS-CoV-2. Two bis-piperazine backbone-based DHHC9 inhibitors inhibit SARS-CoV-2 spike protein palmitoylation and the resulting progeny virion particles released are defective in fusion and infection.
 
The study findings indicate that palmitoyltransferase inhibitors could be potential new intervention strategies against the SARS-CoV-2 coronavirus.
 
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2021.10.07.463402v1
 
It is already known that the spike protein of SARS-CoV-2 coronavirus is responsible for binding with the human angiotensin-converting enzyme 2 (ACE2) receptor to facilitate host cell entry, achieved firstly by bonding via the receptor-binding domain and undergoing subsequent conformational changes that allow membrane fusion. The cytosolic side of this membrane-spanning protein domain is cysteine-rich and capable of undergoing protein palmitoylation, the reversible addition of palmitic acid.
 
The process of palmitoylation is a common post-translational modification that occurs in over 30% of all cellular proteins to encourage protein stability, regulate membrane trafficking, and undertake vesical fusion. Palmitoylation of viral proteins has been shown to impact viral replication and fusion, playing an essential role in some cases.
 
The murine coronavirus spike protein for example, must undergo palmitoylation to assemble and enter host cells properly.
 
Similarly, previous reports have demonstrated that modifications to the SARS-CoV spike protein that prevents palmitoylation also reduces the ability of the virus to cause the formation of syncytia.
 
The study findings showed that palmitoylation of the first five cysteine residues of the C-terminal domain of the SARS-CoV-2 spike protein is essential to SARS-CoV-2, faci litating cell entry and the formation of syncytia. Endogenous enzymes facilitate palmitoylation of the spike protein, and thus inhibition of these enzymes could be a viable strategy against COVID-19. The group also put forward two drug candidates that demonstrate inhibition of SARS-CoV-2 in treated cell culture.  
 
It has been found that the C terminal cysteine-rich domain of the SARS-CoV-2 spike protein bears ten highly conserved cysteine residues. To investigate the role of palmitoylation at these sites, the group first exchanged them with serine, finding that some of the residue clusters are more prone to palmitoylation than others.
 
Mutating the clusters with the greatest preference for palmitoylation also degraded the ability of SARS-CoV-2 spike pseudotyped lentivirus to enter host cells most greatly, highlighting to the group that the first five residues (C1235, 1236, 1240, 1241, and 1243) are the most functionally important.
 
This held true for infected cell-uninfected cell interactions, where mutation of these key clusters caused a marked reduction in fusogenic activity and syncytia formation between cells.
 
Also, once a cell is infected, spike protein trimerization occurs within the endoplasmic reticulum membrane of host cells before virion particle assembly.
 
The study team next compared full-length wild-type spike proteins to those produced with serine mutations. Mutant spike proteins unable to undergo palmitoylation were defective in trimer formation, potentially explaining the reduced capacity to enter host cells observed.
 
Interestingly unlike other betacoronaviruses, SARS-CoV-2 also bears a furin-mediated cleavage site to facilitate host membrane fusion, which was found to be unaffected by the mutations imposed by the group.
 
Similarly, affinity towards the ACE2 receptor was found to be unaffected by the mutations.
 
Importantly the reduced infectivity of mutated spike protein pseudovirus was also found to not be due to a lessened ability to transport virion particles from the host endoplasmic reticulum to plasma membrane for outward expression, as only spike proteins with fully mutated cysteine residues showed a lesser expression, not those with the first five key residues.
 
Hence the study findings established that palmitoylation of the full cysteine chain is important to proper spike trimerization, with the first five residues playing a less important role in this regard.
 
The findings showed that palmitoyl acyltransferase enzymes induce palmitoylation of the thiol group of cysteine residues.
 
The study team next sought to identify the enzyme responsible for palmitoylation of the SARS-CoV-2 spike protein.
 
The proteins DHHC5 and DHHC9 were knocked down in HEK293T cells using siRNA, finding that knockdown of the former caused a compensatory upregulation in the latter, which was not reciprocated.
 
Hence, only knockdown of DHHC9 was seen to reduce spike protein palmitoylation in infected cells significantly.
 
The DHHC9 knockdown cells were then exposed to pseudovirus particles bearing the SARS-CoV-2 spike protein.
 
It was found that these cells were more resistant to infection and significantly less prone to engage in syncytia formation.
 
However, these cells were no less resistant to infection originating from neighboring untreated cells with palmitoylated spike proteins, implying that palmitoylation of the spike protein is only necessary for initial infection and not any other downstream event.
 
Further immunofluorescence experiments confirmed that DHHC5 localized with the spike protein in the endoplasmic reticulum and Golgi.
 
In order to test whether drug inhibition of DHHC9 could lessen SARS-CoV-2 infectivity to group assembled several palmitoyl acyltransferase inhibitors that were computationally screened for affinity.
 
The toxicity of a selection of these compounds was tested in HEK293T and Caco-2 cells and sufficiently tolerated compounds utilized in a SARS-CoV-2 spike palmitoylation assay.
 
Two unnamed compounds, termed compounds 13 and 25, were found to inhibit SARS-CoV-2 infection in cell culture. Cells pretreated with either compound and subsequently infected with SARS-CoV-2 exhibited a dose-dependent reduction in SARS-CoV-2 viral load and reduction in syncytia size.
 
Viral progeny collected from the supernatant of the treated cells then demonstrated significantly reduced infectivity towards uninfected untreated cells, demonstrating that preventing palmitoylation during the generation of new viral particles can impair their ability to infect new host cells.
 
The study findings indicate that palmitoyltransferase inhibitors could be potential new intervention strategies against the SARS-CoV-2 coronavirus.
 
Example of existing palmitoyltransferase inhibitors include 2BP, cerulenin and tunicamycin while phytochemicals like curcumin and piperidine also possess similar properties to a certain degree.
https://www.mdpi.com/2218-273X/10/8/1118/htm
https://pubmed.ncbi.nlm.nih.gov/27213958/
 
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