Palmitoylation of SARS-CoV-2 S protein is essential for viral infectivity

Wu, Zhonghu; Zhang, Zhaoying; Wang, Xin; Zhang, Jing; Ren, Caiyue; Li, Yuming; Gao, Lifen; Liang, Xiaohong; Wang, Pei‐Hui; Hong, Chun Pyo

doi:10.1038/s41392-021-00651-y

Cited by 68 publications

(80 citation statements)

References 7 publications

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“…Moreover, S protein palmitoylation was mainly mediated by ZDHHC8,9,20 via analyzing spike-incorporated radioactivity in Hela cells cotransfected with individual siRNAs targeting all human ZDHHCs [ 25 ], while Puthenveetil et al reported that ZDHHC2,3,6,11,20,21,24 were as putative palmitoylation enzymes for SARS-CoV-2 S protein modification by click-chemistry-based analyses of coexpression of S protein with individual ZDHHC [ 26 ]. Our results are consistent with the report of Mesquita et al However, in other reports, the interactions between SARS-CoV-2 S protein and ZDHHC5/GOLGA7 were confirmed by co-IP and the two host protein overexpressions in HEK293T cells enhanced S protein palmitoylation synergistically and ZDHHC5 knockdown decreased S protein palmitoylation and pseudovirus infection [ 27 , 28 ]. All these distinct results were likely caused by different analytic systems, such as cell lines and detection methods.…”

Section: Discussionsupporting

confidence: 93%

The interactions of ZDHHC5/GOLGA7 with SARS-CoV-2 spike (S) protein and their effects on S protein’s subcellular localization, palmitoylation and pseudovirus entry

Zeng

Cheng

2021

Virol J

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Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein determines virus entry and the palmitoylation of S protein affects virus infection. An acyltransferase complex ZDHHC5/GOGAL7 that interacts with S protein was detected by affinity purification mass spectrometry (AP-MS). However, the palmitoylated cysteine residues of S protein, the effects of ZDHHC5 or GOLGA7 knockout on S protein’s subcellular localization, palmitoylation, pseudovirus entry and the enzyme for depalmitoylation of S protein are not clear. Methods The palmitoylated cysteine residues of S protein were identified by acyl-biotin exchange (ABE) assays. The interactions between S protein and host proteins were analyzed by co-immunoprecipitation (co-IP) assays. Subcellular localizations of S protein and host proteins were analyzed by fluorescence microscopy. ZDHHC5 or GOGAL7 gene was edited by CRISPR-Cas9. The entry efficiencies of SARS-CoV-2 pseudovirus into A549 and Hela cells were analyzed by measuring the activity of Renilla luciferase. Results In this investigation, all ten cysteine residues in the endodomain of S protein were palmitoylated. The interaction of S protein with ZDHHC5 or GOLGA7 was confirmed. The interaction and colocalization of S protein with ZDHHC5 or GOLGA7 were independent of the ten cysteine residues in the endodomain of S protein. The interaction between S protein and ZDHHC5 was independent of the enzymatic activity and the PDZ-binding domain of ZDHHC5. Three cell lines HEK293T, A549 and Hela lacking ZDHHC5 or GOLGA7 were constructed. Furthermore, S proteins still interacted with one host protein in HEK293T cells lacking the other. ZDHHC5 or GOLGA7 knockout had no significant effect on S protein’s subcellular localization or palmitoylation, but significantly decreased the entry efficiencies of SARS-CoV-2 pseudovirus into A549 and Hela cells, while varying degrees of entry efficiencies may be linked to the cell types. Additionally, the S protein interacted with the depalmitoylase APT2. Conclusions ZDHHC5 and GOLGA7 played important roles in SARS-CoV-2 pseudovirus entry, but the reason why the two host proteins affected pseudovirus entry remains to be further explored. This study extends the knowledge about the interactions between SARS-CoV-2 S protein and host proteins and probably provides a reference for the corresponding antiviral methods.

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Section: Discussionsupporting

confidence: 93%

The interactions of ZDHHC5/GOLGA7 with SARS-CoV-2 spike (S) protein and their effects on S protein’s subcellular localization, palmitoylation and pseudovirus entry

Zeng

Cheng

2021

Virol J

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“…A subset screen, in near-haploid HAP-1 cells knocked out (KO) for individual ZDHHCs, confirmed these findings and indicated a principal role for ZDHHC20 ( Figures 1 F and S1 C). These screens did not pick up ZDHHC5, which during the course of this study was proposed as a spike acyltransferase based on overexpression experiments ( Wu et al., 2021 ). Spike S-acylation upon depletion of the ZDHHC8, 9, or 20 could be restored by expression of the correspondent siRNA-resistant myc-tagged human enzyme, but not a random acyltransferase ( Figure S1 D).…”

Section: Resultsmentioning

confidence: 99%

S-acylation controls SARS-CoV-2 membrane lipid organization and enhances infectivity

et al. 2021

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SARS-CoV-2 virions are surrounded by a lipid bilayer that contains membrane proteins such as spike, responsible for target-cell binding and virus fusion. We found that during SARS-CoV-2 infection, spike becomes lipid modified, through the sequential action of the S-acyltransferases ZDHHC20 and 9. Particularly striking is the rapid acylation of spike on 10 cytosolic cysteines within the ER and Golgi. Using a combination of computational, lipidomics, and biochemical approaches, we show that this massive lipidation controls spike biogenesis and degradation, and drives the formation of localized ordered cholesterol and sphingolipid-rich lipid nanodomains in the early Golgi, where viral budding occurs. Finally, S-acylation of spike allows the formation of viruses with enhanced fusion capacity. Our study points toward S-acylating enzymes and lipid biosynthesis enzymes as novel therapeutic anti-viral targets.

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“…A previous study shows that the palmitoylation modification of the SARS-CoV-2 spike protein is essential for viral infectivity 34 . FAS inhibitors reduced cellular levels of free fatty acids and palmitoylated proteins (Extended Data Fig.…”

Section: Mainmentioning

confidence: 99%

Pharmacological inhibition of fatty acid synthesis blocks SARS-CoV-2 replication

Chu

Xing

et al. 2021

Nat Metab

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Caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), COVID-19 is a virus-induced inflammatory disease of the airways and lungs that leads to severe multi-organ damage and death. Here we show that cellular lipid synthesis is required for SARS-CoV-2 replication and offers an opportunity for pharmacological intervention. Screening a short-hairpin RNA sublibrary that targets metabolic genes, we identified genes that either inhibit or promote SARS-CoV-2 viral infection, including two key candidate genes, ACACA and FASN , which operate in the same lipid synthesis pathway. We further screened and identified several potent inhibitors of fatty acid synthase (encoded by FASN ), including the US Food and Drug Administration-approved anti-obesity drug orlistat, and found that it inhibits in vitro replication of SARS-CoV-2 variants, including more contagious new variants, such as Delta. In a mouse model of SARS-CoV-2 infection (K18-hACE2 transgenic mice), injections of orlistat resulted in lower SARS-CoV-2 viral levels in the lung, reduced lung pathology and increased mouse survival. Our findings identify fatty acid synthase inhibitors as drug candidates for the prevention and treatment of COVID-19 by inhibiting SARS-CoV-2 replication. Clinical trials are needed to evaluate the efficacy of repurposing fatty acid synthase inhibitors for severe COVID-19 in humans.

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Palmitoylation of SARS-CoV-2 S protein is essential for viral infectivity

Cited by 68 publications

References 7 publications

The interactions of ZDHHC5/GOLGA7 with SARS-CoV-2 spike (S) protein and their effects on S protein’s subcellular localization, palmitoylation and pseudovirus entry

The interactions of ZDHHC5/GOLGA7 with SARS-CoV-2 spike (S) protein and their effects on S protein’s subcellular localization, palmitoylation and pseudovirus entry

S-acylation controls SARS-CoV-2 membrane lipid organization and enhances infectivity

Pharmacological inhibition of fatty acid synthesis blocks SARS-CoV-2 replication

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