key: cord-0928916-erxerif5
authors: Li, Daoqun; Liu, Yihan; Lu, Yue; Gao, Shan; Zhang, Leiliang
title: Palmitoylation of SARS‐CoV‐2 S protein is critical for S‐mediated syncytia formation and virus entry
date: 2021-09-23
journal: J Med Virol
DOI: 10.1002/jmv.27339
sha: 1831a0fcc0b51ae82dcfce4878a5df7cdd7d9453
doc_id: 928916
cord_uid: erxerif5

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the cause of the ongoing coronavirus disease 2019 (COVID‐19) pandemic. The S protein is the key viral protein for associating with ACE2, the receptor for SARS‐CoV‐2. There are many kinds of posttranslational modifications in S protein. However, the detailed mechanism of palmitoylation of SARS‐CoV‐2 S remains to be elucidated. In our current study, we characterized the palmitoylation of SARS‐CoV‐2 S. Both the C15 and cytoplasmic tail of SARS‐CoV‐2 S were palmitoylated. Fatty acid synthase inhibitor C75 and zinc finger DHHC domain‐containing palmitoyltransferase (ZDHHC) inhibitor 2‐BP reduced the palmitoylation of S. Interestingly, palmitoylation of SARS‐CoV‐2 S was not required for plasma membrane targeting of S but was critical for S‐mediated syncytia formation and SARS‐CoV‐2 pseudovirus particle entry. Overexpression of ZDHHC2, ZDHHC3, ZDHHC4, ZDHHC5, ZDHHC8, ZDHHC9, ZDHHC11, ZDHHC14, ZDHHC16, ZDHHC19, and ZDHHC20 promoted the palmitoylation of S. Furthermore, those ZDHHCs were identified to associate with SARS‐CoV‐2 S. Our study not only reveals the mechanism of S palmitoylation but also will shed important light into the role of S palmitoylation in syncytia formation and virus entry.

C terminal cysteines. Palmitoylation of S is not essential for the plasma membrane localization of S but is critical for S-mediated formation of syncytia and virus entry. We identified that multiple ZDHHCs associated with SARS-CoV-2 S and mediated its palmitoylation. Taken together, our findings identified the key enzymes for the palmitoylation of SARS-CoV-2 S and revealed that palmitoylation of S contributes to syncytia formation and virus entry. 

The mouse antibodies used in this study have been described pre- 

Cells were washed three times with ice-cold phosphate-buffered saline (PBS) (Solarbio, P1010) and lysed in radioimmunoprecipitation assay lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, and 1% Triton X-100, 1% sodium deoxycholate, 1% sodium dodecyl sulfate [SDS], without inhibitors) (Beyotime, P0013K) supplemented with a protease inhibitor cocktail for mammalian cell and tissue extracts (100×) (Beyotime, P1010). Both the cell lysate and the antibody need to be bound to protein A + G agarose beads (Beyotime, P2055). The beads need to be equilibrated (washed three times with PBS stored at 4°C) before use to remove the preservatives in the bead storage solution.

The cell lysate was incubated with the equilibrated protein A + G agarose beads slurry at 4°C for 30 min and centrifuged at 12 000 rpm at 4°C for 15 min. Then the cell lysate was incubated with protein A + G agarose beads bound to the antibody for 1.5 h and washed three times with PBS at 4°C followed by centrifugation at 500 g for 5 min. The proteins bound to the beads were boiled in two-times loading buffer (Beyotime, P0015B) for 5 min and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% separation gel in the lower layer, 5% concentrated gel in the upper layer).

Palmitoylation sites of S from SARS-CoV-2 were predicted in CSS-palm 4.0 (http://csspalm.biocuckoo.org/online.php).

Detection of S palmitoylation by an ABE assay was performed based on a previous study. 12 The cell lysate buffer contained 10% glycerol, 

The plasma membrane was isolated using a plasma membrane protein isolation and cell fractionation kit (Bestbio, BB-31161-2), and the assay was performed according to the manufacturer's protocol.

The protein samples were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane. After transferring the membrane, we sealed it with a solution of 1×TBST (pH 8.0, CWBIO, CW0043S) and 5% skimmed milk powder, and then probed LI ET AL. | 343 with appropriate primary and secondary antibodies. In the ABE experiment, 3% BSA (Beyotime, ST023) was used for blocking instead of 5% skimmed milk powder.

The 293T cells in 100 mm plates were transfected using PEI with | 345 mutant also reduced the palmitoylation level compared with that in wild-type S ( Figure 1E ). When 10 predicted cysteines are all mutated to serine (S), the palmitoylation signal is even less than those in C1 or C9 mutant ( Figure 1E ). Thus, the cysteines at sites C15, and C terminal cytoplasmic tail are two key sites for SARS-CoV-2 S palmitoylation.

Fatty acid synthase generates PA, while protein palmitoylation is mediated by ZDHHC. To further confirm the palmitoylation of SARS-CoV-2, we applied fatty acid synthase inhibitor C75 and ZDHHC inhibitor 2-BP. As shown in Figure 1F ,G, both C75 and 2-BP reduced the palmitoylation of S.

As palmitoylation increases protein hydrophobicity and could facilitate protein trafficking to cellular membranes, next we investigated whether Figure 2B , incorporation of SARS-CoV-2 S mutants into pseudovirions is less efficient compared with that of wild-type S. Consistent with that, the entry abilities of all three mutant S based SARS-CoV-2pp were reduced, indicating that palmitoylation of S is critical for SARS-CoV-2pp entry ( Figure 2C) . Interestingly, the C1, C9, and C10 mutants of SARS-CoV-2 S-mediated fewer syncytia formation, indicating less virus entry ( Figure 2D ). Thus, we concluded that the cysteines at sites C15, C1235, C1236, C1240, C1243, C1247, C1248, C1250, C1253, and C1254 of S are crucial for S-mediated syncytia formation and SARS-CoV-2pp entry.

Protein palmitoylation is mediated mainly by ZDHHCs. Therefore, we 

As multiple ZDHHCs have been identified to promote S palmitoylation, we hypothesized that those ZDHHCs participated in the palmitoylation of S associated with S. To test this hypothesis, the plasmids expressing ZDHHC2, ZDHHC3, ZDHHC4, ZDHHC5, ZDHHC8, ZDHHC9, ZDHHC11, ZDHHC14, ZDHHC16, ZDHHC19, or ZDHHC20 were individually cotransfected with the plasmid expressing GFP-S in 293T for 24 h (Figure 4) . We used mouse IgG as a negative control to perform the co-IP experiment. As shown in Figure 4 , those ZDHHCs interacted with S. The results of interaction between S and ZDHHCs assayed by IP are summarized in Table 1 .

Previous studies showed the palmitoylation of S from other coronaviruses including SARS-CoV, MHV, and PEDV. 8-10 Consistent with our findings, two recent studies found that the palmitoylation of 

Note: Z1-Z24, ZDHHC1-ZDHHC24. The interactions in the same row were scored relative to each other from not detectable (−) and strong to very strong (from + to ++). /, not tested.

Abbreviations: ABE, acyl-biotinyl exchange; Co-IP, co-immunoprecipitation; ZDHHCs, zinc finger DHHC domain-containing palmitoyltransferases.

LI ET AL.

| 347 SARS-CoV-2 S protein is essential for viral infectivity. 13, 14 However, they identified the key palmitoylation sites are restricted in the C terminal of S. In the current study, we have shown not only the C terminal cysteines of S but also the N-terminal C15 is palmitoylated.

The discrepancy between Wu's work and our study is due to the interpretation of their results. In Wu's paper, the palmitoylation signals of wild-type S (S-WT), C terminus (S-ΔC-Palm), and N terminus (S-C15A) were 1.0, 0.1, and 0.9 using densitometric analysis with Image-Pro Plus software. 14 However, the protein level of S-WT was lower than that of S-C15A. When the palmitoylation levels were normalized with protein levels, their results also indicated the palmitoylation in the N terminus of S protein.

A previous study only discovered ZDHHC5 as the major ZDHHC for SARS-CoV-2 S palmitoylation. 14 In the current study, we not only expanded the palmitoylation sites in SARS-CoV-2 S but also found that multiple ZDHHCs could palmitoylate SARS-CoV-2 S. As multiple ZDHHCs are involved in S palmitoylation, we proposed that the following three scenarios may occur. One possible scenario is that ZDHHCs are located in different cellular compartment palmitoylate S in different intracellular locations, such as Golgi or plasma membrane.

The second scenario is that different ZDHHCs may contribute to different palmitoylation sites in S. The third scenario is that multiple ZDHHCs are redundant for the palmitoylation of S.

Palmitoylation of S from SARS-CoV, MHV, or PEDV is not critical for plasma membrane localization. [8] [9] [10] For SARS-CoV-2, it is the same case. Plasma membrane isolation experiment showed that the protein level in the plasma membrane of palmitoylation deficient mutant of C10 was the same as that in wild-type S. However, the syncytia formation was reduced by palmitoylation deficient mutation of S. Interestingly, palmitoylation of SARS-CoV-2 is critical for the incorporation of S to pseudovirus particles, which could explain the important role for S palmitoylation in SARS-CoV-2pp entry. The detailed mechanism of how palmitoylation affects S-mediated fusion is unknown, which needs to be explored in the future.

In conclusion, we confirmed the palmitoylation of SARS-CoV-2 S and its function, proposing that palmitoylation of SARS-CoV-2 S is critical for S-mediated syncytia formation and virus entry. C15 and C terminal multiple sites of cysteines are the key sites for palmitoylation. ZDHHC2, ZDHHC3, ZDHHC4, ZDHHC5, ZDHHC8, ZDHHC9, ZDHHC11, ZDHHC14, ZDHHC16, ZDHHC19, and ZDHHC20 are the major PATs for S.

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This study was supported by grants from the National Natural 

The authors declare that there are no conflict of interests.

Daoqun Li, Y Liu, and Y Lu performed the experiments. Shan Gao revised the manuscript. Leiliang Zhang conceived the work and wrote the manuscript. All authors read and approved the final manuscript.

http://orcid.org/0000-0002-7015-9661