key: cord-0726210-7ypo4k17
authors: Puthenveetil, Robbins; Lun, Cheng Man; Murphy, R. Elliot; Healy, Liam B.; Vilmen, Geraldine; Christenson, Eric T.; Freed, Eric O.; Banerjee, Anirban
title: S-acylation of SARS-CoV-2 Spike Protein: Mechanistic Dissection, In Vitro Reconstitution and Role in Viral Infectivity
date: 2021-08-21
journal: J Biol Chem
DOI: 10.1016/j.jbc.2021.101112
sha: c01d4158a6f5cd9b5a93f69e3a2518083359a2b6
doc_id: 726210
cord_uid: 7ypo4k17

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long chain fatty acids get attached to cytosol-facing cysteines. 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 or the impact of this modification 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 compromises viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate the 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 towards developing future strategies that could aid in the identification of targeted small-molecule modulators.

The Coronavirus disease 2019 pandemic, the most recent epidemic caused by an outbreak of zoonotic coronaviruses in the past two decades, was preceded closely by MERS in 2012 and SARS in 2003 (1, 2) . Since the onset of COVID-19, over 160 million cases have been recorded, resulting in more than 3.3 million deaths globally (3) . These trends undoubtably suggest that coronaviruses represent an ongoing threat to human health and economic stability for years to come (4) . As the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rightly received an unprecedented amount of attention from the scientific community and been the focus of intense investigation (5). However, several aspects about this virus remains poorly understood. These gaps in knowledge underscore the need to investigate the chemistry and biology of SARS-CoV-2 in a manner that could lead to a greater comprehension of the unknown aspects of the disease.

As a global initiative to eradicate COVID-19 and fortify a future against similar pandemics, not only is it essential to focus research on the discovery of antivirals, but it is also important to obtain insights into the cell biology of SARS-CoV-2 and identify pathways with the highest potential for the discovery of novel therapeutics. SARS-CoV-2 belongs to the -Coronavirus family of enveloped, positive-strand RNA viruses (6) . Upon infection, the Spike (S) protein ( Fig. 1A) , resident in the viral membrane, directly mediates the critical process of membrane fusion, which is initiated upon binding to the angiotensin-converting enzyme 2 (ACE2) receptor located on the host cell plasma membrane (7) . Post infection, the viral genetic material encodes for several proteins alongwith the S protein which transverse the ER-Golgi intermediate compartment (ERGIC) and Golgi where they get cleaved into the S1 and S2

proteins, now noncovalently attached to each other (8) . Virions move through these organelles as membrane bound carriers and eventually get trafficked out of the cell (9) . Consequently, SARS-CoV-2 S protein has been the single most active target of various research in the last several months. Though most of the attention has been concentrated on the extracellular domain of S J o u r n a l P r e -p r o o f protein resulting in a large body of structural and biochemical data, very little is known about the flexible C-terminal domain, located on the intracellular/intraviral side of the membrane.

It has been previously demonstrated that the C-terminal segment of the S protein from pathogenic viruses including closely related coronaviruses, such as SARS-CoV and MERS-CoV, are targets of posttranslational S-acylation (10) (11) (12) , the most prevalent form of covalent lipid modification of proteins (13) . Protein S-acylation, defined by the attachment of long chain fatty acids to cysteine residues proximal to the cytosolic face of the membrane, is catalyzed by 23 members of the zDHHC family of integral membrane enzymes in humans (14) (15) (16) . Both intracellular as well as transmembrane proteins are targets of S-acylation, which is important in a wide range of physiological processes; nearly 4000 proteins have been identified as substrates for zDHHC enzymes (13) .

Interestingly, S-acylation was first discovered in viral S proteins (17) and is a well conserved feature of the S proteins in many enveloped RNA viruses (18) . S-acylation has been conclusively shown to be a crucial determinant for multiple facets of the viral replication cycle across a wide range of virus families [reviewed in (19) ]. Notably, mutation of S-acylation sites in the S protein of SARS-CoV severely compromises viral fusion. A sequence alignment between the cytoplasmic domains of SARS-CoV and SARS-CoV-2 shows that they are virtually identical with an additional cysteine residue in the SARS-CoV-2 sequence (Fig. 1C) . Given the prevalence and importance of S-acylation in viruses, it is surprising that there is currently no biochemical dissection of the S-acylation of any viral protein that can offer a framework for the mechanistic understanding of this important PTM. Here, we demonstrate that the S protein of SARS-CoV-2 (hereafter referred to as S protein) is S-acylated, identify the sites of S-acylation, and unravel their role in viral infectivity. To accomplish this, we have developed a library of human zDHHC enzymes in mammalian expression vectors in order to identify putative members of the zDHHC family that are involved in S protein S-acylation. Finally, we reconstitute in vitro J o u r n a l P r e -p r o o f the S-acylation of an S protein fragment with purified zDHHC enzymes, which provides valuable insight into the mechanisms of substrate specificity, allows for further biochemical dissection, and ultimately lays the groundwork for the development of novel assays for the identification of small-molecule inhibitors (20, 21) .

Protein S-acylation involves cysteine residues that face the cytosol. The C-terminal cytoplasmic tail of SARS-CoV-2 S protein contains a series of membrane-proximal cysteines that lie adjacent to the transmembrane domain ( Fig 1A) . To determine the S-acylation status of the cytoplasmic tail, we first utilized an in cellulo click chemistry assay; metabolic labeling of wild-type S protein expressed in HEK293T cells with 17-octadecynoic acid (17-ODYA), a fatty acid alkyne, followed by the conjugation and subsequent detection of a rhodamine fluorophore (22) , revealed robust S-acylation (Fig. 1B) . A cys construct, in which all the cysteines in the intracellular Cterminus were mutated, showed no detectable S-acylation. The use of 2-bromopalmitate (2BP), a global inhibitor of protein S-acylation, completely abolished palmitoylation of S protein indicating the role of zDHHCs in this posttranslational modification (PTM) (Fig. 1B) . Together, these experiments confirmed that the S protein is S-acylated by zDHHC enzymes at one or more cysteine residues within its cytoplasmic tail.

Sequence alignment of the cytoplasmic tail of the S proteins of SARS-CoV-2 together with those of SARS-CoV, MERS-CoV, and other closely related coronaviruses revealed that, in addition to the highly conserved cysteines in the C-terminus, SARS-CoV-2 S protein contains an additional cysteine (Fig. 1C) . Consequently, the C-terminus of SARS-CoV-2 S protein appears to be the most cysteine-rich in sequence amongst the known human coronaviruses. In SARS-CoV-2, these cysteines can be broadly divided into six groups, with four groups each containing two cysteine residues and two groups with a single cysteine ( Fig. 2A) . To interrogate which of J o u r n a l P r e -p r o o f these sites are targets for S-acylation, we used the previously described click chemistry experiments with S protein constructs (M1 through M6) that have had each of the putative Sacylation site removed by mutagenesis of the cysteine residue to alanine ( Fig. 2A ). An additional mutant, M7, represents a construct where the cysteine unique to SARS-CoV-2 S protein was mutated to mimic the sequence of SARS-CoV S protein ( Fig. 1C and Fig. 2A) . Expression of the S protein mutants in HEK293T cells, followed by click chemistry, revealed diminished Sacylation of the M1 and M2 mutants compared to wild-type, implicating the involvement of those cysteines in S-acylation (Fig. 2C) . Comparison of the double mutant, M1+M2, showed a complete lack of detectable palmitoylation compared to the wild-type S protein ( Fig 2D) .

To gain further understanding, we performed the reciprocal experiment, in which we started from a construct in which all the cysteines on the intracellular C-terminus were mutated to alanine (cys) and, on this background, each of the six groups of cysteine was reintroduced individually (C1 through C6) (Fig. 2B) . Thus, in each of the constructs from C1 through C6, only one of the six groups of cysteines was retained and the rest mutated to alanine. Consistent with our previous experiments, C1 and C2 showed the most prominent levels of S-acylation, thus reinforcing findings from the aforementioned experiments with the corresponding M constructs ( Fig. 2E) . Additionally, modest level of palmitoylation was also detected at position C4. We also conducted experiments for C1 through C6 mutants with 15-yne, a fatty acid alkyne that more closely resembles palmitic acid (23) and is gaining traction in the field as a replacement for 17-ODYA. Similar results to those obtained with 17-ODYA were observed with C1 and C2 being the major site of S-acylation together with C4 being a third minor site (Fig. 2F) . These experiments altogether revealed that the S protein is S-acylated and that the S-acylation status of the individual groups of cysteines within the cytosolic tail is markedly heterogeneous.

Effect of the S protein mutations on particle infectivity, incorporation into virus particles, subcellular localization, and cell-surface expression J o u r n a l P r e -p r o o f

To determine the effects of the S protein mutations on particle infectivity, HIV-1 pseudotypes bearing WT and mutant S protein were generated and their infectivities measured in HEK293T cells expressing human TMPRESS2 and ACE2 (see Methods). The mutations were observed to elicit a range of effects on particle infectivity in this system. The M7 mutant showed WT levels of infectivity. M3, M5, and C1 were reduced by around 50% relative to WT; M1 was reduced by about 30% while M2, M6, C3, and C4 exhibited ~20% WT levels of infectivity. The M4, Cys, C2, C5, and C6 showed a nearly complete loss of infectivity (Fig. 3A) . Likewise, the M1+M2 mutation largely abrogated pseudotype particle infectivity (Fig. S1A ).

To investigate the effects of the mutations on S protein expression, processing, and particle incorporation, we performed western blotting analysis of cell and virus lysates obtained from cells cotransfected with the HIV-1 reporter vector and the S protein expression vectors. A range of phenotypes was observed. The Cys mutant showed a marked reduction in levels of S1 in virions (approximately 20% of WT levels) ( Fig. 3B and C); however, levels of particleassociated S2 were close to those of WT ( Fig. 3B and D) . The presence of WT levels of particleassociated S2 while having severely reduced incorporation of S1 suggests the possibility of increased S1 shedding from the surface of cells and/or particles. The M6 mutant showed a similar phenotype. Several of the mutants (e.g., C5 and C6) displayed severe reductions in the levels of both S1 and S2 in particles. The M2 mutant showed a pattern of S protein expression and incorporation that was similar to that of the WT, despite severely compromised infectivity ( Fig. 3A-D) . Thus, mutation of Cys residues in the cytoplasmic tail of the S protein imposed a range of defects; in some cases, reduced infectivity was associated with reductions in the levels of S1 and/or S2 in virions, whereas for other mutants, reduced infectivity was not linked to defects in expression or incorporation. The M1+M2 mutant exhibited essentially WT levels of particle-associated S1 and S2 protein ( Fig. S1B-D) .

We next examined the effect of a set of S protein mutations -Cys, M2, M4, and C2on the subcellular localization of the S protein by confocal microscopy. Cells transfected with S protein expression vectors were stained with antibodies specific for the S protein and the lysosomal marker LAMP-1. The S protein showed a high level of colocalization with LAMP-1 (Pearson correlation coefficient r~0.8). For the mutants tested, the degree of colocalization between the S protein and LAMP-1 was similar to that of the WT (Fig. S2 ). We also investigated the cell-surface expression of WT and the Cys, M2, and M4 mutants by flow cytometry using antibodies specific for the S1 and S2 subunits of the S protein complex. The results indicated that these mutations did not significantly affect S protein expression at the cell surface ( Fig. S3 ).

Bacterial and viral pathogens do not encode for enzymes that catalyze S-acylation. Instead, they rely on the S-acylation machinery of the host. In humans, protein S-acylation is carried out by 23 members of the zDHHC family of integral membrane enzymes that reside in various organellar membranes as well as the plasma membrane. zDHHC family of enzymes are promiscuous in their substrates with no defined consensus motif for S-acylation. Consequently, the zDHHC enzymes that S-acylate a target substrate need to be determined empirically. The most prevalent method, by far, has been to individually coexpress a target with each zDHHC member (24) . The zDHHC members that increase the levels of S-acylation of a target can be reliably interpreted as the zDHHC enzymes which canonically act on that target, or at the very least, are capable of acting on that target. Historically, this has been achieved with a library of murine zDHHCs originally constructed by the Fukata lab (24). Presumably, this was because of the generous availability of the "Fukata library" as a reagent. However, in the context of SARS-CoV-2, we deemed it imperative to develop a generic screen (which we call the HeaTil screen named after two of the co-authors of this manuscript) with human zDHHCs that, at the outset, could provide more pertinent information to human disease. All 23 ZDHHC genes were inserted into the J o u r n a l P r e -p r o o f Bacmam vector (25) and optimized for overexpression with a terminal YFP fusion. Click chemistry-based analyses of coexpression of S protein with each human zDHHC clearly identified multiple enzymes that palmitoylate S protein (Fig. 4A) . Remarkably, S protein expression varied over a wide range when coexpressed with different zDHHC members ( Figure   4A ). Although such variation is uncommon in the vast majority of cases where zDHHC enzymes for a specific target have been identified by this method, S-acylation has indeed been shown, in certain cases, to modulate protein levels by affecting protein turnover rates (26) .

To further validate our findings, we tested the coexpression of the S protein together with each candidate zDHHC enzyme and its catalytically inactive zDHHS mutant (Fig. 4B ). This experiment clearly demonstrated the ability of the selected zDHHC members to S-acylate the S protein and helped further substantiate our findings from the initial screen. For example, coexpression of S protein with zDHHC2 increased S protein S-acylation levels, whereas coexpression with the catalytically inactive zDHHS2 mutant did not (Fig. 4B ). On the contrary, coexpression of either zDHHC15 or the catalytically inactive zDHHS15 did not change the Sacylation levels of S protein (Fig. 4B) . Altogether, these experiments revealed zDHHCs 2,3,6,11,20,21 and 24 as putative S-acylation enzymes for the SARS-CoV-2 S protein.

Due to the intrinsic complexity of the system used in the cell-based assays, an essential step towards mechanistic dissection of S-acylation of the S protein is the development of an in vitro assay using purified proteins. However, remarkably few reports of in vitro reconstitution of substrate S-acylation by zDHHC enzymes exist in the literature (27, 28) , most of them being carried out with very small peptide fragments of substrates (29) . S protein, on the other hand, is a complex protein with a very large N-terminal ectodomain leading into a single transmembrane helix, followed by the relatively small C-terminal cytoplasmic domain, which contains the sites for S-acylation. To simplify the design of our in vitro assay, we focused on the C-terminal J o u r n a l P r e -p r o o f cytosolic domain, which harbors the sites for S-acylation (Fig. 5A, left panel) . To facilitate detection, we appended a C-terminal GFP and replaced the transmembrane and the extracellular domain with an N-terminal myristoylation sequence (Fig. 5A, right panel) . A cysteineless construct was generated on this backbone, S-CTD-Cys. Subsequently, individual clusters of cysteines were introduced onto this background to generate the S protein substrates, S-CTD-C1 through CT-C6, as designed previously for the C-mutants (Fig. 2B ). These substrates were purified from E. coli and tested in a click-chemistry based in vitro assay (see Methods) with palmitoyl alkyne-coenzyme A and purified zDHHCs to append an alkyne analog of palmitic acid onto the substrate followed by conjugation of a rhodamine fluorophore to the alkyne moiety for detection. Thus, the rhodamine channel revealed the extent of S-acylation while the GFP channel enabled independent assessment of total substrate protein levels in the assay (Fig. 5B ).

Based on the zDHHCs identified from our in cellulo experiments (Fig. 4 ) and the availability of an established protocol for the purification of human zDHHC20 (28), we first focused on zDHHC20 for the in vitro assay. zDHHC20 showed robust S-acylation of the S-CTD-C1 through S-CTD-C6 constructs with a clear difference from the level of S-acylations detected with the catalytically inactive DHHS20 (Fig. 5B ). There is a striking heterogeneity between the levels of acylation in S-CTD-C1 through S-CTD-C6 with the highest levels of Sacylation in S-CTD-C1, C2, and C4 (Fig. 5C) . Intriguingly, these are the same cysteines that showed the highest levels of S-acylation in our in cellulo experiments with full length S protein ( Fig. 2) , indicating that we are recapitulating salient aspects of the in cellulo S-acylation in our in vitro assay. We next tested both human zDHHC2 and human zDHHC3 using the same in vitro Sacylation assay. Again, we observed robust S-acylation of the S protein substrates in comparison with the respective catalytically inactive mutants and a similar level of heterogeneity among the levels of S-acylation in the different substrates (Fig. 5D, 5E) . However, the different zDHHC members showed a distinct spectrum of activities among the individual cysteines. Notably, S-CTD-C1, C2, and C4 showed the highest activity with zDHHC2, while S-CTD-C2 and C4 J o u r n a l P r e -p r o o f showed the highest activity with zDHHC3. Taken together, these experiments show that between zDHHC2, zDHHC3, and zDHHC20, S-CTD-C1, C2 and C4 showed the highest level of Sacylation activity, in agreement with our in cellulo experiments.

S-acylation of viral proteins has been known since the discovery of this post-translational modification (17). SARS-CoV-2 S protein harbors the most cysteine-rich C-terminal cytosolic tail compared to other related coronaviruses such as SARS-CoV and MERS-CoV (Fig. 1) .

However, it has been unclear whether some or all of these cysteines are S-acylated and how that might impact viral infectivity. Herein, we demonstrate conclusively that the S protein of SARS-CoV-2 is S-acylated and identify the sites of S-acylation. We also identify specific cysteines that are targets for this modification. Although the clusters of cysteines are all positioned in the membrane-proximal segment of the C-terminus within a stretch of 20 residues, yet our in cellulo assay demonstrated a remarkable heterogeneity in the ability of different cysteines to become Sacylated. Our initial analysis identified cysteines corresponding to positions C1 and C2 as the major site of palmitoylation along with C4, which demonstrated modest S-acylation.

Remarkably, mutating cysteines at these positions selectively reduced viral infectivity. Even the mutation of a single cluster of cysteines, such as the M2 construct, led to an 80% reduction in comparison to the wild-type S protein. This reduction is not due to aberrant S protein expression, processing, or maturation. Notably, C6 is a fourth cysteine cluster whose mutation (construct M6) showed a similar effect on viral infectivity. We did not identify C6 as a target site for Sacylation. However, mutating C6 (construct M6) results in aberrant S protein maturation and was one of the prominent enzymes that palmitoylates S protein as identified both in the present and another recent study (33) . A ready purification protocol for zDHHC20 was available to us since we solved high-resolution structures of human zDHHC20 (28) . With these considerations, we chose to first focus on developing an in vitro assay for S protein S-acylation with the entire cytosolic fragment and purified zDHHC20. Remarkably, the pattern of S-acylation activity on the individual clusters of cysteines was distinctly heterogeneous. Moreover, the highest activity was seen with C2 and C4, two of the three sites identified by our in cellulo S-acylation assays.

These are also the same two sites, that when mutated, severely compromised viral infectivity. In sharp contrast, when we reconstituted the S-acylation of S protein with zDHHC2 or zDHHC3, the pattern of reactivity, although similarly heterogeneous, was notably different. Most striking was the fact that zDHHC3 activity on the C1 site was greatly diminished compared to that of zDHHC2 and zDHHC20. Interestingly, previous studies analyzing the acyl chain length selectivities of zDHHC 2, 3 and 20 demonstrated that, while zDHHC2 and zDHHC20 are capable of utilizing acyl-CoAs consisting of chain lengths greater than 16 carbons, zDHHC3 J o u r n a l P r e -p r o o f discriminates against acyl chains of such length (28, 34) . This observation provides insight into our results when considering studies that demonstrated that the viral spike protein for influenza A (heamagglutinin) is primarily modified with a 16-carbon fatty acid at most sites on its cytoplasmic tail accept for the site closest to the transmembrane domain, which is predominantly modified with an 18-carbon fatty acid (30) . Our data suggest a mechanism for such heterogeneity by providing evidence that certain zDHHCs which cannot utilize 18-carbon fatty acids (like (36, 37) . A recent study showed that cholesterol and S protein association plays a crucial role in SARS-CoV-2 infection (38) . Furthermore, we also find that the cys-less (Cys) form of S protein was compromised in its ability to incorporate into 0100-01). Imaging was performed with a Leica TCS SP8 microscope (Leica Microsystems Inc.) using a 63x oil-immersion objective. 3-D images were generated using ImageJ (NIH) from z-stack images. Background was subtracted using ImageJ's built-in 'rolling ball' background subtraction process. Colocalization analyses were performed using the colocalization test within ImageJ.

Untransfected cells were excluded from analyses.

J o u r n a l P r e -p r o o f Flow cytometry. HEK293T cells transfected with vectors expressing WT or mutant SARS-CoV-2 S protein were seeded in 6-well plates and collected. Cells were pellected and stained with antibodies targeting the S1 subunit (Sinobiological, #40589-T62) or S2 subunit (Invitrogen, #MA5-35946) at 1g/ul, Alexa Fluor 647 at 2g/ul was used for secondary antibody. Cells were fixed with 4% paraformaldehyde prior to flow cytometry analysis with a FACSCalibur. cells in 6-well dishes were cotransfected with pNL4-3.Luc.R-E-(3 mg) and vectors expressing WT or mutant SARS-CoV-2 S (300 ng). Virus supernatants were collected two days posttransfection, normalized for RT activity, and infectivity was measured by luciferase assay using TMPRESS2-transfected HEK293T cells stably expressing hACE2. Infectivity of HIV-1 particles pseudotyped with WT SARS-CoV-2 S protein was set to 100%. Data are derived from 4 independent experiments. B) Western blotting of cell-and particle-associated S protein.

HEK293T cells were cotransfected with pNL4-3.Luc.R-E-(3 mg) and vectors expressing WT or mutant SARS-CoV-2 S (300 ng). Cell and virus lysates were prepared and subjected to western blot analysis with anti-SARS-CoV-2 S1 or S2 Ab to detect S protein expression. HIV-Ig was used to detect HIV-1 Gag proteins. Mobility of molecular mass standards is shown on the left side of the blots. The levels of S1 C) and S2 D) in virus was quantified and normalized to p24 (CA) and set to 100% for WT S protein. P values using two-tailed unpaired t-test: *p < 0.01, § p< 0.001, ° p < 0.0001. Values that are not statistically significantly different from WT are not labeled. Histograms represent average ± SD (n = 3).

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The following reagent was obtained through BEI Resources, NIAID, NIH: Monoclonal Anti- 

All data are available upon request from the authors and are included in the article.

The authors declare that they have no conflicts of interest with the contents of this article.J o u r n a l P r e -p r o o f