Inhibition of SARS-CoV-2 spike protein palmitoylation reduces virus infectivity

Abstract

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. Here, we report 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. This establishes these palmitoyltransferase inhibitors as potential new intervention strategies against SARS-CoV-2.

SciScore for 10.1101/2021.10.07.463402: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Thereafter, the cells were washed two times with DMEM with 5% FBS and incubated with anti-spike antibody diluted (1:200) in DMEM with 5% FBS and 0.1% Sodium Azide for 1 h at 37°C.	anti-spike suggested: None
Experimental Models: Cell Lines
Sentences	Resources
HEK293T cells constitutively expressing human ACE2 (HEK293T-ACE2 cells) obtained from BEI Resources (#NR52511) were grown in the same media supplemented with hygromycin (100 μg/ml).	HEK293T suggested: None
For the syncytia formation assay, …

SciScore for 10.1101/2021.10.07.463402: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Thereafter, the cells were washed two times with DMEM with 5% FBS and incubated with anti-spike antibody diluted (1:200) in DMEM with 5% FBS and 0.1% Sodium Azide for 1 h at 37°C.	anti-spike suggested: None
Experimental Models: Cell Lines
Sentences	Resources
HEK293T cells constitutively expressing human ACE2 (HEK293T-ACE2 cells) obtained from BEI Resources (#NR52511) were grown in the same media supplemented with hygromycin (100 μg/ml).	HEK293T suggested: None
For the syncytia formation assay, HEK293T-GFP cells were transiently transfected with wild-type and mutant spike plasmids using TransIT-X2 transfection reagent (Mirus #MIR 6000) according to the manufacturer’s instructions, and fresh media was added after 6 hours.	HEK293T-GFP suggested: None
Briefly, 96-Flat bottom well plates were coated with poly-D-lysine (Gibco, #A3890401) and seeded with 1.25x104 HEK293T-ACE2 or Caco-2 cells per each well.	Caco-2 suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)
6 h after transfection, the HEK293T-ACE2 cells were treated with accutase cell detachment reagent and added to the HEK293T-EGFP cells transfected with spike protein, at a 1:1 ratio.	HEK293T-ACE2 suggested: None HEK293T-EGFP suggested: None
All viral stocks were produced and isolated from supernatants of Vero-E6 ACE2 cells, cultured in T175 culture flasks to a confluency of 80-90%, and infected with an original passage 2 (P2) SARS-CoV-2 or SARS-CoV-2-mNG (SARS-CoV-2 stably encoding mNeonGreen) virus, at MOI of 0.1 for 72 h, in 10 ml MEM supplemented with 5% FBS.	Vero-E6 ACE2 suggested: None
In brief, viral stocks were serially diluted (10-fold) in serum-free medium and inoculated on 1x 105 Vero E6-ACE2 cells in triplicates in a 48 well plate.	Vero E6-ACE2 suggested: None
Recombinant DNA
Sentences	Resources
3rd generation lentivirus were produced using the flowing packaging plasmids, which were a gift from Didier Trono’s lab, pMDL (Addgene #12251), pRev (Addgene #12253) and pMD2.G (Addgene #12259) (61)	pMDL suggested: None pMD2 . G suggested: None
Briefly, plasmids expressing the HIV-1 gag and pol (pHDM-Hgpm2), HIV-1 rev (pRC-CMV-rev1b), HIV-1 Tat (pHDM-tat1b), the SARS CoV2 spike (pHDM-SARS-CoV-2 Spike) and a luciferase/ZsGreen reporter (pHAGE-CMV-Luc2-IRES-ZsGreen-W) were co-transfected into HEK293T cells at a 1:1:1:1.6:4.6 ratio using CalPhos mammalian transfection kit (TaKaRa Clontech, Mountain View, CA #631312) according to manufacturer’s instructions.	pRC-CMV-rev1b suggested: RRID:Addgene_164443) pHAGE-CMV-Luc2-IRES-ZsGreen-W suggested: RRID:Addgene_164432)
HEK293T cells constitutively expressing EGFP (HEK293T-EGFP) were established by transducing HEK293T cells with the pLJM1-EGFP containing lentivirus.	pLJM1-EGFP suggested: RRID:Addgene_19319)
Lentivirus was produced by transfecting 106 HEK293T cells on a 60 mm plate with pMDL, pRev and pVSVG using CalPhos mammalian transfection kit (TaKaRa Clontech).	pRev suggested: None pVSVG suggested: RRID:Addgene_85140)
Software and Algorithms
Sentences	Resources
Alignment of these sequences was done using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/).	Clustal Omega suggested: (Clustal Omega, RRID:SCR_001591)
Images were quantified using ImageJ.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
Statistical analysis and reproducibility: Statistical analysis was performed using GraphPad Prism 9 (San Diego, CA).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 56, 61 and 55. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

Results from rtransparent:

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No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

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Inhibition of SARS-CoV-2 spike protein palmitoylation reduces virus infectivity

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