SARS- CoV-2 viroporins: A multi-omics insight from nucleotides to amino acids

  1. Manish Sarkar
  2. Paul Etheimer
  3. Victor Hannothiaux
  4. Soham Saha

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Abstract

COVID-19 is caused by SARS-CoV-2 which has so far affected more than 500 million people worldwide and killed over 6 million as of 1st May, 2022. The approved emergency-use vaccines were lifesaving to such a devastating pandemic. Viroporins are important players of the life cycle of SARS-CoV-2 and are primary to its pathogenesis. We studied the two prominent viroporins of SARS-CoV-2 (i) Orf3a and (ii) Envelope (E) protein from a sequential and structural point of view. Orf3a is a cation selective viral ion channel which has been shown to disrupt the endosomal pathways. E protein is one of the most conserved proteins among the SARS-CoV proteome which affects the ERGIC related pathways. The aqueous medium through the viroporins mediates the non-selective translocation of cations, affecting ionic homeostasis in the host cellular compartments. This ionic imbalance could potentially lead to increased inflammatory response in the host cell. Our results shed light into the mechanism of viroporin action, which can be potentially leveraged for the development of antiviral therapeutics. Our results corroborate with previously published transcriptomic data from COVID-19 infected lung alveolar cells where inflammatory responses and molecular regulators directly impacted by ion channelling were upregulated.

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  1. Version published to 10.1101/2021.09.26.461873v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.09.26.461873: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Experimental Models: Organisms/Strains
    SentencesResources
    To identify the processes encoded by the upregulated genes, we used the publicly available protocol in Metascape (www.metascape.org/; [55]).
    www.metascape.org/
    suggested: None
    Software and Algorithms
    SentencesResources
    2.1 Multiple sequence alignment: In order to assess the conservation among the coronavirus Orf3a-proteins, a protein-protein BLAST (
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    Using a threshold of 75% sequence identity as filters, we aligned the resulting sequences using the online alignment tool Clustal Omega [31] and was visualized with Jalview (www.jalview.org).
    Clustal Omega
    suggested: (Clustal Omega, RRID:SCR_001591)
    Jalview
    suggested: (Jalview, RRID:SCR_006459)
    All the homology modelling procedures have been performed using MODELLER [36] and SWISS-MODEL [37].
    MODELLER
    suggested: (MODELLER, RRID:SCR_008395)
    Model refinement and further structural fine-tuning of unreliable structural regions were done using the GalaxyWeB server [38].
    GalaxyWeB
    suggested: (GalaxyWEB, RRID:SCR_018558)
    The structures obtained were validated by scores obtained from the MolProbity [39,40].
    MolProbity
    suggested: (MolProbity, RRID:SCR_014226)
    The structural models of proteins and lipids were presented using the CHARMM36 force field parameters [44,45] and NAMD 2.12 [46,47] was used to run the molecular dynamics (MD) simulations.
    NAMD
    suggested: (NAMD, RRID:SCR_014894)
    , RMSD, RMSF and solvent accessible surface area (SASA) of specific pore forming residues were analysed with respect to time steps as obtained from the results of the NAMD simulation in VMD [51] interface and snapshots of the timesteps were represented and visualized using Chimera 1.10 [52].
    Chimera
    suggested: (Chimera, RRID:SCR_002959)
    Data was downloaded from Gene Expression Omnibus library [ID: GSE152586] and preprocessing of the fastq files were performed.
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    The DESeq2 package in R BioConductor (http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html) was used to analyze the data.
    BioConductor
    suggested: (Bioconductor, RRID:SCR_006442)
    For dimensional reduction and outlier identification, we performed a principal component analysis (PCA) on the DESeq2 data class of count reads.
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    To identify the processes encoded by the upregulated genes, we used the publicly available protocol in Metascape (www.metascape.org/; [55]).
    Metascape
    suggested: (Metascape, RRID:SCR_016620)
    We annotated the functions encoded by the genes using the following gene ontology enrichment: Biological processes, Cellular components, Molecular components, KEGG pathway and Reactome pathways.
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    Reactome
    suggested: (Reactome, RRID:SCR_003485)

    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: We detected the following sentences addressing limitations in the study:
    Indeed, our studies are in silico and have limitations of being non-experimental from an in vitro and in vivo standpoint. However, the impact of ionic imbalances in cellular micro-environment as a result of viral infections and viroporins have been studied in great detail earlier. One immediate observation comes from the previous SARS strain, the SARS-CoV-1. It was shown that E protein localized in the ERGIC membrane, and facilitated the movement of Ca2+ ions into the cytosol [69]. On the other hand, the Orf3a localized at the Golgi apparatus and the plasma membrane, transported K+ ions [70]. A proper regulation of airway epithelial ionic balance is indispensable for healthy homoeostasis in the lungs. A tight regulation of cationic and anionic ion channels controls the ionic homeostasis in the airways which can be correlated to complex pathological features in lung diseases [71]. Viroporins localized in the subcellular membranes of these lung airway epithelial cells is the primary cause of ionic imbalance and thus can be potential therapeutic targets against ARDS, which is the primary reason for fatality in SARS-CoV-2 infection [72,73]. Transcriptomics analysis from patient samples affected with SARS-CoV-2 showed an upregulation in inflammatory response mediated by several interleukins and interferons [74] which are probably regulated by NFκB [75]. Increase in CD40 [76], IL-6 [77], IL-12 [78] and IL-33 [79] transcripts strongly correlate with similar expression patterns of di...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 41 and 43. 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:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

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

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  3. Version published to 10.1101/2021.09.26.461873v1 on bioRxiv