Mutating novel interaction sites in NRP1 reduces SARS-CoV-2 spike protein internalization

  1. Debjani Pal
  2. Kuntal De
  3. Timothy B. Yates
  4. Wellington Muchero

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Abstract

The global pandemic of Coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 has become a severe global health problem because of its rapid spread( 1 ). Both angiotensin-converting enzyme 2 and neuropilin 1 provide initial viral binding sites for SARS-CoV-2 ( 2, 3 ). Here, we show that three cysteine residues located in a1/a2 and b1 domains of neuropilin 1 are necessary for SARS-CoV-2 spike protein internalization in human cells. Mutating cysteines C82, C104 and C147 altered neuropilin 1 stability and binding ability as well as cellular internalization and lysosomal translocation of the spike protein. This resulted in up to 4 times reduction in spike protein load in cells for the original, alpha and delta SARS-CoV-2 variants even in the presence of the endogenous angiotensin-converting enzyme 2 receptor. Transcriptome analysis of cells transfected with mutated NRP1 revealed significantly reduced expression of genes involved in viral infection and replication, including eight members of the ribosomal protein L, ten members of ribosomal protein S and five members of the proteasome β subunit family proteins. We also observed higher expression of genes involved in suppression of inflammation and endoplasmic reticulum associated degradation. These observations suggest that these cysteines offer viable targets for therapies against COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2021.10.11.463689: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    HA antibody (HA.C5 #18181; 1:1000) and LAMP1 antibody (#24170) were purchased from Abcam.
    LAMP1
    suggested: (Abcam Cat# ab24170, RRID:AB_775978)
    M2 anti Flag Mouse antibody (#SLBT7654; 1:5000) and Actin (#087M4850; 1:10,000) were purchased from Sigma.
    M2 anti Flag Mouse antibody (#SLBT7654; 1:5000)
    suggested: None
    anti Flag Mouse antibody
    suggested: None
    Actin
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Mammalian cell culture, transfection, and drug treatment: HeLa and HEK 293T cells were obtained from ATCC and maintained in a humidified atmosphere at 5% CO2 in Dulbecco’s Modified Eagle’s (DMEM) complete medium (Corning) supplemented with 10% fetal bovine serum (FBS; Seradigm) in 37°C.
    HeLa
    suggested: None
    Western Blotting and immunoprecipitation: For immunoprecipitation, either HA-tagged NRP1 (or mutants) and Flag-tagged SARS-CoV-2-S (or S1493-685) mutant or HA-tagged NRP1 (or mutants) and Flag-Plexin-A1 were expressed where indicated in 293T cells for 30 hours.
    293T
    suggested: None
    RNA-seq analysis: For RNA-seq analysis, Total RNA was extracted from HEK293T cell line 30 hours post transfection (HA-NRP1 and SARS-CoV-2-S1493-685 and or HA-NRP1-4Cys-4Ala and SARS-CoV-2-S1493-685) using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions.
    HEK293T
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids and recombinant proteins: HA-NRP1 and different mutants of HA-NRP1, Flag-Plexin-A1, Flag-SARS-CoV-2-S-original (NC_045512.2), Flag-SARS-CoV-2-B.1.1.7-S, Flag-SARS-CoV-B.1.351-S were all obtained from GenScript (Cloned into pcDNA 3.1 vector).
    pcDNA 3.1
    suggested: RRID:Addgene_20407)
    For cycloheximide stability assay, 293T cells were transfected with the constructs encoding HA-NRP1 or HA-NRP1 mutants.
    HA-NRP1
    suggested: None
    Software and Algorithms
    SentencesResources
    The PAN domain coordinates from Uniprot were used to extract the PAN domain sequences from the full-length proteins which were then aligned with MAFFT linsi(54).
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    The alignment was visualized with Geneious.
    Geneious
    suggested: (Geneious, RRID:SCR_010519)
    The raw RNA-seq reads have been deposited at NCBI under BioProject ID PRJNA767890.
    BioProject
    suggested: (NCBI BioProject, RRID:SCR_004801)
    Reads were mapped with bowtie2 v2.2.5. (55).
    bowtie2
    suggested: (Bowtie 2, RRID:SCR_016368)
    Differential expression analysis was performed with DESeq2, genes with an adjusted p-value less than 0.05 were considered differentially expressed (56).
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    Statistical analysis: Statistical analyses were performed on individual experiments, as indicated, with GraphPad Prism 8 Software using an unpaired t-Test, equal variance for comparison between two groups and one-way ANOVA for comparisons between more than two groups.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    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 did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.10.11.463689v1 on bioRxiv