A Rare Deletion in SARS-CoV-2 ORF6 Dramatically Alters the Predicted Three-Dimensional Structure of the Resultant Protein

  1. Marco A. Riojas
  2. Andrew M. Frank
  3. Nikhita P. Puthuveetil
  4. Beth Flores
  5. Michael Parker
  6. Stephen P. King
  7. Malik Peiris
  8. Daniel K. W. Chu
  9. Briana Benton
  10. Rebecca Bradford
  11. Manzour Hernando Hazbón
  12. Sujatha Rashid

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Abstract

The function of the SARS-CoV-2 accessory protein p6, encoded by ORF6, is not fully known. Based upon its similarity to p6 from SARS-CoV, it may play a similar role, namely as an antagonist of type I interferon (IFN) signaling. Here we report the sequencing of a SARS-CoV-2 strain passaged six times after original isolation from a clinical patient in Hong Kong. The genome sequence shows a 27 nt in-frame deletion (Δ27,264-27,290) within ORF6, predicted to result in a 9 aa deletion ( ΔFKVSIWNLD ) from the central portion of p6. This deletion is predicted to result in a dramatic alteration in the three-dimensional structure of the resultant protein (p6 Δ22-30 ), possibly with significant functional implications. Analysis of the original clinical sample indicates that the deletion was not present, while sequencing of subsequent passages of the strain identifies the deletion as a majority variant. This suggests that the deletion originated ab initio during passaging and subsequently propagated into the majority, possibly due to the removal of selective pressure through the IFN-deficient Vero E6 cell line. The specific function of the SARS-CoV-2 p6 N-terminus, if any, has not yet been determined. However, this deletion is predicted to cause a shift from N-endo to N-ecto in the transmembrane localization of the SARS-CoV-2 p6 Δ22-30 N-terminus, possibly leading to the ablation of its native function.

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    SciScore for 10.1101/2020.06.09.134460: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableViral Isolation and Passaging: SARS-CoV-2 Hong Kong/VM20001061/2020 was isolated from a nasopharyngeal aspirate and throat swab from 39-year-old male patient in Hong Kong on January 22, 2020.15 The complete genome of the SARS-CoV-2 Hong Kong/VM20001061/2020 clinical isolate (Passage 0) has been sequenced (GISAID: EPI_ISL_412028).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Viral Growth and Extraction: Using the virus deposited with BEI Resources (hereinafter referred to as “HKU Passage 5”), we inoculated Vero E6 cells (ATCC® CRL-1586™) using an multiplicity of infection (MOI) of 0.01, adsorbed for one hour at 37°C before adding viral growth media consisting of Eagle’s Minimum Essential Medium (ATCC® 30-2003™) supplemented with 2% fetal bovine serum (ATCC® 30-2020™).
    Vero E6
    suggested: None
    Software and Algorithms
    SentencesResources
    For de novo assembly, reads were assembled into contigs using SPAdes v3.12.0 under “RNA-seq” mode, and the resulting de novo contigs were filtered based on taxonomy using the One Codex Database version a76e9f6d3b23445d.
    SPAdes
    suggested: (SPAdes, RRID:SCR_000131)
    Reference-based assembly was then performed by mapping the reads to the genome sequence of the clinical isolate (GISAID: EPI_ISL_412028) using minimap2 default parameters and short read preset mode -x sr, and mapping statistics were generated by the bamqc feature of QualiMap v2.2.1.
    QualiMap
    suggested: (QualiMap, RRID:SCR_001209)
    We first employed the One Codex SARS-CoV-2 pipeline (https://github.com/onecodex/sars-cov-2), which also uses minimap2 to map reads to the SARS-CoV-2 reference genome (GenBank MN908947.3, RefSeq NC_045512.2), but uses iVar16 for viral variant calling and consensus generation.
    RefSeq
    suggested: (RefSeq, RRID:SCR_003496)
    We aligned the One Codex consensus sequence, the IRMA consensus sequence, and our de novo and consensus sequences using Muscle 3.8.425.
    Muscle
    suggested: (MUSCLE, RRID:SCR_011812)

    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 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 11 and 12. 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.

    About SciScore

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  2. Version published to 10.1101/2020.06.09.134460v1 on bioRxiv