SARS-CoV-2 variant spike and accessory gene mutations alter pathogenesis

  1. Marisa E. McGrath
  2. Yong Xue
  3. Carly Dillen
  4. Lauren Oldfield
  5. N. Assad-Garcia
  6. Jayshree Zaveri
  7. Natasha Singh
  8. Lauren Baracco
  9. Louis J. Taylor
  10. Sanjay Vashee
  11. Matthew B. Frieman

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Abstract

The ongoing COVID-19 pandemic is a major public health crisis. Despite the development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pandemic persists. The continued spread of the virus is largely driven by the emergence of viral variants, which can evade the current vaccines through mutations in the spike protein. Although these differences in spike are important in terms of transmission and vaccine responses, these variants possess mutations in the other parts of their genome that may also affect pathogenesis. Of particular interest to us are the mutations present in the accessory genes, which have been shown to contribute to pathogenesis in the host through interference with innate immune signaling, among other effects on host machinery. To examine the effects of accessory protein mutations and other nonspike mutations on SARS-CoV-2 pathogenesis, we synthesized both viruses possessing deletions in the accessory genes as well as viruses where the WA-1 spike is replaced by each variant spike gene in a SARS-CoV-2/WA-1 infectious clone. We then characterized the in vitro and in vivo replication of these viruses and compared them to both WA-1 and the full variant viruses. Our work has revealed that the accessory proteins contribute to SARS-CoV-2 pathogenesis and the nonspike mutations in variants can contribute to replication of SARS-CoV-2 and pathogenesis in the host. This work suggests that while spike mutations may enhance receptor binding and entry into cells, mutations in accessory proteins may alter clinical disease presentation.

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  1. Version published to 10.1073/pnas.2204717119
  2. ScreenIT

    SciScore for 10.1101/2022.05.31.494211: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: Infection of BALB/c and hACE2/k18 Mice: All animals were cared for according to the standards set forth by the Institutional Animal Care and Use Committee at the University of Maryland-Baltimore.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    BlindingH/E Staining of Lungs and Pathological Scoring: Lungs were scored in a blinded fashion with a 0-5 score given, 0 being no inflammation and 5 being the highest degree of inflammation.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Virus Reconstitution: 24 hours prior to transfection, 5e4 VeroE6 cells (ATCC,Manassas, VA) were plated per well in 1mL of VeroE6 media (DMEM (Quality Biological, Gaithersburg, MD), 10% FBS (Gibco, Waltham, MA), 1% Penicillin-Streptomycin (Gemini Bio Products, Sacramento, CA), 1% L-Glutamine (Gibco, Waltham, MA)).
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Experimental Models: Organisms/Strains
    SentencesResources
    Infection of BALB/c and hACE2/k18 Mice: All animals were cared for according to the standards set forth by the Institutional Animal Care and Use Committee at the University of Maryland-Baltimore.
    BALB/c
    suggested: None
    The K18-hACE2 mice were inoculated with 1e3 PFU of each virus in 50μL PBS.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Recombinant DNA
    SentencesResources
    To assemble DNA fragment clones, the TAR vectors were PCR amplified from pCC1BAC-his3 with KOD Xtreme Hot Start DNA polymerase (Millipore, Burlington, MA) using the construction primers (labeled “Con”, Table S1).
    pCC1BAC-his3
    suggested: None
    50 fmol of each amplicon and 15 fmol of YCpBAC vector were assembled using a standard Gibson assembly reaction (New England Biolabs, Ipswich, MA), transformed into E. coli DH10B competent cells (Thermo Fisher, Waltham, MA), and plated on LB medium with 12.5 mg/ml chloramphenicol.
    YCpBAC
    suggested: None
    Full-length genome assembly: The TAR vector for assembly of the full-length genome was amplified from pCC1BAC-ura3 using primers ConCMVpR and ConBGHtermF with KOD Xtreme Hot Start DNA polymerase (Millipore, Burlington, MA).
    pCC1BAC-ura3
    suggested: None
    Software and Algorithms
    SentencesResources
    Virus Reconstitution: 24 hours prior to transfection, 5e4 VeroE6 cells (ATCC,Manassas, VA) were plated per well in 1mL of VeroE6 media (DMEM (Quality Biological, Gaithersburg, MD), 10% FBS (Gibco, Waltham, MA), 1% Penicillin-Streptomycin (Gemini Bio Products, Sacramento, CA), 1% L-Glutamine (Gibco, Waltham, MA)).
    Quality Biological
    suggested: None
    The cells were rocked every 15 minutes for 1 hour at 37°C prior to overlay with 2mL of a solid agarose overlay (EMEM (Quality Biological, Gaithersburg, MD), 10% FBS, 1% Penicillin-Streptomycin, 1% L-Glutamine, 0.4% w/v SeaKem agarose (Lonza Biosciences,Morrisville, NC).
    Lonza Biosciences
    suggested: (Science Exchange, RRID:SCR_010620)
    Statistical Analysis: All statistical analyses were carried out using the GraphPad Prism software.
    GraphPad Prism
    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 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.

  3. Version published to 10.1101/2022.05.31.494211v1 on bioRxiv