Contribution of SARS-CoV-2 accessory proteins to viral pathogenicity in K18 hACE2 transgenic mice

  1. Jesus Silvas
  2. Desarey Morales-Vasquez
  3. Jun-Gyu Park
  4. Kevin Chiem
  5. Jordi B. Torrelles
  6. Roy Neal Platt
  7. Tim Anderson
  8. Chengjin Ye
  9. Luis Martinez-Sobrido

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Abstract

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. To date, it is estimated that over 113 million individuals have been infected with SARS-CoV-2 and over 2.5 million human deaths have been recorded worldwide. Currently, three vaccines have been approved by the Food and Drug Administration for emergency use only. However much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse genetics system approach to successfully engineer recombinant (r)SARS-CoV-2, where we individually removed viral 3a, 6, 7a, 7b, and 8 ORF proteins, and characterized these recombinant viruses in vitro and in vivo . Our results indicate differences in plaque morphology, with ORF deficient (ΔORF) viruses producing smaller plaques than those of the wild-type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2 identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b and ΔORF8 rSARS-CoV-2 induced comparable pathology to rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse genetics system to generate rSARS-CoV-2 and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live-attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19.

IMPORTANCE

Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and contribution of viral proteins to disease outcome remains elusive. Our study aims to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins in viral pathogenesis and disease outcome, and develop a synergistic platform combining our robust reverse genetics system to generate recombinant (r)SARS-CoV-2 with a validated rodent model of infection and disease. We demonstrated that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as the foundation to generate attenuated forms of the virus to develop live-attenuated vaccines for the treatment of SARS-CoV-2.

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  1. ScreenIT

    SciScore for 10.1101/2021.03.09.434696: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Biosafety (IBC) and Animal Care and Use (IACUC) committees.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableMice: Specific-pathogen-free, 4–8 week-old, female B6.Cg-Tg(K18-ACE2)2Prlmn/J (Stock No: 034860
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells were incubated overnight with 1 μg/ml of a SARS-CoV N protein cross-reactive polyclonal antibody at 4°C and a SARS-CoV-2 spike (S) cross-reactive monoclonal antibody (3B4), washed with PBS, and stained with a FITC-labeled goat anti-mouse IgG (1:200) and Rhodamine-labeled goat anti-rabbit IgG (1:200).
    anti-mouse IgG
    suggested: None
    anti-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells: African green monkey kidney epithelial cells (Vero E6, CRL-1586) were obtained from the American Type Culture Collection (ATCC, Bethesda, MD) and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% (v/v)
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: Specific-pathogen-free, 4–8 week-old, female B6.Cg-Tg(K18-ACE2)2Prlmn/J (Stock No: 034860
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    K18 hACE2 transgenic mice were purchased from The Jackson Laboratory (Bar Harbor, ME).
    K18 hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Software and Algorithms
    SentencesResources
    Statistical analysis: Statistical analysis was performed using GraphPad Prism 8.3.
    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 34. 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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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

    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 10.1101/2021.03.09.434696 on bioRxiv