Identification of a critical horseshoe-shaped region in the nsp5 (Mpro, 3CLpro) protease interdomain loop (IDL) of coronavirus mouse hepatitis virus (MHV)

  1. Benjamin C. Nick
  2. Mansi C. Pandya
  3. Xiaotao Lu
  4. Megan E. Franke
  5. Sean M. Callahan
  6. Emily F. Hasik
  7. Sean T. Berthrong
  8. Mark R. Denison
  9. Christopher C. Stobart

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Abstract

Human coronaviruses are enveloped, positive-strand RNA viruses which cause respiratory diseases ranging in severity from the seasonal common cold to SARS and COVID-19. Of the 7 human coronaviruses discovered to date, 3 emergent and severe human coronavirus strains (SARS-CoV, MERS-CoV, and SARS-CoV-2) have recently jumped to humans in the last 20 years. The COVID-19 pandemic spawned by the emergence of SARS-CoV-2 in late 2019 has highlighted the importance for development of effective therapeutics to target emerging coronaviruses. Upon entry, the replicase genes of coronaviruses are translated and subsequently proteolytically processed by virus-encoded proteases. Of these proteases, nonstructural protein 5 (nsp5, Mpro, or 3CLpro), mediates the majority of these cleavages and remains a key drug target for therapeutic inhibitors. Efforts to develop nsp5 active-site inhibitors for human coronaviruses have thus far been unsuccessful, establishing the need for identification of other critical and conserved non-active-site regions of the protease. In this study, we describe the identification of an essential, conserved horseshoe-shaped region in the nsp5 interdomain loop (IDL) of mouse hepatitis virus (MHV), a common coronavirus replication model. Using site-directed mutagenesis and replication studies, we show that several residues comprising this horseshoe-shaped region either fail to tolerate mutagenesis or were associated with viral temperature-sensitivity. Structural modeling and sequence analysis of these sites in other coronaviruses, including all 7 human coronaviruses, suggests that the identified structure and sequence of this horseshoe regions is highly conserved and may represent a new, non-active-site regulatory region of the nsp5 (3CLpro) protease to target with coronavirus inhibitors.

Importance

In December 2019, a novel coronavirus (SARS-CoV-2) emerged in humans and triggered a pandemic which has to date resulted in over 8 million confirmed cases of COVID-19 across more than 180 countries and territories (June 2020). SARS-CoV-2 represents the third emergent coronavirus in the past 20 years and the future emergence of new coronaviruses in humans remains certain. Critically, there remains no vaccine nor established therapeutics to treat cases of COVID-19. The coronavirus nsp5 protease is a conserved and indispensable virus-encoded enzyme which remains a key target for therapeutic design. However, past attempts to target the active site of nsp5 with inhibitors have failed stressing the need to identify new conserved non-active-site targets for therapeutic development. This study describes the discovery of a novel conserved structural region of the nsp5 protease of coronavirus mouse hepatitis virus (MHV) which may provide a new target for coronavirus drug development.

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    SciScore for 10.1101/2020.06.18.160671: (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

    Antibodies
    SentencesResources
    Lysates were separated on a 4 - 15% polyacrylamide gel, transferred to a PVDF membrane, and blotted using an MHV nsp8-specific rabbit primary antibody, anti-nsp8 (VU123) (29).
    anti-nsp8
    suggested: (Acris Antibodies GmbH Cat# AP09089SU-N, RRID:AB_2035808)
    VU123
    suggested: None
    Western blots were resolved using an HRP-conjugated goat anti-rabbit secondary antibody and Western ECL substrate (Bio-Rad).
    anti-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Virus experiments were performed in murine delayed brain tumor 9 (DBT-9) cells, which are naturally permissive to MHV-A59 infection, and baby hamster kidney cells which express the MHV receptor (BHK-R) under selection with 0.8 mg/ml of G418. Complete Dulbecco’s Modified Eagle medium (DMEM, VWR) supplemented with 10% fetal bovine serum (FBS), 1% HEPES, and an antibiotic-antimycotic solution (Corning) containing penicillin, streptomycin, and amphotericin B, was used for growth of both DBT-9 and BHK-R cells.
    BHK-R
    suggested: None
    Viral replication assays and efficiency of plating (EOP) analysis: To evaluate viral replication kinetics, DBT-9 cells were grown to near confluency (~90 - 100%) in 6-well plates prior to infection with a virus multiplicity of infection (MOI) of 0.01 PFU/cell at either 37°C or 40°C.
    DBT-9
    suggested: None
    Software and Algorithms
    SentencesResources
    Sequence and structural analyses: The nsp5 IDL sequences of MHV-A59 and all 7 HCoVs were aligned using CLUSTALW and analyzed for sequence conservation using WebLogo (31).
    CLUSTALW
    suggested: (ClustalW, RRID:SCR_017277)
    WebLogo
    suggested: (WEBLOGO, RRID:SCR_010236)
    Structural analysis of coronavirus nsp5 proteases was performed using PyMol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.) using the following protease structures available in the Protein Data Bank (PDB) or the Protein Modeling Data Base (
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    All statistical analysis were performed using JMP (SAS Institute, Cary, NC).
    SAS Institute
    suggested: (Statistical Analysis System, RRID:SCR_008567)

    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:
    • 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.

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  2. Version published to 10.1101/2020.06.18.160671v1 on bioRxiv