Unification of the M/ORF3-related proteins points to a diversified role for ion conductance in pathogenesis of coronaviruses and other nidoviruses

  1. Yongjun Tan
  2. Theresa Schneider
  3. Prakash K. Shukla
  4. Mahesh B. Chandrasekharan
  5. L Aravind
  6. Dapeng Zhang

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Abstract

The new coronavirus, SARS-CoV-2, responsible for the COVID-19 pandemic has emphasized the need for a better understanding of the evolution of virus-host conflicts. ORF3a in both SARS-CoV-1 and SARS-CoV-2 are ion channels (viroporins) and involved in virion assembly and membrane budding. Using sensitive profile-based homology detection methods, we unify the SARS-CoV ORF3a family with several families of viral proteins, including ORF5 from MERS-CoVs, proteins from beta-CoVs (ORF3c), alpha-CoVs (ORF3b), most importantly, the Matrix (M) proteins from CoVs, and more distant homologs from other nidoviruses. By sequence analysis and structural modeling, we show that these viral families utilize specific conserved polar residues to constitute an ion-conducting pore in the membrane. We reconstruct the evolutionary history of these families, objectively establish the common origin of the M proteins of CoVs and Toroviruses. We show that the divergent ORF3a/ORF3b/ORF5 families represent a duplication stemming from the M protein in alpha- and beta-CoVs. By phyletic profiling of major structural components of primary nidoviruses, we present a model for their role in virion assembly of CoVs, ToroVs and Arteriviruses. The unification of diverse M/ORF3 ion channel families in a wide range of nidoviruses, especially the typical M protein in CoVs, reveal a conserved, previously under-appreciated role of ion channels in virion assembly, membrane fusion and budding. We show that the M and ORF3 are under differential evolutionary pressures; in contrast to the slow evolution of M as core structural component, the CoV-ORF3 clade is under selection for diversification, which indicates it is likely at the interface with host molecules and/or immune attack.

IMPORTANCE

Coronaviruses (CoVs) have become a major threat to human welfare as the causative agents of several severe infectious diseases, namely Severe Acute Respiratory Syndrome (SARS), Middle Eastern Respiratory Syndrome (MERS), and the recently emerging human coronavirus disease 2019 (COVID-19). The rapid spread, severity of these diseases, as well as the potential re-emergence of other CoV-associated diseases have imposed a strong need for a thorough understanding of function and evolution of these CoVs. By utilizing robust domain-centric computational strategies, we have established homologous relationships between many divergent families of CoV proteins, including SARS-CoV/SARS-CoV-2 ORF3a, MERS-CoV ORF5, proteins from both beta-CoVs (ORF3c) and alpha-CoVs (ORF3b), the typical CoV Matrix proteins, and many distant homologs from other nidoviruses. We present evidence that they are active ion channel proteins, and the Cov-specific ORF3 clade proteins are under selection for rapid diversification, suggesting they might have been involved in interfering host molecules and/or immune attack.

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    SciScore for 10.1101/2020.11.10.377366: (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

    Software and Algorithms
    SentencesResources
    Similarity-based clustering was conducted by BLASTCLUST, a BLAST score-based single-linkage clustering method (ftp://ftp.ncbi.nih.gov/blast/documents/blastclust.html).
    BLASTCLUST
    suggested: (BLASTClust, RRID:SCR_016641)
    MUSCLE (30) and PROMALS3D (31) programs, followed by careful manual adjustments based on the profile–profile alignment and predicted structural information.
    MUSCLE
    suggested: (MUSCLE, RRID:SCR_011812)
    Secondary structure was predicted using the JPRED program (32).
    JPRED
    suggested: (Jpred, RRID:SCR_016504)
    The alignments were visualized using CHROMA program (33) and further modified using adobe illustrator.
    adobe illustrator
    suggested: (Adobe Illustrator, RRID:SCR_010279)
    Structural analysis and comparison were conducted using the molecular visualization program PyMOL (38)
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    Molecular phylogenetic analysis: Based on the super-alignment of the β-sandwich domains of nine M/ORF3 families, we conducted phylogenetic analysis using three robust methods, including the Maximum Likelihood (ML) analysis implemented in the MEGA7 program (21), an approximately-maximum-likelihood method implemented in the FastTree 2.1 program (20), and Bayesian Inference implemented in the BEAST 1.8.3 program (22).
    MEGA7
    suggested: None
    BEAST
    suggested: (BEAST, RRID:SCR_010228)
    For FastTree analysis, default parameters were applied, which include the WAG evolutionary model and the discrete gamma model with 20 rate categories.
    FastTree
    suggested: (FastTree, RRID:SCR_015501)
    The tree with the highest log likelihood from the ML analysis was visualized using the FigTree 1.4.4 program (http://tree.bio.ed.ac.uk/).
    FigTree
    suggested: (FigTree, RRID:SCR_008515)

    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.

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  2. Version published to 10.1101/2020.11.10.377366v1 on bioRxiv