Emergence of new subgenomic mRNAs in SARS-CoV-2

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Two mutations occurred in SARS-CoV-2 early during the COVID-19 pandemic that have come to define circulating virus lineages 1 : first a change in the spike protein (D614G) that defines the B.1 lineage and second, a double substitution in the nucleocapsid protein (R203K, G204R) that defines the B.1.1 lineage, which has subsequently given rise to three Variants of Concern: Alpha, Gamma and Omicron. While the latter mutations appear unremarkable at the protein level, there are dramatic implications at the nucleotide level: the GGG→AAC substitution generates a new Transcription Regulatory Sequence (TRS) motif, driving SARS-CoV-2 to express a novel subgenomic mRNA (sgmRNA) encoding a truncated C-terminal portion of nucleocapsid (N.iORF3), which is an inhibitor of type I interferon production. We find that N.iORF3 also emerged independently within the Iota variant, and further show that additional TRS motifs have convergently evolved to express novel sgmRNAs; notably upstream of Spike within the nsp16 coding region of ORF1b, which is expressed during human infection. Our findings demonstrate that SARS-CoV-2 is undergoing evolutionary changes at the functional RNA level in addition to the amino acid level, reminiscent of eukaryotic evolution. Greater attention to this aspect in the assessment of emerging strains of SARS-CoV-2 is warranted.

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  1. SciScore for 10.1101/2022.04.20.488895: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Membranes were probed with anti-Nucleocapsid (MA5-35943, Invitrogen, 1:400), anti-HSP90 (MA5-35624, Invitrogen, 1:2000) or anti-Membrane (MRC PPU & CVR Coronavirus Toolkit, Sheep No. DA107, 1:800), followed by IRdye secondary antibodies (Li-Cor), allowing visualisation on an Odyssey CLx imaging system (Li-Cor).
    anti-Nucleocapsid (MA5-35943
    suggested: None
    anti-Nucleocapsid (MA5-35943, Invitrogen, 1:400), anti-HSP90 (MA5-35624, Invitrogen, 1:2000)
    suggested: None
    suggested: (Thermo Fisher Scientific Cat# MA5-35624, RRID:AB_2849524)
    suggested: (Thermo Fisher Scientific Cat# MA5-35624, RRID:AB_2849524)
    Experimental Models: Cell Lines
    Cell Lines, Viruses, and In Vitro Infections: Vero E6 (Pasteur), Vero V1 (a gift from Stephen Goodbourn) and VeroE6 ACE2-TMPRSS2 cells36 (a gift from Suzannah Rihn) were maintained in Dulbecco’s Modified Eagle Medium, supplemented with 10% foetal calf serum and penicillin-streptomycin (100 U/mL each).
    Vero E6
    suggested: None
    Virus stocks were propagated in Vero V1 cells by infection at an MOI of 0.01 in Dulbecco’s Modified Eagle Medium, supplemented with 1% foetal calf serum and penicillin-streptomycin (100 U/mL each), harvested when CPE was visible, and stocks were titrated on Vero E6 cells.
    Vero V1
    suggested: None
    To determine innate immune responses, RT-qPCR was carried out using the Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent) with 0.8 µM primers, against IFNb, IFIT1 or GAPDH (see “Primer Sequences”, below), and cDNA from 5 ng of RNA extracted from transfected HEK293T cells.
    suggested: RRID:CVCL_HA71)
    Recombinant DNA
    Plasmids: Sequences for N and N.iORF3 were amplified from cDNA from B.1.1.7-infected cells, to include 5′ NheI and 3′ NotI sites, then ligated into pCDNA3-T2A-mCherry, to generate pCDNA3-B117-N-T2A-mCherry and pCDNA3-B117-Nstar-T2A-mCherry.
    suggested: None
    suggested: None
    pCDNA3-NS1 was a kind gift from Caetano Reis e Sousa.
    suggested: None
    Transfection: HEK293T cells were transfected at 70 % confluency in 24-well plates, with 25, 50 or 100 fmol pCDNA3-B117-N-T2A-mCherry or pCDNA3-B117-Nstar-T2A-mCherry from B.1.1.7, or 100 fmol pCDNA-NS1, or mock transfected, using Lipofectamine 2000 (Invitrogen) at a 2:1 ratio according to the manufacturer’s instructions, in antibiotic-free DMEM.
    suggested: None
    suggested: None
    Software and Algorithms
    Alignments were visualised in Integrative Genomics Viewer40, and statistics were extracted using the samtools flagstat command.
    suggested: None
    Quantification of read statistics were plotted using GraphPad Prism.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    As the ‘--keeplength’ flag to MAFFT carries the potential to create artefactual alignments by removing nucleotides inserted relative to the reference, for each novel location of either sequence motif, the corresponding genomes were extracted and re-aligned to the Wuhan-Hu-1 reference using MAFFT without this flag to ensure that reported sites were valid.
    suggested: (MAFFT, RRID:SCR_011811)
    To screen for possible convergent evolution, we examined the Audacity tree made available by the GISAID Initiative34, using a mutation-annotated tree inferred by UShER43.
    suggested: None

    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.