hnRNPA1 regulates early translation to replication switch in SARS-CoV-2 life cycle

  1. Ram Kumar
  2. Yogesh Chander
  3. Nitin Khandelwal
  4. Himanshu Nagori
  5. Assim Verma
  6. Yash Pal
  7. Baldev R. Gulati
  8. Bhupendra N. Tripathi
  9. Sanjay Barua
  10. Naveen Kumar

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Abstract

Our study suggests that methylation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is essential for its optimal replication in the target cells. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1, an RNA-binding protein) was shown to mediate deposition of N6-methyladenosine (m6A) in internal SARS-CoV-2 RNA. The levels of hNRNPA1 expression and extent of methylation varied, depending on the course of SARS-CoV-2 life cycle. The recruitment of eIF4E (translational initiation factor) facilitated viral RNA translation at 1 hour post infection (1 hpi). However, at 2 hpi, methylation of internal SARS-CoV-2 RNA recruited hNRNPA1 which facilitated viral RNA transcription but resulted in translational repression, a phenomenon contributing in understanding the early translation to replication switch in the viral life cycle. Besides, the abrogation of methylation also produced a defective 5’ cap of viral RNA which failed to interact with eIF4E, thereby resulting in a decreased synthesis of viral proteins. To conclude, methylation of the internal and 5’ cap of SARS-CoV-2 RNA was shown to regulate transcription and translation of SARS-CoV-2 in a time dependent manner.

IMPORTANCE

RNA modifications are found in all life forms and have been linked to development, health and diseases. Our study reveals that internal SARS-CoV-2 RNA methylation (m6A) is essential for interaction with hNRNPA1 to effectively synthesize viral genome. Besides, m6A-marked RNA and hRNPA1 interaction was also shown to regulate early translation to replication switch in SARS-CoV-2 life cycle. Blocking SARS-CoV-2 RNA methylation resulted in reduced virus yield, suggesting epitranscriptomic machinery (methylation) facilitates SARS-CoV-2 replication and might represent potential target for new antiviral drugs against COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2021.07.13.452288: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Samples were received via civil surgeon (District Medical Officer) Hisar, India who granted the permission to collect the biological specimens.
    Consent: A due consent was also taken from the patient before collection of the specimens.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies: N6-Methyladenosine (m6A) (D9D9W
    Antibodies: N6-Methyladenosine
    suggested: None
    m6A
    suggested: (Cell Signaling Technology Cat# 56593, RRID:AB_2799515)
    ) Rabbit mAb and hnRNP A1 (D21H11) monoclonal antibodies were received from Cell Signalling Technology (Massachusetts, USA)
    hnRNP A1
    suggested: (Cell Signaling Technology Cat# 8443, RRID:AB_10828725)
    eIF4E monoclonal antibody (5D11) and Phospho-eIF4E (Ser209) polyclonal antibodies were received from Invitrogen (South San Francisco, CA, USA)
    Phospho-eIF4E
    suggested: None
    Ser209
    suggested: None
    Glyceraldehyde 3-phosphate dehydrogenase, house-keeping control protein) primary antibody, Anti-Mouse IgG (whole molecule)-Alkaline Phosphatase antibody (produced in goat) and Anti-Rabbit IgG (whole molecule)–Peroxidase antibody (produced in goat) was received from Sigma-Aldrich (St. Louis, USA)
    Glyceraldehyde 3-phosphate dehydrogenase , house-keeping control protein
    suggested: None
    Anti-Mouse IgG
    suggested: None
    molecule)-Alkaline Phosphatase
    suggested: None
    Anti-Rabbit IgG ( whole molecule)–Peroxidase
    suggested: None
    The pellet was discarded and the clarified cytosolic fraction was mixed with 10 units of RiboLock RNase, protease and phosphatase inhibitor cocktail and incubated with the Protein A Sepharose-bound m6A specific primary antibody (reactive antibody), Protein A Sepharose bound-phospho ERK antibody (non-reactive antibody) or an equivalent volume of IP buffer (beads control) overnight at 4°C on a rotary platform.
    ERK
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS-CoV-2 was propagated in Vero cells in the Biosafety level 3 (BSL-3) laboratory of ICAR-National Research Centre on Equines (NRCE), Hisar, India.
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Software and Algorithms
    SentencesResources
    Statistical analysis: Pairwise statistical comparisons were performed by two-tailed Student’s t-test in GraphPad Prism 8 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

    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.

  2. Version published to 10.1101/2021.07.13.452288v1 on bioRxiv