SARS-CoV-2 genomes from Saudi Arabia implicate nucleocapsid mutations in host response and increased viral load

  1. Tobias Mourier
  2. Muhammad Shuaib
  3. Sharif Hala
  4. Sara Mfarrej
  5. Fadwa Alofi
  6. Raeece Naeem
  7. Afrah Alsomali
  8. David Jorgensen
  9. Amit Kumar Subudhi
  10. Fathia Ben Rached
  11. Qingtian Guan
  12. Rahul P. Salunke
  13. Amanda Ooi
  14. Luke Esau
  15. Olga Douvropoulou
  16. Raushan Nugmanova
  17. Sadhasivam Perumal
  18. Huoming Zhang
  19. Issaac Rajan
  20. Awad Al-Omari
  21. Samer Salih
  22. Abbas Shamsan
  23. Abbas Al Mutair
  24. Jumana Taha
  25. Abdulaziz Alahmadi
  26. Nashwa Khotani
  27. Abdelrahman Alhamss
  28. Ahmed Mahmoud
  29. Khaled Alquthami
  30. Abdullah Dageeg
  31. Asim Khogeer
  32. Anwar M. Hashem
  33. Paula Moraga
  34. Eric Volz
  35. Naif Almontashiri
  36. Arnab Pain

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Abstract

Monitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia from March to August 2020. We show that two consecutive mutations (R203K/G204R) in the nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found increased interaction of GSK3A kinase simultaneously with hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein. Furthermore, the host cell transcriptome analysis suggests that the mutant N protein produces dysregulated interferon response genes. Here, we provide crucial information in linking the R203K/G204R mutations in the N protein to modulations of host-virus interactions and underline the potential of the nucleocapsid protein as a drug target during infection.

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  1. Version published to 10.1038/s41467-022-28287-8
  2. Version published to 10.1101/2021.05.06.21256706v2 on medRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.05.06.21256706: (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
    For affinity confirmation, bound proteins were eluted using buffer BXT (IBA Lifesciences) and after running on SDS-PAGE were subjected to silver staining and western-blot using anti-strep-II antibody (ab76949).
    anti-strep-II
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The raw reads from HEK293T RNA-sequencing were processed and trimmed using trimmomatic 60 and mapped to annotated ENSEMBL transcripts from the human genome (hg19) 79,80 using kallisto81.
    HEK293T
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmid and cloning: The pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro was a gift from Nevan Krogan (Addgene plasmid # 141391; http://n2t.net/addgene:141391; RRID:Addgene_141391)36.
    detected: RRID:Addgene_141391)
    Software and Algorithms
    SentencesResources
    Genome assembly, SNP and indel calling: Illumina adapters and low quality sequences were trimmed using Trimmomatic v0.38 60.
    Trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    Reads were mapped to SARS-CoV-2 Wuhan-Hu-1 NCBI reference sequence NC_045512.2 using BWA 61
    BWA
    suggested: (BWA, RRID:SCR_010910)
    Mapped reads were processed using GATK v 4.1.7 pipeline commands MarkDuplicatesSpark, HaplotypeCaller, VariantFiltration, SelectVariants, BaseRecalibrator, ApplyBQSR, and HaplotypeCaller to identify variants 62.
    GATK
    suggested: (GATK, RRID:SCR_001876)
    Differential expression analysis was performed after normalization using EdgeR integrated in the NetworkAnalyst 82.
    EdgeR
    suggested: (edgeR, RRID:SCR_012802)

    Results from OddPub: Thank you for sharing your code and 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:
    • 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.

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  4. Version published to 10.1101/2021.05.06.21256706v1 on medRxiv