SARS-CoV-2 is an intensively investigated virus from the order Nidovirales (Coronaviridae family) that causes COVID-19 disease in humans. Through enormous scientific effort, thousands of viral strains have been sequenced to date, thereby creating a strong background for deep bioinformatics studies of the SARS-CoV-2 genome. In this study, we inspected high-frequency mutations of SARS-CoV-2 and carried out systematic analyses of their overlay with inverted repeat (IR) loci and CpG islands. The main conclusion of our study is that SARS-CoV-2 hot-spot mutations are significantly enriched within both IRs and CpG island loci. This points to their role in genomic instability and may predict further mutational drive of the SARS-CoV-2 genome. Moreover, CpG islands are strongly enriched upstream from viral ORFs and thus could play important roles in transcription and the viral life cycle. We hypothesize that hypermethylation of these loci will decrease the transcription of viral ORFs and could therefore limit the progression of the disease.

Department of Biology and Ecology Faculty of Science University of Ostrava Ostrava Czech Republic

Department of Biophysical Chemistry and Molecular Oncology Institute of Biophysics of the Czech Academy of Sciences Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Department of Mathematics Faculty of Science University of Ostrava Ostrava Czech Republic

Department of Physics Faculty of Science University of Ostrava Ostrava Czech Republic

Faculty of Informatics Masaryk University Brno Czech Republic

Global Change Research Institute of the Czech Academy of Sciences Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno Czech Republic

See more in PubMed

Wu F, Zhao S, Yu B, et al. . A new coronavirus associated with human respiratory disease in China. Nature 2020;579:265–9. PubMed PMC

Naqvi AAT, Fatima K, Mohammad T, et al. . Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: structural genomics approach. Biochim Biophys Acta Mol Basis Dis 1866;2020:165878. PubMed PMC

Shu Y, McCauley J. GISAID: global initiative on sharing all influenza data—from vision to reality. Euro Surveill 2017;22:13. PubMed PMC

Andersen KG, Rambaut A, Lipkin WI, et al. . The proximal origin of SARS-CoV-2. Nat Med 2020;26:450–2. PubMed PMC

Wu A, Niu P, Wang L, et al. . Mutations, recombination and insertion in the evolution of 2019-nCoV. Preprint. bioRxiv 2020;2020.02.29.971101. Published 2020 Mar 2. doi: 10.1101/2020.02.29.971101. DOI

Matyášek R, Kovařík A. Mutation patterns of human SARS-CoV-2 and bat RaTG13 coronavirus genomes are strongly biased towards C>U transitions, indicating rapid evolution in their hosts. Genes 2020;11:761. PubMed PMC

Zhou P, Yang X-L, Wang X-G, et al. . A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020;579:270–3. PubMed PMC

Zhang T, Wu Q, Zhang Z. Probable pangolin origin of SARS-CoV-2 associated with the COVID-19 outbreak. Curr Biol 2020;30:1346, e2–51. PubMed PMC

Chan JF-W, Kok K-H, Zhu Z, et al. . Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerg Microbes Infect 2020;9:221–36. PubMed PMC

Chen Y, Guo Y, Pan Y, et al. . Structure analysis of the receptor binding of 2019-nCoV. Biochem Biophys Res Commun 2020;525:135–40. PubMed PMC

Luan J, Lu Y, Jin X, et al. . Spike protein recognition of mammalian ACE2 predicts the host range and an optimized ACE2 for SARS-CoV-2 infection. Biochem Biophys Res Commun 2020;526:165–9. PubMed PMC

Othman H, Bouslama Z, Brandenburg J-T, et al. . Interaction of the spike protein RBD from SARS-CoV-2 with ACE2: similarity with SARS-CoV, hot-spot analysis and effect of the receptor polymorphism. Biochem Biophys Res Commun 2020;527:702–8. PubMed PMC

Lu R, Zhao X, Li J, et al. . Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. Lancet 2020;395:565–74. PubMed PMC

Zeng W, Liu G, Ma H, et al. . Biochemical characterization of SARS-CoV-2 nucleocapsid protein. Biochem Biophys Res Commun 2020;527:618–23. PubMed PMC

Kim D, Lee J-Y, Yang J-S, et al. . The architecture of SARS-CoV-2 transcriptome. Cell 2020;181:914–21.e10. PubMed PMC

Wang C, Liu Z, Chen Z, et al. . The establishment of reference sequence for SARS-CoV-2 and variation analysis. J Med Virol 2020;99:667–74. doi: 10.1002/jmv.25762. PubMed DOI PMC

Kim J-S, Jang J-H, Kim J-M, et al. . Genome-wide identification and characterization of point mutations in the SARS-CoV-2 genome. Osong Public Health Res Perspect 2020;11:101–11. PubMed PMC

Pachetti M, Marini B, Benedetti F, et al. . Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant. J Transl Med 2020;18:179. PubMed PMC

Brázda V, Laister RC, Jagelská EB, et al. . Cruciform structures are a common DNA feature important for regulating biological processes. BMC Mol Biol 2011;12:33. PubMed PMC

Nelson LD, Bender C, Mannsperger H, et al. . Triplex DNA-binding proteins are associated with clinical outcomes revealed by proteomic measurements in patients with colorectal cancer. Mol Cancer 2012;11:38. PubMed PMC

Métifiot M, Amrane S, Litvak S, et al. . G-quadruplexes in viruses: function and potential therapeutic applications. Nucleic Acids Res 2014;42:12352–66. PubMed PMC

Zhao J, Bacolla A, Wang G, et al. . Non-B DNA structure-induced genetic instability and evolution. Cell Mol Life Sci 2010;67:43–62. PubMed PMC

Bartas M, Brazda V, Bohálová N, et al. . In-depth bioinformatic analyses of Nidovirales including human SARS-CoV-2, SARS-CoV, MERS-CoV viruses suggest important roles of noncanonical nucleic acid structures in their lifecycles. Front Microbiol 2020;11:1583. PubMed PMC

Pearson CE, Zorbas H, Price GB, et al. . Inverted repeats, stem-loops, and cruciforms: significance for initiation of DNA replication. J Cell Biochem 1996;63:1–22. PubMed

Bikard D, Loot C, Baharoglu Z, et al. . Folded DNA in action: hairpin formation and biological functions in prokaryotes. Microbiol Mol Biol Rev 2010;74:570–88. PubMed PMC

Svoboda P, Di Cara A. Hairpin RNA: a secondary structure of primary importance. Cell Mol Life Sci 2006;63:901–8. PubMed PMC

Lu J, Gao F, Wei Z, et al. . A 5′-proximal stem-loop structure of 5′ untranslated region of porcine reproductive and respiratory syndrome virus genome is key for virus replication. Virol J 2011;8:172. PubMed PMC

Wakida H, Kawata K, Yamaji Y, et al. . Stability of RNA sequences derived from the coronavirus genome in human cells. Biochem Biophys Res Commun 2020;527:993–9. PubMed PMC

Lu S, Wang G, Bacolla A, et al. . Short inverted repeats are hotspots for genetic instability: relevance to cancer genomes. Cell Rep 2015;10:1674–80. PubMed PMC

Xia X. Extreme genomic CpG deficiency in SARS-CoV-2 and evasion of host antiviral defense. Mol Biol Evol 2020;37:2699–705. doi: 10.1093/molbev/msaa094. PubMed DOI PMC

Cheng X, Virk N, Chen W, et al. . CpG usage in RNA viruses: data and hypotheses. PLOS One 2013;8:e74109. PubMed PMC

Alinejad-Rokny H, Anwar F, Waters SA, et al. . Source of CpG depletion in the HIV-1 genome. Mol Biol Evol 2016;33:3205–12. PubMed

Gu H, Fan RLY, Wang D, et al. . Dinucleotide evolutionary dynamics in influenza a virus. Virus Evol 2019;5:vez038. PubMed PMC

Trus I, Udenze D, Berube N, et al. . CpG-recoding in Zika virus genome causes host-age-dependent attenuation of infection with protection against lethal heterologous challenge in mice. Front Immunol 2020;10:3077. PubMed PMC

Burns CC, Campagnoli R, Shaw J, et al. . Genetic inactivation of poliovirus infectivity by increasing the frequencies of CpG and UpA dinucleotides within and across synonymous capsid region codons. J Virol 2009;83:9957–69. PubMed PMC

Balakrishnan L, Milavetz B. Epigenetic regulation of viral biological processes. Viruses 2017;9:346. PubMed PMC

Dorp L, Acman M, Richard D, et al. . Emergence of genomic diversity and recurrent mutations in SARS-CoV-2. Infect Genet Evol 2020;83:104351. PubMed PMC

Varshney D, Spiegel J, Zyner K, et al. . The regulation and functions of DNA and RNA G-quadruplexes. Nat Rev Mol Cell Biol 2020;21:459–74. PubMed PMC

Jaubert C, Bedrat A, Bartolucci L, et al. . RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand. Sci Rep 2018;8:8120. PubMed PMC

Ji D, Juhas M, Tsang CM, Kwok CK, Li Y, Zhang Y.. Discovery of G-quadruplex-forming sequences in SARS-CoV-2 [published online ahead of print, 2020 Jun 1]. Brief Bioinform 2020;bbaa114. doi: 10.1093/bib/bbaa114. PubMed DOI PMC

Xie J, Mao Q, Tai PW, et al. . Short DNA hairpins compromise recombinant adeno-associated virus genome homogeneity. Mol Ther 2017;25:1363–74. PubMed PMC

Bridges R, Correia S, Wegner F, et al. . Essential role of inverted repeat in Epstein–Barr virus IR-1 in B cell transformation; geographical variation of the viral genome. Philos T R Soc B 2019;374:20180299. PubMed PMC

Ishimaru D, Plant EP, Sims AC, et al. . RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus. Nucleic Acids Res 2013;41:2594–608. PubMed PMC

Goorha R, Granoff A, Willis DB, et al. . The role of DNA methylation in virus replication: inhibition of frog virus 3 replication by 5-azacytidine. Virology 1984;138:94–102. PubMed

Tsai K, Jaguva Vasudevan AA, Martinez Campos C, et al. . Acetylation of cytidine residues boosts HIV-1 gene expression by increasing viral RNA stability. Cell Host & Microbe 2020;28:306–312.e6. PubMed PMC

Girelli D, Martinelli N, Pizzolo F, et al. . The interaction between MTHFR 677 C→T genotype and folate status is a determinant of coronary atherosclerosis risk. J Nutr 2003;133:1281–5. PubMed

Leclerc D, Sibani S, Rozen R. Molecular biology of methylenetetrahydrofolate reductase (MTHFR) and overview of mutations/polymorphisms. In: MTHFR Polymorphisms and Disease. Georgetown, TX: Landes Bioscience/Eurekah.com, 2005, 1–20.

Cui S, Li W, Lv X, et al. . Folic acid supplementation delays atherosclerotic lesion development by modulating MCP1 and VEGF DNA methylation levels in vivo and in vitro. Int J Mol Sci 2017;18:990. PubMed PMC

Piyathilake CJ, Macaluso M, Alvarez RD, et al. . A higher degree of methylation of the HPV 16 E6 gene is associated with a lower likelihood of being diagnosed with cervical intraepithelial neoplasia. Cancer 2011;117:957–63. PubMed PMC

Piyathilake CJ, Macaluso M, Chambers MM, et al. . Folate and vitamin B12 may play a critical role in lowering the HPV 16 methylation-associated risk of developing higher grades of CIN. Cancer Prev Res (Phila) 2014;7:1128–37. PubMed PMC

McNulty H, Strain JJ, Hughes CF, et al. . Riboflavin, MTHFR genotype and blood pressure: a personalized approach to prevention and treatment of hypertension. Mol Aspects Med 2017;53:2–9. PubMed

Paschos K, Allday MJ. Epigenetic reprogramming of host genes in viral and microbial pathogenesis. Trends Microbiol 2010;18:439–47. PubMed PMC

Biswas B, Kandpal M, Vivekanandan P. A G-quadruplex motif in an envelope gene promoter regulates transcription and virion secretion in HBV genotype B. Nucleic Acids Res 2017;45:11268–80. PubMed PMC

Brázda V, Hároníková L, Liao JCC, et al. . DNA and RNA quadruplex-binding proteins. Int J Mol Sci 2014;15:17493–517. PubMed PMC

Ruggiero E, Richter SN. Viral G-quadruplexes: new frontiers in virus pathogenesis and antiviral therapy. Annu Rep Med Chem 2020;54:101–31. PubMed PMC

Bohálová N, Cantara A, Bartas M, et al. . How to be invisible? Viruses causing acute infections are significantly depleted for G-quadruplex forming sequences. Genomics Submitted for publication. 2020.

Page AJ, Taylor B, Delaney AJ, et al. . SNP-sites: rapid efficient extraction of SNPs from multi-FASTA alignments. Microb Genom 2016;2:e000056. PubMed PMC

Altschul SF, Gish W, Miller W, et al. . Basic local alignment search tool. J Mol Biol 1990;215:403–10. PubMed

Brown NP, Leroy C, Sander C. MView: a web-compatible database search or multiple alignment viewer. Bioinformatics 1998;14:380–1. PubMed

Brázda V, Kolomazník J, Lýsek J, et al. . Palindrome analyser—a new web-based server for predicting and evaluating inverted repeats in nucleotide sequences. Biochem Biophys Res Commun 2016;478:1739–45. PubMed

Afgan E, Baker D, Batut B, et al. . The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res 2018;46:W537–44. PubMed PMC

Rice P, Longden I, Bleasby A. EMBOSS: the European molecular biology open software suite. Trends Genet 2000;16:276–7. PubMed

Stothard P. The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques 2000;28:1102–4. PubMed

Asymmetric distribution of G-quadruplex forming sequences in genomes of retroviruses

Special Issue "Bioinformatics of Unusual DNA and RNA Structures"

Variability of Inverted Repeats in All Available Genomes of Bacteria

G-quadruplexes in the evolution of hepatitis B virus

Beyond the Primary Structure of Nucleic Acids: Potential Roles of Epigenetics and Noncanonical Structures in the Regulations of Plant Growth and Stress Responses

Genomic Analysis of Non-B Nucleic Acids Structures in SARS-CoV-2: Potential Key Roles for These Structures in Mutability, Translation, and Replication?

Inverted repeats in the monkeypox virus genome are hot spots for mutation

Interaction of Proteins with Inverted Repeats and Cruciform Structures in Nucleic Acids

Letter to the Editor: Significant mutation enrichment in inverted repeat sites of new SARS-CoV-2 strains

Mutational Asymmetries in the SARS-CoV-2 Genome May Lead to Increased Hydrophobicity of Virus Proteins