Hepatitis B virus (HBV) is one of the most dangerous human pathogenic viruses found in all corners of the world. Recent sequencing of ancient HBV viruses revealed that these viruses have accompanied humanity for several millenia. As G-quadruplexes are considered to be potential therapeutic targets in virology, we examined G-quadruplex-forming sequences (PQS) in modern and ancient HBV genomes. Our analyses showed the presence of PQS in all 232 tested HBV genomes, with a total number of 1258 motifs and an average frequency of 1.69 PQS per kbp. Notably, the PQS with the highest G4Hunter score in the reference genome is the most highly conserved. Interestingly, the density of PQS motifs is lower in ancient HBV genomes than in their modern counterparts (1.5 and 1.9/kb, respectively). This modern frequency of 1.90 is very close to the PQS frequency of the human genome (1.93) using identical parameters. This indicates that the PQS content in HBV increased over time to become closer to the PQS frequency in the human genome. No statistically significant differences were found between PQS densities in HBV lineages found in different continents. These results, which constitute the first paleogenomics analysis of G4 propensity, are in agreement with our hypothesis that, for viruses causing chronic infections, their PQS frequencies tend to converge evolutionarily with those of their hosts, as a kind of 'genetic camouflage' to both hijack host cell transcriptional regulatory systems and to avoid recognition as foreign material.

Faculty of Chemistry Brno University of Technology Purkyňova 118 612 00 Brno Czech Republic

Institute of Biophysics of the Czech Academy of Sciences Brno Czech Republic

Laboratoire d'Optique et Biosciences Ecole Polytechnique CNRS INSERM Institut Polytechnique de Paris 91120 Palaiseau France

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Bock C.T., Schranz P., Schröder C.H., Zentgraf H.. Hepatitis B virus genome is organized into nucleosomes in the nucleus of the infected cell. Virus Genes. 1994; 8:215–229. PubMed

Newbold J.E., Xin H., Tencza M., Sherman G., Dean J., Bowden S., Locarnini S.. The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes. J. Virol. 1995; 69:3350–3357. PubMed PMC

Summers J., Mason W.S.. Replication of the genome of a hepatitis B–like virus by reverse transcription of an RNA intermediate. Cell. 1982; 29:403–415. PubMed

Tiollais P., Pourcel C., Dejean A.. The hepatitis B virus. Nature. 1985; 317:489–495. PubMed

MacDonald D.M., Holmes E.C., Lewis J.C., Simmonds P.. Detection of hepatitis B virus infection in wild-born chimpanzees (Pan troglodytes verus): phylogenetic relationships with human and other primate genotypes. J. Virol. 2000; 74:4253–4257. PubMed PMC

Drexler J.F., Geipel A., König A., Corman V.M., van Riel D., Leijten L.M., Bremer C.M., Rasche A., Cottontail V.M., Maganga G.D.et al. .. Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 2013; 110:16151–16156. PubMed PMC

Lauber C., Seitz S., Mattei S., Suh A., Beck J., Herstein J., Börold J., Salzburger W., Kaderali L., Briggs J.A.G.et al. .. Deciphering the origin and evolution of hepatitis B viruses by means of a family of non-enveloped fish viruses. Cell Host Microbe. 2017; 22:387–399. PubMed PMC

de Carvalho Dominguez Souza B.F., König A., Rasche A., de Oliveira Carneiro I., Stephan N., Corman V.M., Roppert P.L., Goldmann N., Kepper R., Müller S.F.et al. .. A novel hepatitis B virus species discovered in capuchin monkeys sheds new light on the evolution of primate hepadnaviruses. J. Hepatol. 2018; 68:1114–1122. PubMed

Kocher A., Papac L., Barquera R., Key F.M., Spyrou M.A., Hübler R., Rohrlach A.B., Aron F., Stahl R., Wissgott A.et al. .. Ten millennia of hepatitis B virus evolution. Science. 2021; 374:182–188. PubMed

Perrone R., Butovskaya E., Daelemans D., Palù G., Pannecouque C., Richter S.N.. Anti-HIV-1 activity of the G-quadruplex ligand BRACO-19. J. Antimicrob. Chemother. 2014; 69:3248–3258. PubMed

Frasson I., Soldà P., Nadai M., Tassinari M., Scalabrin M., Gokhale V., Hurley L.H., Richter S.N.. Quindoline-derivatives display potent G-quadruplex-mediated antiviral activity against herpes simplex virus 1. Antiviral Res. 2022; 208:105432. PubMed PMC

Zhai L.-Y., Su A.-M., Liu J.-F., Zhao J.-J., Xi X.-G., Hou X.-M.. Recent advances in applying G-quadruplex for SARS-CoV-2 targeting and diagnosis: a review. Int. J. Biol. Macromol. 2022; 221:1476–1490. PubMed PMC

Puig Lombardi E., Londoño-Vallejo A.. A guide to computational methods for G-quadruplex prediction. Nucleic Acids Res. 2020; 48:1–15. PubMed PMC

Ruggiero E., Richter S.N.. Viral G-quadruplexes: new frontiers in virus pathogenesis and antiviral therapy. Annu. Rep. Med. Chem. 2020; 54:101–131. PubMed PMC

Teng Y., Zhu M., Chi Y., Li L., Jin Y.. Can G-quadruplex become a promising target in HBV therapy?. Front. Immunol. 2022; 13:1091873. PubMed PMC

Chakraborty D., Ghosh S.. The epsilon motif of hepatitis B virus RNA exhibits a potassium-dependent ribonucleolytic activity. FEBS J. 2017; 284:1184–1203. PubMed

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; 45:11268–11280. PubMed PMC

Saranathan N., Vivekanandan P.. G-Quadruplexes: more Than Just a Kink in Microbial Genomes. Trends Microbiol. 2019; 27:148–163. PubMed PMC

Meier-Stephenson V., Badmalia M.D., Mrozowich T., Lau K.C.K., Schultz S.K., Gemmill D.L., Osiowy C., van Marle G., Coffin C.S., Patel T.R.. Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus covalently closed circular DNA. J. Biol. Chem. 2021; 296:100589. PubMed PMC

Fleming A.M., Nguyen N.L.B., Burrows C.J.. Colocalization of m6A and G-quadruplex-forming sequences in viral RNA (HIV, zika, hepatitis B, and SV40) suggests topological control of adenosine N6-methylation. ACS Cent. Sci. 2019; 5:218–228. PubMed PMC

Somkuti J., Molnár O.R., Grád A., Smeller L.. Pressure perturbation studies of noncanonical viral nucleic acid structures. Biology. 2021; 10:1173. PubMed PMC

Molnár O.R., Végh A., Somkuti J., Smeller L.. Characterization of a G-quadruplex from hepatitis B virus and its stabilization by binding TMPyP4, BRACO19 and PhenDC3. Sci. Rep. 2021; 11:23243. PubMed PMC

Sun J., Wu G., Pastor F., Rahman N., Wang W.-H., Zhang Z., Merle P., Hui L., Salvetti A., Durantel D.et al. .. RNA helicase DDX5 enables STAT1 mRNA translation and interferon signalling in hepatitis B virus replicating hepatocytes. Gut. 2022; 71:991–1005. PubMed PMC

Nurk S., Koren S., Rhie A., Rautiainen M., Bzikadze A.V., Mikheenko A., Vollger M.R., Altemose N., Uralsky L., Gershman A.et al. .. The complete sequence of a human genome. Science. 2022; 376:44–53. PubMed PMC

Okonechnikov K., Golosova O., Fursov M., Team U.. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics. 2012; 28:1166–1167. PubMed

Crooks G.E., Hon G., Chandonia J.-M., Brenner S.E.. WebLogo: a sequence logo generator. Genome Res. 2004; 14:1188–1190. PubMed PMC

Harkins K.M., Stone A.C.. Ancient pathogen genomics: insights into timing and adaptation. J. Hum. Evol. 2015; 79:137–149. PubMed

de-Dios T., Scheib C.L., Houldcroft C.J.. An adagio for viruses, played out on ancient DNA. Genome Biol. Evol. 2023; 15:evad047. PubMed PMC

Taubenberger J.K., Baltimore D., Doherty P.C., Markel H., Morens D.M., Webster R.G., Wilson I.A.. Reconstruction of the 1918 influenza virus: unexpected rewards from the past. Mbio. 2012; 3:e00201-12. PubMed PMC

Sudhan S.S., Sharma P.. Human viruses: emergence and evolution. Emerg. Reemerg. Viral Pathog. 2020; 2020:53–68.

Irving-Pease E.K., Muktupavela R., Dannemann M., Racimo F.. Quantitative human paleogenetics: what can ancient DNA tell us about complex trait evolution?. Front. Genet. 2021; 12:703541. PubMed PMC

Schädler S., Hildt E.. HBV life cycle: entry and morphogenesis. Viruses. 2009; 1:185–209. PubMed PMC

Bohálová N., Cantara A., Bartas M., Kaura P., Šťastný J., Pečinka P., Fojta M., Brázda V.. Tracing dsDNA virus-host coevolution through correlation of their G-quadruplex-forming sequences. Int. J. Mol. Sci. 2021; 22:3433. PubMed PMC

Bartas M., Brázda V., Bohálová N., Cantara A., Volná A., Stachurová T., Malachová K., Jagelská E.B., Porubiaková O., Červeň J.et al. .. In-depth bioinformatic analyses of nidovirales including human SARS-CoV-2, SARS-CoV, MERS-CoV viruses suggest important roles of non-canonical nucleic acid structures in their lifecycles. Front. Microbiol. 2020; 11:1583. PubMed PMC

Lavigne M., Helynck O., Rigolet P., Boudria-Souilah R., Nowakowski M., Baron B., Brülé S., Hoos S., Raynal B., Guittat L.et al. .. SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction. Nucleic Acids Res. 2021; 49:7695–7712. PubMed PMC

Ely B. Genomic GC content drifts downward in most bacterial genomes. PLoS One. 2021; 16:e0244163. PubMed PMC

Šmarda P., Bureš P., Horová L., Leitch I.J., Mucina L., Pacini E., Tichý L., Grulich V., Rotreklová O.. Ecological and evolutionary significance of genomic GC content diversity in monocots. Proc. Natl. Acad. Sci. U.S.A. 2014; 111:E4096–E4102. PubMed PMC

Wang Y., Mao J.-M., Wang G.-D., Luo Z.-P., Yang L., Yao Q., Chen K.-P.. Human SARS-CoV-2 has evolved to reduce CG dinucleotide in its open reading frames. Sci. Rep. 2020; 10:12331. PubMed PMC

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 (Basel). 2020; 11:761. PubMed PMC

Goswami P., Bartas M., Lexa M., Bohálová N., Volná A., Červeň J., Červeňová V., Pečinka P., Špunda V., Fojta M.et al. .. SARS-CoV-2 hot-spot mutations are significantly enriched within inverted repeats and CpG island loci. Brief Bioinform. 2021; 22:1338–1345. PubMed PMC

Brázda V., Porubiaková O., Cantara A., Bohálová N., Coufal J., Bartas M., Fojta M., Mergny J.-L.. G-quadruplexes in H1N1 influenza genomes. BMC Genomics [Electronic Resource]. 2021; 22:77. PubMed PMC

Bohálová N., Cantara A., Bartas M., Kaura P., Šťastný J., Pečinka P., Fojta M., Mergny J.-L., Brázda V.. Analyses of viral genomes for G-quadruplex forming sequences reveal their correlation with the type of infection. Biochimie. 2021; 186:13–27. PubMed

Brown J.C. High G+C content of herpes simplex virus DNA: proposed role in protection against retrotransposon insertion. Open Biochem. J. 2007; 1:33–42. PubMed PMC

Vinogradov A.E., Anatskaya O.V.. DNA helix: the importance of being AT-rich. Mamm. Genome. 2017; 28:455–464. PubMed

Calvignac-Spencer S., Düx A., Gogarten J.F., Patrono L.V.. Kielian M., Mettenleiter T.C., Roossinck M.J.. Chapter Two - Molecular archeology of human viruses. Advances in Virus Research. 2021; 111:Academic Press; 31–61. PubMed

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

Abundance of G-Quadruplex Forming Sequences in the Hepatitis Delta Virus Genomes