We have identified seven putative guanine quadruplexes (G4) in the RNA genome of tick-borne encephalitis virus (TBEV), a flavivirus causing thousands of human infections and numerous deaths every year. The formation of G4s was confirmed by biophysical methods on synthetic oligonucleotides derived from the predicted TBEV sequences. TBEV-5, located at the NS4b/NS5 boundary and conserved among all known flaviviruses, was tested along with its mutated variants for interactions with a panel of known G4 ligands, for the ability to affect RNA synthesis by the flaviviral RNA-dependent RNA polymerase (RdRp) and for effects on TBEV replication fitness in cells. G4-stabilizing TBEV-5 mutations strongly inhibited RdRp RNA synthesis and exhibited substantially reduced replication fitness, different plaque morphology and increased sensitivity to G4-binding ligands in cell-based systems. In contrast, strongly destabilizing TBEV-5 G4 mutations caused rapid reversion to the wild-type genotype. Our results suggest that there is a threshold of stability for G4 sequences in the TBEV genome, with any deviation resulting in either dramatic changes in viral phenotype or a rapid return to this optimal level of G4 stability. The data indicate that G4s are critical elements for efficient TBEV replication and are suitable targets to tackle TBEV infection.

Department of Biophysical Chemistry and Molecular Oncology Institute of Biophysics of the Czech Academy of Sciences Brno CZ 61200 Czech Republic

Department of Biophysics of Nucleic Acids Institute of Biophysics of the Czech Academy of Sciences Brno CZ 61200 Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University CZ 62500 Brno Czech Republic

Faculty of Tropical Agrisciences Czech University of Life Sciences Prague CZ 16500 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy ofSciences CZ 16000 Prague Czech Republic

Institute of Parasitology Biology Centre of the Czech Academy of Sciences CZ 37005 Ceske Budejovice Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University CZ 62500 Brno Czech Republic

Veterinary Research Institute Emerging Viral Diseases Brno CZ 62100 Czech Republic

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RNA G-quadruplex formation in biologically important transcribed regions: can two-tetrad intramolecular RNA quadruplexes be formed?