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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.<\/jats:p>","DOI":"10.1093\/nar\/gkac225","type":"journal-article","created":{"date-parts":[[2022,3,25]],"date-time":"2022-03-25T08:30:20Z","timestamp":1648197020000},"page":"4574-4600","source":"Crossref","is-referenced-by-count":24,"title":["Guanine quadruplexes in the RNA genome of the tick-borne encephalitis virus: their role as a new antiviral target and\u00a0in virus biology"],"prefix":"10.1093","volume":"50","author":[{"given":"Ji\u0159\u00ed","family":"Holoubek","sequence":"first","affiliation":[{"name":"Veterinary Research Institute, Emerging Viral Diseases, Brno\u00a0CZ-62100, Czech Republic"},{"name":"Department of Experimental Biology, Faculty of Science, Masaryk University, CZ-62500\u00a0Brno, Czech Republic"},{"name":"Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, CZ-37005\u00a0Ceske Budejovice, Czech 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