title: Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus.
abstract: We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem-loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.
authors.affiliation: School of Life Sciences, University of Warwick, Coventry, CV4 7AL, Cranfield Health, Cranfield University, Cranfield, Bedfordshire MK43 0AL, UK.
authors.forename: A
authors.initials: A
authors.lastname: Tuplin
authors.forename: D J
authors.initials: DJ
authors.lastname: Evans
authors.forename: A
authors.initials: A
authors.lastname: Buckley
authors.forename: I M
authors.initials: IM
authors.lastname: Jones
authors.forename: E A
authors.initials: EA
authors.lastname: Gould
authors.forename: T S
authors.initials: TS
authors.lastname: Gritsun
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004669:Encephalitis Viruses, Tick-Borne / Q000235:genetics* / Q000502:physiology; D004742:Enhancer Elements, Genetic; D019143:Evolution, Molecular; D005416:Flavivirus / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D012367:RNA, Viral / Q000737:chemistry*; D014779:Virus Replication
chemical_list: D012367:RNA, Viral