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Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus.

Tuplin A, Evans DJ, Buckley A, Jones IM, Gould EA, Gritsun TS - Nucleic Acids Res. (2011)

Bottom Line: Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses.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.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Warwick, Coventry, CV4 7AL, Cranfield Health, Cranfield University, Cranfield, Bedfordshire MK43 0AL, UK.

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.

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Effect of point mutations on SL6 conformation as predicted by MFold. Point mutations are shown in circles. Names of the mutants are indicated on the top. Free dG energy of folding is indicated underneath each structure.
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Figure 4: Effect of point mutations on SL6 conformation as predicted by MFold. Point mutations are shown in circles. Names of the mutants are indicated on the top. Free dG energy of folding is indicated underneath each structure.

Mentions: The predicted impact of each substitution (Figure 3) on the secondary structure of SL6 is shown in Figure 4. The plaque characteristics, cpe and growth dynamics of each mutant compared with those of original pGGVs virus (Table 1 and Figure 5). Single-step growth curves revealed differences of ∼1 log10 between the mutants early after infection (12–16 h p.i.) which were reproducible and statistically significant (Figure 5).Figure 4.


Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus.

Tuplin A, Evans DJ, Buckley A, Jones IM, Gould EA, Gritsun TS - Nucleic Acids Res. (2011)

Effect of point mutations on SL6 conformation as predicted by MFold. Point mutations are shown in circles. Names of the mutants are indicated on the top. Free dG energy of folding is indicated underneath each structure.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3303483&req=5

Figure 4: Effect of point mutations on SL6 conformation as predicted by MFold. Point mutations are shown in circles. Names of the mutants are indicated on the top. Free dG energy of folding is indicated underneath each structure.
Mentions: The predicted impact of each substitution (Figure 3) on the secondary structure of SL6 is shown in Figure 4. The plaque characteristics, cpe and growth dynamics of each mutant compared with those of original pGGVs virus (Table 1 and Figure 5). Single-step growth curves revealed differences of ∼1 log10 between the mutants early after infection (12–16 h p.i.) which were reproducible and statistically significant (Figure 5).Figure 4.

Bottom Line: Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses.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.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Warwick, Coventry, CV4 7AL, Cranfield Health, Cranfield University, Cranfield, Bedfordshire MK43 0AL, UK.

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.

Show MeSH
Related in: MedlinePlus