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Virus Genes

, Volume 52, Issue 3, pp 309–316 | Cite as

Expression of a second open reading frame present in the genome of tick-borne encephalitis virus strain Neudoerfl is not detectable in infected cells

  • Jiří ČernýEmail author
  • Martin Selinger
  • Martin Palus
  • Zuzana Vavrušková
  • Hana Tykalová
  • Lesley Bell-Sakyi
  • Ján Štěrba
  • Libor Grubhoffer
  • Daniel Růžek
Article

Abstract

A short upstream open reading frame (uORF) was recently identified in the 5′ untranslated region of some tick-borne encephalitis virus (TBEV) strains. However, it is not known if the peptide encoded by TBEV uORF (TuORF) is expressed in infected cells. Here we show that TuORF forms three phylogenetically separated clades which are typical of European, Siberian, and Far-Eastern TBEV subtypes. Analysis of selection pressure acting on the TuORF area showed that it is under positive selection pressure. Theoretically, TuORF may code for a short hydrophobic peptide embedded in a biological membrane. However, expression of TuORF was detectable neither by immunoblotting in tick and mammalian cell lines infected with TBEV nor by immunofluorescence in TBEV-infected mammalian cell lines. These results support the idea that TuORF is not expressed in TBEV-infected cell or expressed in undetectably low concentrations. Therefore we can assume that TuORF has either minor or no biological role in the TBEV life cycle.

Keywords

TBEV uORF TuORF Immunoblotting Immunofluorescence 

Notes

Acknowledgments

We would like to thank B. Černá Bolfíková for her help with phylogenetic analyses, F. X. Heinz (Medical University of Vienna, Austria) for TBEV strain Neudoerfl, T. Eckschlager (Chales University in Prague, Czech Republic) for human neural cell lines, and M. Bloom (Rocky Mountain Laboratories, USA) for anti-NS3 antibody. The tick cell line IRE/CTVM19 was provided by the Tick Cell Biobank. This work was supported by the Czech Science Foundation [P502/11/2116 and GA14-29256S to D. R. and 15-03044S to L. G.), Grant Agency of University of South Bohemia [155/2013/P to L. G.], the Ministry of Education, Youth, and Sports of the Czech Republic [Z60220518 to D. R.], ANTIGONE [278976 to L. G.], and by Project LO1218, with financial support from the MEYS of the Czech Republic under the NPU I program. J. C. and J. S. are the postdoctoral fellows supported by the Project Postdok BIOGLOBE (CZ. 1.07/2.3.00/30.0032) co-financed by the European Social Fund and state budget of the Czech Republic. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors contribution

JC did all the bioinformatics predictions and phylogenetic calculations; he participated in the immunofluorescence and Western blot experiments and he drafted the manuscript. JC, MS, MP, HT, and ZV grew the cells and did TBEV infections. JS, MS, and ZV carried out the immunofluorescence and Western blot experiments, assisted by JC, HT, and MP. LBS provided the tick cell line and critically revised the manuscript. LG and DR supervised all work and participated in the manuscript revisions.

Compliance with ethical standards

Conflict of interest

The authors have declared no competing interests.

Supplementary material

11262_2015_1273_MOESM1_ESM.docx (23 kb)
Supplementary material 1 Sequence analysis of uORFs detected in other Flavivirus species: Full length sequence of 5´ UTRs of Flavivirus species with detected uORFs (A). uORF sequences are marked in color, while remaining part of the 5´ UTR is in grey. uORF start and stop codons as well as major ORF start codons are underlined. Sequence of putative peptides encoded by detected uORFs (B). Alignment of putative peptides encoded by detected Flavivirus uORFs (C). GenBank accession numbers of all nucleotide sequences used in this study are listed in Supplementary Table 2. Protein sequences of hypothetical TuORF peptides were deduced from nucleotide sequences as indicated in Methods. (DOCX 22 kb)
11262_2015_1273_MOESM2_ESM.jpg (2.2 mb)
Supplementary material 2 Phylogenetic analysis of TuORF relationships: Phylogenetic analysis based on nucleotide (A) and protein (B) sequences of TuORF showed existence of three clearly separated phylogenetic clades. Only bootstrap values on which the tree separation into three clades is based are shown. The first clade unites European subtype TBEV strains (encircled in red). The second clade includes Siberian subtype TBEV strains (encircled in blue). The third clade comprises Far Eastern subtype TBEV strains (encircled in green). (JPEG 2232 kb)
11262_2015_1273_MOESM3_ESM.jpg (62 kb)
Supplementary material 3 Detection limit of the synthetic TuORF peptide by immunoblotting: To estimate the detection limit of the TuORF peptide by immunoblotting, we tested different concentrations of the synthetic TuORF in ten-fold dilutions from 100μg to 10pg. The lowest detectable amount of the synthetic TuORF was 10pg. (JPEG 61 kb)
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Supplementary material 4 (XLSX 13 kb)
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Supplementary material 5 (XLSX 11 kb)
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Supplementary material 6 (XLSX 18 kb)
11262_2015_1273_MOESM7_ESM.xlsx (10 kb)
Supplementary material 7 (XLSX 9 kb)

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Jiří Černý
    • 1
    • 2
    • 3
    Email author
  • Martin Selinger
    • 1
    • 2
  • Martin Palus
    • 1
    • 2
    • 3
  • Zuzana Vavrušková
    • 1
  • Hana Tykalová
    • 1
    • 2
  • Lesley Bell-Sakyi
    • 4
  • Ján Štěrba
    • 1
    • 2
  • Libor Grubhoffer
    • 1
    • 2
  • Daniel Růžek
    • 1
    • 3
  1. 1.Institute of ParasitologyBiology Centre of the Czech Academy of SciencesČeské BudějoviceCzech Republic
  2. 2.Faculty of ScienceUniversity of South Bohemia in České BudějoviceČeské BudějoviceCzech Republic
  3. 3.Veterinary Research InstituteBrnoCzech Republic
  4. 4.The Pirbright InstituteSurreyUK

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