Expression of a second open reading frame present in the genome of tick-borne encephalitis virus strain Neudoerfl is not detectable in infected cells
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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.
KeywordsTBEV uORF TuORF Immunoblotting Immunofluorescence
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.
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.
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Conflict of interest
The authors have declared no competing interests.
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