Production of TBEV VLP as a tool for studying of flavivirus protein-gRNA interactions

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60461373%3A22330%2F25%3A43932641" target="_blank" >RIV/60461373:22330/25:43932641 - isvavai.cz</a>

Result on the web

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DOI - Digital Object Identifier

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Result language

angličtina

Original language name

Production of TBEV VLP as a tool for studying of flavivirus protein-gRNA interactions

Original language description

Background:Tick-Borne Encephalitis Virus (TBEV) is an RNA virus belonging to the flavivirus genus that causes tick-borne encephalitis (TBE). The virus is primarily transmitted through the bite of infected ticks, most commonly from the species Ixodes ricinus. It can also be transmitted through the consumption of unpasteurized milk or dairy products.Objectives:We are studying the incorporating of gRNA into TBEV viral particles, as the mechanism of this specificity remains unknown. In Dengue and Zika viruses, this binding is mediated by nonstructural protein NS2A and the 3&apos; UTR of gRNA. To investigate the mechanism in TBEV, we use virus-like particles (VLPs), which are non-infectious and easy to handle.Methods:To produce TBE virus-like particles (VLPs) we utilized a combination of two vectors: one encoding structural proteins: capsid (C), membrane (M), envelope (E) and nonstructural proteins (NS1 and NS2A) and the second encoding TBEV protease (NS3) and its cofactor NS2B. This combination is sufficient for the assembly and released of the VLPs. To determine the specificity of the genomic RNA (gRNA) incorporation, we used vectors encoding either the 5&apos; or 3&apos; untranslated regions (UTRs) or their combination under the control of the CMV promoter. By combining imunoassays, ultracentrifugation, transmission electron microscopy and quantitative PCR, we identified preferences for gRNA segment interaction.Results:To test which region is critical for gRNA recognition and binding, we generated a series of mutations within C and NS2A proteins. We observed that incorporation of the 5′ UTR into VLPs was approximately twice as effective compared to the 3′ UTR alone, as well as compared to the combination of both the 5′ and 3′ UTRs.Conclusions:Our findings suggest that the 5′ UTR plays a key role in gRNA incorporation into TBEV VLPs. A better understanding of the specific components involved in gRNA packaging is essential for elucidating the mechanisms underlying the formation of infectious virus particles.This work was supported by the project National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103) - Funded by the European Union - Next Generation EU.

Czech name

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Czech description

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Type

O - Miscellaneous

CEP classification

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OECD FORD branch

10607 - Virology

Project

<a href="/en/project/LX22NPO5103" target="_blank" >LX22NPO5103: National Institute of Virology and Bacteriology</a><br>

Continuities

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Publication year

2025

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů