Endosomal ions and serum promote enterovirus uncoating

Enterovirusten B ryhmään kuuluva Echovirus 1 on RNA-virus, jonka tiedetään infektoivan soluja ei-happamien endosomien välityksellä ja kerääntyvän neutraaleihin multivesikulaarisiin kappaleisiin. Tämän tutkimuksen alussa oli olemassa hyvin rajallisesti tietoa fysiologisista tekijöistä, jotka johtavat...

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Päätekijä: Haapakoski, Marjo
Muut tekijät: Matemaattis-luonnontieteellinen tiedekunta, Faculty of Sciences, Bio- ja ympäristötieteiden laitos, Department of Biological and Environmental Science, Jyväskylän yliopisto, University of Jyväskylä
Aineistotyyppi: Pro gradu
Kieli:eng
Julkaistu: 2020
Aiheet:
Linkit: https://jyx.jyu.fi/handle/123456789/71029
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author Haapakoski, Marjo
author2 Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science Jyväskylän yliopisto University of Jyväskylä
author_facet Haapakoski, Marjo Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science Jyväskylän yliopisto University of Jyväskylä Haapakoski, Marjo Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science Jyväskylän yliopisto University of Jyväskylä
author_sort Haapakoski, Marjo
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description Enterovirusten B ryhmään kuuluva Echovirus 1 on RNA-virus, jonka tiedetään infektoivan soluja ei-happamien endosomien välityksellä ja kerääntyvän neutraaleihin multivesikulaarisiin kappaleisiin. Tämän tutkimuksen alussa oli olemassa hyvin rajallisesti tietoa fysiologisista tekijöistä, jotka johtavat enterovirusten avautumiseen. Tutkimuksen tarkoituksena oli selvittää in vitro millainen yhdistelmä ioneja edistää echovirus 1:n avautumista fysiologisessa lämpötilassa. Päämääränä oli myös tutkia seerumin vaikutusta viruksen avautumiseen. Spektroskooppisissa mittauksissa käytettiin hyväksi SYBR Green II:n kykyä sitoutua viruksen RNA:han ja saada aikaan voimakasta fluoresenssia siinä tilanteessa, kun virus on avautunut vähän tai RNA on kokonaan vapautunut viruksen ulkopuolelle. Viruksen infektiivisyyttä erilaisten käsittelyjen jälkeen arvioitiin muun muassa sytopaattisen määrityksen avulla. Radioaktiivisesti leimatun viruksen avautumisen taso varmistettiin lopulta sakkaroosi- ja cesiumkloridigradienttierottelulla. Matala natriumpitoisuus yhdistettynä korkeaan kaliumpitoisuuteen edisti viruksen avautumista tämän tutkimuksen tulosten mukaan. Seerumilla havaittiin olevan myös viruspartikkelin avautumista ja genomin vapautumista edistävä vaikutus. Lisäksi tutkimuksessa tuli ilmi, että MgCl2 ja CaCl2 vakauttavat viruksen rakennetta. Tämä tutkimus paljasti uutta tietoa fysiologisista tekijöistä, jotka edistävät enterovirusten avautumista. Tutkimuksen tulosten pohjalta on mahdollista suunnitella uusia aihetta syventäviä jatkotutkimuksia. Echovirus 1 (E1) belonging to the enterovirus B species is an RNA virus that enters non-acidic endosomes and accumulates in neutral multivesicular bodies. There was limited information about physiological triggers leading to uncoating of enteroviruses in the beginning of this study. The aim of this study was to evaluate in vitro what combinations of key ions promote uncoating of echovirus 1 at physiological temperature. Another aim of the study was to investigate the effect of serum on uncoating of the virus. Real-time fluorescence spectroscopy was used to measure the binding of SYBR Green II to viral RNA indicating the possible uncoating. End point dilution and cytopathic effect assay were performed to estimate viral infectivity after different treatments. Finally, sucrose and caesium chloride gradient separation of radioactively labeled virus was performed to confirm the state of uncoating. Low sodium concentration together with high potassium concentration induced the uncoating process according to our results. Addition of a small amount of serum enhanced the uncoating leading to the formation of the uncoating intermediate particle and genome egress. In addition, MgCl2 and CaCl2 prevented the uncoating process and, thus had a stabilizing effect in the assay. This study offered new information about molecular cues that trigger the uncoating under physiological conditions. Further studies can be designed based on these findings.
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spellingShingle Haapakoski, Marjo Endosomal ions and serum promote enterovirus uncoating viral uncoating a-particle echovirus 1 potassium RNA release sodium Solu- ja molekyylibiologia Cell and molecular biology 4013 virukset enterovirukset infektiot seerumi ionit virologia viruses enteroviruses infections serum ions virology
title Endosomal ions and serum promote enterovirus uncoating
title_full Endosomal ions and serum promote enterovirus uncoating
title_fullStr Endosomal ions and serum promote enterovirus uncoating Endosomal ions and serum promote enterovirus uncoating
title_full_unstemmed Endosomal ions and serum promote enterovirus uncoating Endosomal ions and serum promote enterovirus uncoating
title_short Endosomal ions and serum promote enterovirus uncoating
title_sort endosomal ions and serum promote enterovirus uncoating
title_txtP Endosomal ions and serum promote enterovirus uncoating
topic viral uncoating a-particle echovirus 1 potassium RNA release sodium Solu- ja molekyylibiologia Cell and molecular biology 4013 virukset enterovirukset infektiot seerumi ionit virologia viruses enteroviruses infections serum ions virology
topic_facet 4013 Cell and molecular biology RNA release Solu- ja molekyylibiologia a-particle echovirus 1 enterovirukset enteroviruses infections infektiot ionit ions potassium seerumi serum sodium viral uncoating virologia virology virukset viruses
url https://jyx.jyu.fi/handle/123456789/71029 http://www.urn.fi/URN:NBN:fi:jyu-202007025210
work_keys_str_mv AT haapakoskimarjo endosomalionsandserumpromoteenterovirusuncoating