PKR phosphorylation as an Indicator for echovirus 1 genome egress from endosomes

Isäntäsolut ovat kehittäneet useita keinoja tunnistaa viruksia ja estää niiden lisääntyminen. Yksi näistä keinoista on translaatioon vaadittavan tukiproteiini eukaryotic intiation factor 2:n (eIF2) fosforylaatio, joka estää translaation tehokkaasti. Kaksijuosteisesta RNA:a (dsRNA) aktivoituva protei...

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Päätekijä: Aspelin, William
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: 2019
Aiheet:
Linkit: https://jyx.jyu.fi/handle/123456789/67530
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author Aspelin, William
author2 Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science Jyväskylän yliopisto University of Jyväskylä
author_facet Aspelin, William Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science Jyväskylän yliopisto University of Jyväskylä Aspelin, William Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science Jyväskylän yliopisto University of Jyväskylä
author_sort Aspelin, William
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description Isäntäsolut ovat kehittäneet useita keinoja tunnistaa viruksia ja estää niiden lisääntyminen. Yksi näistä keinoista on translaatioon vaadittavan tukiproteiini eukaryotic intiation factor 2:n (eIF2) fosforylaatio, joka estää translaation tehokkaasti. Kaksijuosteisesta RNA:a (dsRNA) aktivoituva protein kinase dsRNA-dependent-proteiini (PKR) on eIF2 kinaasi, joka itsefosforyloituu aktivoituessaan. Sekä PKR:N että eIF2:n fosforylaatio yhdistetään usein antiviraaliseen soluviestintään. Echovirus 1 (EV1) kuuluu enterovirusten B-alaryhmään, jonka jäsenistä useat on yhdistetty tyypin 1 diabeteksen puhkeamiseen sille altistuneissa lapsissa. Se tunkeutuu isäntäsoluihin endosominkaltaisissa rakkuloissa ja vapauttaa genominsa proteiinikuoresta näissä rakkuloissa. Ei ole täysin varmaa miten EV1 genomi pääsee rakkulakalvon läpi solulimaan, jossa translaatio ja replikaatio tapahtuvat. Tässä työssä osoitamme, että EV1-infektoituihin soluihin transfektoidut virusgenomille komplementaariset oligonukleotidikoettimet aktivoivat PKR:n fosforylaatiota muodostaessaan kompleksin virusgenomin kanssa solulimassa. Tätä fosforylaatiota voi käyttää osoittamaan, että EV1 genomi on vapautunut endosomirakkulasta solulimaan, mikä tapahtui A549 soluissa noin 2,5−3,5 tuntia infektion jälkeen. Pidemmälle kehitettynä tämä uusi metodi voi tulevaisuudessa auttaa selvittämään, miten EV1:n genomi vapautuu endosomista, löytämään siihen liittyviä proteiineja sekä kehittämään lääkkeitä ja rokotteita EV1:ä vastaan. Pathogens may trigger various antiviral responses in eukaryotic cells. One such response is preventing viral translation by eukaryotic initiation factor 2 (eIF2) phosphorylation. Protein kinase double-stranded RNA-dependent (PKR) is an eIF2 kinase activated by phosphorylation after exposure to double-stranded RNA (dsRNA). Though they act as hub elements for several stress signalling pathways, PKR and eIF2 phosphorylation is strongly associated with antiviral responses. Echovirus 1 (EV1) belongs to the enterovirus subgroup B, many members of which have been implicated in the development of type 1 diabetes in individuals exposed to the virus as infants, sparking interest in their biology and the development of drugs and vaccines. The major entry route of EV1 is via endosomal-like vesicles in which the uncoating of the genome occurs. How and when echoviral genomes cross this membrane barrier to the cytoplasm is not well known. Here we demonstrate that the phosphorylation of PKR can be induced by dsRNA composed of released EV1 genomes and transfected oligonucleotide probes. Furthermore, this phosphorylation can be detected by Western blot and used to demonstrate that echovirus 1 genomes cross the endosomal membrane barrier approximately 2.5−3.5 hours post-infection in A549 cells. If developed further, this new method may help characterise the mechanism of endosomal egress and any proteins involved as well as provide a tool for anti-echoviral drug and vaccine development.
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Yksi n\u00e4ist\u00e4 keinoista on translaatioon vaadittavan tukiproteiini eukaryotic intiation factor 2:n (eIF2) fosforylaatio, joka est\u00e4\u00e4 translaation tehokkaasti. Kaksijuosteisesta RNA:a (dsRNA) aktivoituva protein kinase dsRNA-dependent-proteiini (PKR) on eIF2 kinaasi, joka itsefosforyloituu aktivoituessaan. Sek\u00e4 PKR:N ett\u00e4 eIF2:n fosforylaatio yhdistet\u00e4\u00e4n usein antiviraaliseen soluviestint\u00e4\u00e4n. Echovirus 1 (EV1) kuuluu enterovirusten B-alaryhm\u00e4\u00e4n, jonka j\u00e4senist\u00e4 useat on yhdistetty tyypin 1 diabeteksen puhkeamiseen sille altistuneissa lapsissa. Se tunkeutuu is\u00e4nt\u00e4soluihin endosominkaltaisissa rakkuloissa ja vapauttaa genominsa proteiinikuoresta n\u00e4iss\u00e4 rakkuloissa. Ei ole t\u00e4ysin varmaa miten EV1 genomi p\u00e4\u00e4see rakkulakalvon l\u00e4pi solulimaan, jossa translaatio ja replikaatio tapahtuvat. T\u00e4ss\u00e4 ty\u00f6ss\u00e4 osoitamme, ett\u00e4 EV1-infektoituihin soluihin transfektoidut virusgenomille komplementaariset oligonukleotidikoettimet aktivoivat PKR:n fosforylaatiota muodostaessaan kompleksin virusgenomin kanssa solulimassa. T\u00e4t\u00e4 fosforylaatiota voi k\u00e4ytt\u00e4\u00e4 osoittamaan, ett\u00e4 EV1 genomi on vapautunut endosomirakkulasta solulimaan, mik\u00e4 tapahtui A549 soluissa noin 2,5\u22123,5 tuntia infektion j\u00e4lkeen. Pidemm\u00e4lle kehitettyn\u00e4 t\u00e4m\u00e4 uusi metodi voi tulevaisuudessa auttaa selvitt\u00e4m\u00e4\u00e4n, miten EV1:n genomi vapautuu endosomista, l\u00f6yt\u00e4m\u00e4\u00e4n siihen liittyvi\u00e4 proteiineja sek\u00e4 kehitt\u00e4m\u00e4\u00e4n l\u00e4\u00e4kkeit\u00e4 ja rokotteita EV1:\u00e4 vastaan.", "language": "fi", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Pathogens may trigger various antiviral responses in eukaryotic cells. 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spellingShingle Aspelin, William PKR phosphorylation as an Indicator for echovirus 1 genome egress from endosomes eIF2 kinase oligonucleotide stress timeline translation Solu- ja molekyylibiologia Cell and molecular biology 4013 virukset RNA perimä molekyylibiologia solubiologia proteiinit infektiot fosforylaatio viruses genome molecular biology cell biology proteins infections phosphorylation
title PKR phosphorylation as an Indicator for echovirus 1 genome egress from endosomes
title_full PKR phosphorylation as an Indicator for echovirus 1 genome egress from endosomes
title_fullStr PKR phosphorylation as an Indicator for echovirus 1 genome egress from endosomes PKR phosphorylation as an Indicator for echovirus 1 genome egress from endosomes
title_full_unstemmed PKR phosphorylation as an Indicator for echovirus 1 genome egress from endosomes PKR phosphorylation as an Indicator for echovirus 1 genome egress from endosomes
title_short PKR phosphorylation as an Indicator for echovirus 1 genome egress from endosomes
title_sort pkr phosphorylation as an indicator for echovirus 1 genome egress from endosomes
title_txtP PKR phosphorylation as an Indicator for echovirus 1 genome egress from endosomes
topic eIF2 kinase oligonucleotide stress timeline translation Solu- ja molekyylibiologia Cell and molecular biology 4013 virukset RNA perimä molekyylibiologia solubiologia proteiinit infektiot fosforylaatio viruses genome molecular biology cell biology proteins infections phosphorylation
topic_facet 4013 Cell and molecular biology RNA Solu- ja molekyylibiologia cell biology eIF2 fosforylaatio genome infections infektiot kinase molecular biology molekyylibiologia oligonucleotide perimä phosphorylation proteiinit proteins solubiologia stress timeline translation virukset viruses
url https://jyx.jyu.fi/handle/123456789/67530 http://www.urn.fi/URN:NBN:fi:jyu-202001271794
work_keys_str_mv AT aspelinwilliam pkrphosphorylationasanindicatorforechovirus1genomeegressfromendosomes