Intracellular interplay between canine parvovirus with nuclear pore complex and effect of infection on RNA synthesis

Koiran parvovirus (CPV) on halkaisijaltaan 26 nm vaipaton virus. Ikosahedraalinen kapsidi sisältää yksijuosteisen ~5kb DNA-genomin. CPV sisälleotetaan soluun endosytoosilla, jonka jälkeen se kulkeutuu endosomin sisällä kohti tumaa. CPV vapautuu solulimaan tumaa ympäröivistä lysosomeista. Genomin re...

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Päätekijä: Kleemola, Ari
Muut tekijät: Matemaattis-luonnontieteellinen tiedekunta, Faculty of Sciences, Bio- ja ympäristötieteiden laitos, Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylän yliopisto
Aineistotyyppi: Pro gradu
Kieli:eng
Julkaistu: 2015
Aiheet:
Linkit: https://jyx.jyu.fi/handle/123456789/45796
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author Kleemola, Ari
author2 Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science University of Jyväskylä Jyväskylän yliopisto
author_facet Kleemola, Ari Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science University of Jyväskylä Jyväskylän yliopisto Kleemola, Ari Matemaattis-luonnontieteellinen tiedekunta Faculty of Sciences Bio- ja ympäristötieteiden laitos Department of Biological and Environmental Science University of Jyväskylä Jyväskylän yliopisto
author_sort Kleemola, Ari
datasource_str_mv jyx
description Koiran parvovirus (CPV) on halkaisijaltaan 26 nm vaipaton virus. Ikosahedraalinen kapsidi sisältää yksijuosteisen ~5kb DNA-genomin. CPV sisälleotetaan soluun endosytoosilla, jonka jälkeen se kulkeutuu endosomin sisällä kohti tumaa. CPV vapautuu solulimaan tumaa ympäröivistä lysosomeista. Genomin replikaatio ja uusien virionien kasautuminen tapahtuvat tumassa. Aikaisemmin on oletettu, että parvovirukset todennäköisesti pääsisivät tumaan tumahuokosen (NPC) kautta. Uudet tutkimukset kuitenkin viittaavat siihen että siirtyminen tumaan voisi tapahtua toista reittiä käyttäen. CPV ja NPC proteiinien (Nup) kolokalisaatio-kokeissa selvitettiin voidaanko tiettyjä vuorovaikutuksia havaita ja missä mahdolliset vuorovaikutukset sijaitsevat. Näissä kokeissa käytettiin uutta in situ proximity ligation assay (PLA) -menetelmää, joka tuottaa fluoresenssi-signaalin kahteen eri kohdeproteiineihin sitoutuneiden vasta-aineiden sijaitessa lähellä toisiaan. Bromouridiini (BRU) on uridiinin analogi, joka voidaan tunnistaa spesifisti vasta-aineilla. Soluja viljeltiin BRU-mediumissa, jolloin BRU saatiin liitettyä kehittyviin RNA-molekyyleihin. Tämän jälkeen oli mahdollista arvioida infektiosta johtuvia muutoksia RNA-synteesissä. Fluoresenssia mitattiin virtaussytometrian ja konfokaalimikroskopian avulla. Erityistä kolokalisaatiota ei havaittu tumakalvolla Nup358 ja viruskapsidin välillä. Sen sijaan signaali esiintyi levinneenä sytoplasmassa ja näytti siirtyvän kohti tumaa infektion edetessä. Samankaltaista sytoplasmista kolokalisaatiota nähtiin infektoimattomissa soluissa Nup153 ja Nup358 vasta-aineilla suoritetuissa kokeissa. Tämä signaali oli kuitenkin joissain soluissa vahvimmillaan tuman välittömässä läheisyydessä. Infektion myöhäisessä vaiheessa sytoplasmassa oli havaittavissa vahva signaali CPV kapsidi ja NS1 proteiinien välillä. Immunoleimaus ja virtaussytometri mittaukset osoittavat RNA-synteesin vaimentumista infektion seurauksena. Canine parvovirus (CPV) is a non-enveloped virus with a 26 nm diameter icosahedral capsid. The capsid holds a ~5kb single-stranded DNA genome. CPV is internalized by endocytosis followed by intracellular trafficking inside an endosome towards the nucleus. CPV is released from perinuclear lysosomes followed by replication of the genome and assembly of progeny virions inside the nucleus. It has been previously assumed that parvoviruses are likely to enter the nucleus through the nuclear pore complex (NPC). Recent studies however suggest that entry to the nucleus might take place by an NPC-independent mechanism. Colocalization of CPV and NPC proteins (Nups) were examined to find out if specific interactions could be detected and if so, where these interactions would occur. Experiments were carried out by a novel in situ proximity ligation assay (PLA) method that generates fluorescence only when two antibodies bound to different target proteins are situated at close proximity. Bromouridine (BRU) is an uridine analog that can be specifically bound by antibodies. Cells were cultured in the presence of BRU that became incorporated into nascent RNA molecules. It was then possible to estimate changes in RNA synthesis as a result of infection. The fluorescence was measured by flow cytometry and visualized by confocal microscopy. Colocalization at the nuclear envelope was not detected between Nup358 and CPV capsid. Instead, the signal was spread across the cytoplasm and appeared to be shifting towards the nucleus in time-dependent manner. Cytoplasmic colocalization, sometimes located close to perinuclear region, was detected between Nup153 and Nup358 in noninfected cells. In a late phase of infection a very strong colocalization signal was seen between CPV capsid and NS1 proteins. Immunolabeling and flow cytometry assays suggest a decrease in the rate of RNA synthesis as a result of infection.
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Ikosahedraalinen kapsidi sis\u00e4lt\u00e4\u00e4 yksijuosteisen ~5kb DNA-genomin. CPV sis\u00e4lleotetaan soluun endosytoosilla, jonka j\u00e4lkeen se kulkeutuu\r\nendosomin sis\u00e4ll\u00e4 kohti tumaa. CPV vapautuu solulimaan tumaa ymp\u00e4r\u00f6ivist\u00e4 lysosomeista. Genomin replikaatio ja uusien virionien kasautuminen tapahtuvat tumassa. Aikaisemmin on oletettu, ett\u00e4 parvovirukset\r\ntodenn\u00e4k\u00f6isesti p\u00e4\u00e4sisiv\u00e4t tumaan tumahuokosen (NPC) kautta. Uudet tutkimukset kuitenkin viittaavat siihen ett\u00e4 siirtyminen tumaan voisi tapahtua toista reitti\u00e4 k\u00e4ytt\u00e4en.\r\n\r\nCPV ja NPC proteiinien (Nup) kolokalisaatio-kokeissa selvitettiin voidaanko tiettyj\u00e4 vuorovaikutuksia\r\nhavaita ja miss\u00e4 mahdolliset vuorovaikutukset sijaitsevat. 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spellingShingle Kleemola, Ari Intracellular interplay between canine parvovirus with nuclear pore complex and effect of infection on RNA synthesis CPV PLA NPC Nup Solu- ja molekyylibiologia Cell and molecular biology 4013 parvovirukset tuma virukset
title Intracellular interplay between canine parvovirus with nuclear pore complex and effect of infection on RNA synthesis
title_full Intracellular interplay between canine parvovirus with nuclear pore complex and effect of infection on RNA synthesis
title_fullStr Intracellular interplay between canine parvovirus with nuclear pore complex and effect of infection on RNA synthesis Intracellular interplay between canine parvovirus with nuclear pore complex and effect of infection on RNA synthesis
title_full_unstemmed Intracellular interplay between canine parvovirus with nuclear pore complex and effect of infection on RNA synthesis Intracellular interplay between canine parvovirus with nuclear pore complex and effect of infection on RNA synthesis
title_short Intracellular interplay between canine parvovirus with nuclear pore complex and effect of infection on RNA synthesis
title_sort intracellular interplay between canine parvovirus with nuclear pore complex and effect of infection on rna synthesis
title_txtP Intracellular interplay between canine parvovirus with nuclear pore complex and effect of infection on RNA synthesis
topic CPV PLA NPC Nup Solu- ja molekyylibiologia Cell and molecular biology 4013 parvovirukset tuma virukset
topic_facet 4013 CPV Cell and molecular biology NPC Nup PLA Solu- ja molekyylibiologia parvovirukset tuma virukset
url https://jyx.jyu.fi/handle/123456789/45796 http://www.urn.fi/URN:NBN:fi:jyu-201505071751
work_keys_str_mv AT kleemolaari intracellularinterplaybetweencanineparvoviruswithnuclearporecomplexandeffectofinfect