Title ULOGA POLIOVIRUSNOG RECEPTORA
U INFEKCIJI CITOMEGALOVIRUSOM Title (english) THE ROLE OF POLIOVIRUS RECEPTOR
IN CYTOMEGALOVIRUS INFECTION Author Paola Kučan Brlić Mentor Tihana Lenac Roviš (mentor)Committee member Stipan Jonjić (predsjednik povjerenstva)Committee member Magdalena Grce (član povjerenstva)Committee member Đurđica Cekinović Grbeša (član povjerenstva)Committee member Tihana Lenac Roviš (član povjerenstva)Granter University of Rijeka Defense date and country 2018-12-14, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 61 - Medical sciences Abstract Cilj istraživanja: PVR i njegovi receptori imaju važnu ulogu u imunosnom odgovoru
radi čega i tumori i virusi nastoje utjecati na ovaj protein i izbjeći imunosni nadzor. Druga
istraživanja pokazala su da humani citomegalovirus negativno regulira PVR, ali s obzirom na
specifičnost citomegalovirusa za vrstu i kompleksnu interakciju PVR-a i njegovih receptora
na imunosnim stanicama, teško je procijeniti relevantnost tih spoznaja in vivo. Stoga je glavni
cilj ovog istraživanja utvrditi ulogu proteina PVR u imunosnom odgovoru na
citomegalovirusnu infekciju in vivo te proniknuti u kompleksnost sustava PVR/DNAM1/TIGIT/CD96.
Materijali i metode: Kako bih utvrdila učinak MCMV-infekcije na PVR analizirala sam
inficirane stanice metodom kvantitativnog PCR-a te metodama protočne citometrije i
imunoblot analizom. Kako bih identificirala virusni gen važan za regulaciju PVR-a koristila
sam knjižnicu rekombinantnih virusa MCMV-a te konstruirala nove virusne mutante kojima
nedostaju pojedini geni. Za karakterizaciju regulatornog virusnog proteina proizvela sam
protutijelo na taj protein te ga analizirala imunoblot analizom u inficiranim stanicama. Kako
bih utvrdila važnost PVR regulacije in vivo, koristila sam mišji model infekcije MCMV-om. Za
identifikaciju imunosnih populacija i mehanizama odgovornih za PVR-posredovanu kontrolu
virusa koristila sam reagense za uklanjanje ili blokiranje imunosnih populacija in vivo ili
miševe kojima nedostaje ispoljenost određenih receptora. Za fenotipsku i funkcionalnu
analizu imunosnih stanica koristila sam protočnu citometriju.
Rezultati: MCMV uzrokuje pojačano prepisivanje gena za PVR ali smanjenu
površinsku ispoljenost zrelog proteina PVR za što je zaslužan novoidentificirani virusni
protein, m20.1. Uklanjanje ovog proteina odnosno sprječavanje negativnog učinka virusa na
protein PVR omogućuje učinkovitiji imunosni nadzor koji je ovisan o receptoru DNAM-1 te
posredovan stanicama NK i mononulearnim fagocitima, sa značajnom ulogom proupalnih
monocita.
Zaključak: Rezultati ovog istraživanja ukazuju da DNAM-1-PVR interakcija ima
značajnu ulogu u kontroli CMV-a od strane stanica NK i mononuklearnih fagocita. Također,
ovi rezultati pokazuju novi mehanizam virusnog reguliranja imunosnog odgovora ovisnog o
uparenim receptorima, gdje virus smanjenjem ispoljavanja površinskog proteina PVR
zadržava interakciju s inhibicijskim receptorima, ali onemogućuje interakciju s aktivacijskim
receptorom. U konačnici, ovi rezultati pokazuju da mononuklearni fagociti imaju važnu ulogu
u imunosnom odgovoru na viruse, koja je izvan njihove uloge modulatora drugih efektorskih
imunosnih populacija.
Abstract (english) Objectives: PVR and its receptors play a significant role in immune response which is
why both tumors and viruses try to modulate this protein to avoid immune recognition.
Research by others has shown that human cytomegalovirus downregulates PVR protein, but
given the species specificity of cytomegaloviruses and complex interactions between PVR
and its receptors, it is hard to predict the relevance of these findings in vivo. The aim of this
study was to determine the role of PVR in immune response to cytomegalovirus infection in
vivo as well as comprehend the complexity of PVR/DNAM-1/TIGIT/CD96 interactions.
Materials and methods: To assess the effect of MCMV infection on PVR, infected
cells were analysed by quantitative PCR, flow cytometry and by Western blot method. To
identify viral gene involved in PVR regulation, I used recombinant MCMV library and
constructed new viral mutants lacking particular genes. To characterize viral protein in
charge for PVR regulation, I produced antibodies to that protein and analysed it using
Western blot analysis. To assess the significance of PVR regulation in vivo, mouse model of
MCMV infection was used. To identify immune populations and mechanisms responsable for
PVR-mediated viral control I used reagents that deplete or block immune populations in vivo
as well as mice deficient for certain receptors. Analysis of the phenotype and function of
immune cells was performed using flow cytometry.
Results: MCMV induces PVR transcription and at the same time prevents surface
expression of mature PVR protein. Newly identified viral protein, m20.1, is responsible for
this phenomena. Removal of this protein prevents MCMV-caused downregulation of PVR
allowing more efficient immune control which depends on DNAM-1 receptor and is mediated
by NK cells and mononuclear phagocytes, in particular proinflammatory monocytes.
Conclusion: Results of this research show that DNAM-1-PVR interaction has a
significant role in CMV control by NK cells and mononuclear phagocytes. In addition, these
results show novel mechanism of viral regulation of paired receptors in the immune response
where virus by downregulating surface PVR maintains inhibitory interactions but prevents
activating interactions. Finally, these results show important role of mononuclear phagocytes
in the immune response to viruses that goes beyond their role of modulators of other effector
immune cell populations.
Keywords
Imunitet
Mišji CD96 protein
Mišji citomegalovirus
Mišji DNAM-1 protein
Mišji TIGIT protein
Mononuklearni fagociti
Poliovirusni receptor
Keywords (english)
CD96 protein
Mouse
Cytomegalovirus
Mouse
DNAM-1 protein
Mouse
Immunity
Mononuclear phagocytes
Poliovirus receptor
TIGIT protein
Mouse
Language croatian URN:NBN urn:nbn:hr:184:911402 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Type of resource Text File origin Born digital Access conditions Access restricted to students and staff of home institution Terms of use Created on 2023-01-24 14:13:14
