Karakterizacija znotrajceličnega prometa in signaliziranja s herpesvirusnimi s proteinom G sklopljenimi receptorji

Mavri, Maša (author), Kubale Dvojmoč, Valentina (mentor), Spiess, Katja (comentor)

Cilj disertacije sta opredelitev in primerjava molekularnih in bioloških lastnosti ortologov BILF1, kodiranih v treh prašičjih limfotropnih herpesvirusih (PLHV1-3) in humanem virusu Epstein-Barr (EBV). Proučevali smo lokalizacijo, konstitutivno internalizacijo, konstitutivno aktivnost in imunoregulatorno vlogo receptorjev PLHV1-3 BILF1 in te lastnosti primerjali z receptorjem EBV-BILF1. V raziskavi smo prvič opisali lastnosti endocitoze za vse štiri naštete receptorje BILF1. Z encimsko-imunskim testom (ELISA) in fluorescenčno mikroskopijo smo potrdili ohranjeno lokalizacijo na celični površini. Konstitutivno internalizacijo smo dokazali s pristopom, ki obravnava od temperature odvisno endocitozo receptorjev v različnih časovnih intervalih (iz angl. antibody-feeding), in z novim testom internalizacije v realnem času. Z dominantno negativno mutanto (DNM) in kemičnim zaviralcem smo pokazali vključenost klatrinske poti pri internalizaciji receptorjev BILF1. Z analizo bioluminiscenčnega prenosa (BRET2) in bioinformacijskim pristopom smo potrdili, da mehanizem ni odvisen od arestina ß. Dodatno smo potrdili tudi vključenost kaveolina pri internalizaciji receptorjev BILF1. Z luciferaznim testom smo dokazali ohranjenost konstitutivnega znotrajceličnega prenosa signala, odvisnega od G?i, ter različno aktivacijo znotrajceličnih transkripcijskih faktorjev. S prenosom western nismo potrdili aktivacije zunajcelično signalno regulirane kinaze (ERK1/2). Sposobnost receptorjev BILF1 za izmikanje imunskemu sistemu smo potrdili s pretočno citometrijo in metodo fluorescenčne mikroskopije. Pokazali smo, da je sposobnost zniževanja površinske izraženosti molekul poglavitnega histokompatibilnostnega kompleksa (MHC-I) ohranjena za vse receptorje BILF1 v humanih embrionalnih ledvičnih celicah (HEK-293), ne pa tudi v prašičjih ledvičnih celicah (PK-15). Z metodo RT-qPCR smo dokazali povečano izraženost receptorjev PLHV1-BILF1 po razvoju post-transplantacijske limfoproliferativne bolezni (PTLD). Pridobljeni rezultati so prvi korak k vzpostavitvi s PLHV1 okuženega prašičjega modela za bolezen PTLD, ki bi v prihodnje omogočal raziskavo patoloških vidikov bolezni, povezanih z EBV, in raziskavo receptorjev BILF1 kot potencialne tarče za zdravljenje humane oblike bolezni PTLD.

• Language: Slovenian
• Keywords: molekularna biologija, celična membran, človeški herpesvirus 4, prašičji herpesvirus, receptor, sklopljen z G-proteinom, protein transport – fiziologija, prenos signalov, endocitoza, kaveolin, klatrin, dynamin
• Work type: Doctoral dissertation
• Organization: VF - Veterinary Faculty
• Year: 2023
• PID: 20.500.12556/RUL-145139
• Publication date in RUL: 08.04.2023

Secondary language

• Language: English
• Title: Characterization of trafficking and signaling properties of herpesvirus and host encoded G protein coupled receptors

Abstract

The objective of this study was to characterize and compare the molecular and biological properties of BILF1 encoded by the human Epstein-Barr virus (EBV) and BILF1 orthologues encoded by three porcine lymphotropic herpesviruses (PLHV1-3). The localization, constitutive internalization, and signaling, as well as the immunoregulatory role of PLHV1-3 BILFs, were determined and compared to the previously studied molecular properties of EBV-BILF1. Via cell based enzyme-linked immunosorbent assay (ELISA) and fluorescence microscopy, the conserved cell surface expression was confirmed. Moreover, for the first time, detailed endocytic pathways were described in this study of all four BILFs by antibody-feeding and a novel real-time internalization assay in live cells. Dominant negative mutants (DNMs) and chemical inhibitors confirmed clathrin-mediated endocytosis as a mechanism for BILF1 internalization. This mechanism was β-arrestin-independent as was confirmed by bioluminescence resonance energy transfer assay (BRET2) and bioinformatics analysis. Furthermore, caveolin seems to be involved in BILF1 trafficking. Investigating downstream signaling events mediated by the BILF1 receptors, Gαi dependent constitutive BILF1 activity but differential activation of downstream transcription factors were shown by luciferase assay. There was no activation of extracellular signal-regulated kinase (ERK) 1/2 by the BILF1 receptors. The immunoevasive properties of PLHV1-3 BILFs were confirmed, as shown for EBV-BILF1 by flow cytometry and supported by a new method based on fluorescence microscopy. Moreover, downregulation of major histocompatibility complex (MHC-I) was conserved for all BILFs in human embryonic kidney (HEK-293) cells but not in porcine kidney 15 (PK-15) cells. Finally, the association of PLHV1-BILF1 expression with the development of post-transplant lymphoproliferative disease (PTLD) was determined via RT-qPCR showing specific expression of only PLHV1-BILF1 after the disease onset. These results represent a first step toward establishing a PLHV1-associated porcine PTLD model, not only to study the pathological aspects of EBV-mediated disease but also to test BILF1 as a potential drug target relevant for the treatment of EBV-associated PTLD in humans.

• Keywords: molecular biology, cell membrane, herpesvirus 4, human, herpesvirus 1, suid, receptors, G-protein coupled, protein transport – physiology, signal transduction, endocytosis, caveolins, clathrin, dynamins