Title Biogeneza citomegalovirusnog odjeljka za sklapanje viriona preuređivanjem sortiranja u endosomalnim reciklirajućim domenama
Title (english) Biogenesis of cytomegalovirus assembly compartment through rearrangement of sorting mechanisms in endosomal recycling domains
Author Igor Štimac
Mentor Hana Mahmutefendić Lučin (mentor)
Committee member Tihana Lenac Roviš (predsjednik povjerenstva)
Committee member Andreja Ambriović Ristov (član povjerenstva)
Committee member Ivica Pavić (član povjerenstva)
Granter University of Rijeka Faculty of Medicine Rijeka
Defense date and country 2024, Croatia
Scientific / art field, discipline and subdiscipline BIOMEDICINE AND HEALTHCARE Basic Medical Sciences
Universal decimal classification (UDC ) 61 - Medical sciences
Abstract Cilj istraživanja: Infekcija citomegalovirusima (CMV) uzrokuje opsežno remodeliranje i ekspandiranje unutarstaničnih membrana na sučelju ranih endosoma (EE), endosomalnog reciklirajućeg odjeljka (ERC) i Golgija. Cilj istraživanja je bio identificirati stanične proteine na sučelju EE i ERC-a koje mišji citomegalovirus (MCMV) regrutira tijekom infekcije. Također, istražili smo kako regrutirani proteini utječu na replikacijski ciklus virusa i stvaranje odjeljka za sklapanje virusnih čestica (pre-AC). Posebna pažnja posvećena je promjenama u interaktomu proteina Rab10, koji je biljeg pre-AC-a. Materijal i metode: Koristili smo imunofluorescentnu mikroskopiju i western blot analizu za praćenje izražaja staničnih proteina i proteina MCMV-a. Pericentriolarno nakupljanje Rab10 služilo je kao marker nastanka pre-AC-a. Za identifikaciju proteina koji imaju ulogu u nastanku pre-AC-a, proteine sučelja EE-ERC-a utišali smo transfekcijom specifičnom siRNA. Također smo koristili inhibitore i transfekciju dominantno negativnim (DN) konstruktima za istraživanje uloge ključnih proteina. Metoda BioID2 korištena je za analizu interaktoma Rab10, a za to smo proizveli staničnu liniju NIH3T3 Rab10 BioID2-HA sa stabilnim izražajem Rab10-BioID2-HA fuzijskog proteina. Rezultati: Otkrili smo da se većina proteina sučelja EE-ERC-a nakuplja u pericentriolarnom području koje odgovara pre-AC-u, bez promjena njihove količine tijekom infekcije. Utišavanje proteina SNX27, Vps35, SNX1, SNX4 i dinamina 2 je inhibiralo nastanak pre-AC-a. Daljnje istraživanje SNX27 potvrdilo je njegovu ključnu ulogu u nastanku unutarnjeg dijela pre-AC-a, posebno u sintezi proteina MCMV-a odgođene rane i kasne faze infekcije, kao i za sintezu viriona. Inhibitor dinamina, dynasore, te DN konstrukt K44A Dyn2 EGFP također su inhibirali nastanak pre-AC-a i replikacijski ciklus MCMV-a. Interaktom Rab10 u neinficiranim i MCMV-om inficiranim stanicama većinom sačinjavaju proteini EHBP1, MICAL-L1 i Rab11 FIP1. Zaključci: Proteini SNX27:Retromer:ESCPE-1 kompleksa (SNX27, Vps35, SNX1), te dinamin 2 potrebni su za najranije događaje u uspostavljanju pre-AC-a tokom rane faze infekcije MCMV-om. Funkcionalni SNX27,osobito njegova domena F3 FERM, i dinamin 2 su neophodni za odvijanje normalnog replikacijskog ciklusa MCMV-a. Interaktom biljega pre-AC-a, proteina Rab10, ne mijenja se znatnije u ranoj fazi infekcije MCMV-om, a dominiraju interakcije Rab10 s proteinima EHBP1, MICAL-L1 i Rab11 FIP1.
Abstract (english) Objectives: Cytomegalovirus (CMV) infection causes extensive remodeling and expansion of intracellular membranes at the interface of early endosomes (EE), the endosomal recycling compartment (ERC), and the Golgi. The aim of this research was to identify cellular proteins at the EE and ERC interface that murine cytomegalovirus (MCMV) recruits in the early phase of infection. Additionally, we investigated how these proteins affect the viral replication cycle and the formation of the viral assembly compartment (pre-AC). Special attention was given to changes in the interactome of the protein Rab10, which is a marker of the pre-AC. Materials and methods: We used immunofluorescence microscopy and western blot analysis to monitor the expression of cellular and MCMV proteins. Pericentriolar accumulation of Rab10 served as a marker for the formation of the pre-AC. To identify proteins involved in the formation of the pre-AC, proteins at the EE-ERC interface were silenced by transfection with specific siRNA. We also used chemical inhibitors and transfection with dominant negative (DN) constructs for further investigation of the role of key proteins. The BioID2 method was used to analyze the Rab10 interactome, and for this purpose, we generated an NIH3T3 cell line stably expressing the Rab10-BioID2-HA fusion protein. Results: We discovered that most proteins at the EE-ERC interface accumulate in the pericentriolar area corresponding to the pre-AC, without significant changes in their quantity during infection. Knockdown the proteins SNX27, Vps35, SNX1, SNX4 and dynamin 2 significantly inhibited the formation of the pre-AC. Further investigation of SNX27 confirmed its key role in the formation of the inner part of the pre-AC, particularly in the synthesis of MCMV proteins during the delayed early and late phases of infection, as well as in virion synthesis. The dynamin inhibitor dynasore and the DN construct K44A Dyn2 EGFP also inhibited the formation of the pre-AC and the MCMV replication cycle. The Rab10 interactome in uninfected and MCMV-infected cells predominantly consisted of proteins EHBP1, MICAL-L1, and Rab11 FIP1. Conclusions: The SNX27:Retromer:ESCPE-1 complex (proteins SNX27, Vps35, SNX1) and dynamin 2 are required for the earliest events during pre-AC establishment in the early phase of MCMV infection. Furthermore, functional SNX27 (particularly its F3 FERM domain) and dynamin 2 are essential for the normal progression of the MCMV replication cycle. Finally, the Rab10 interactome does not change significantly in the early phase of MCMV infection, with Rab10 primarily interacting with EHBP1, MICAL-L1, and Rab11 FIP1 proteins.
Keywords
Citomegalovirus
Odjeljak za sklapanje virusnih čestica ((V)AC)
SNX27
dinamin 2
Rab10
Keywords (english)
Cytomegalovirus
Viral assembly compartment ((V)AC)
SNX27
dynamin 2
Rab10
Language croatian
URN:NBN urn:nbn:hr:184:344419
Promotion 2025
Project Number: IP-2020-02-2916 Title: Reorganizacija sučelja između ranih endosoma i endosomalnog reciklirajućeg odjeljka u ranoj fazi infekcije citomegalovirusom Title: Cytomegalovirus reorganization of the interface between early endosomes and endosomal recycling compartment Acronym: CREERC Leader: Hana Mahmutefendić Lučin Jurisdiction: Croatia Funder: Hrvatska zaklada za znanost Funding stream: HRZZ
Project Number: IP-2019-04-3582 Title: Sekundarno omatanje i izlazak beta-herpesvirusa iz stanice Title: Beta-herpesvirus secondary envelopment and egress Acronym: AsCoSE&E Leader: Pero Lučin Jurisdiction: Croatia Funder: Hrvatska zaklada za znanost Funding stream: HRZZ
Project Number: uniri-biomed-18-180 Title: Rab10 u biogenezi endosomalnog reciklirajućeg odjeljka Title: Rab10 in the biogenesis of endosomal recycling compartment Leader: Hana Mahmutefendić Lučin Jurisdiction: Croatia Funder: Sveučilište u Rijeci
Study programme Title: Biomedicine Postgraduate (doctoral) study programme Study programme type: university Study level: postgraduate Academic / professional title: doktor/doktorica znanosti, područje biomedicine i zdravstvo (doktor/doktorica znanosti, područje biomedicine i zdravstvo)
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Created on 2025-03-13 09:10:15