EFFECT OF EARLY PHASE MOUSE CYTOMEGALOVIRUS INFECTION ON REMODELLING ENDOSOMAL SYSTEM IN FIBROBLASTS

Abstract

Cilj istraživanja: U ovom radu cilj je bio razotkriti mehanizam kojim virus preuređuje sustav endosoma tijekom rane faze infekcije MCMVom. Stoga smo istražili funkciju proteaza te glavnih endosomalnih putova u inficiranim stanicama, kao i njezin utjecaj na unutarstanični izražaj Rab i Arf obitelji regulatornih bjelančevina. Materijal i metode: U istraživanjima smo koristili dvije mišje fibroblastne linije: primarne mišje embrionalne fibroblaste (MEF) i imortalizirane Balb3T3 stanice te dva rekombinantna soja virusa: Δm138-MCMV i Δ9-MCMV. Smještaj ispitivanih staničnih i virusnih bjelančevina pratili smo imunofluorescencijom i konfokalnom mikroskopijom. Stanične kinetičke parametre smo određivali prvenstveno protočnom citometrijom (uz korištenjem softwarea izrađenog u suradnji sa Tehničkim fakultetom Sveučilišta u Rijeci). Promjene u razini izražaja pojedinih biljega različitih staničnih segmenata određivali smo Western blot analizom. Molekule su praćene bez ili nakon primijene različitih kemijskih inhibitora. Rezultati: Za razvoj infekcije MCMVom potreban je funkcionalan sustav staničnih serinskih proteaza koje djeluju u neutralnom pH, koje uključuju furin. Funkcionalnim testovima smo pokazali da u ranoj fazi infekcije dolazi do promjena u brzinama prijenosa transferina, molekula MHC-I i sfingomijelina kroz stanični sustav endosoma. Njihovo recikliranje je usporeno kao posljedica infekcije, a molekule su zadržane perinuklearno, u virusnom remodeliranom EPERC odjeljku. Matematičkim modeliranjem smo pokazali da je u inficiranim stanicama održano brzo recikliranje, a da je inhibiran izlazak i iz sortirajućih endosoma (SE) i iz endosomalnog reciklirajućeg odjeljka (ERC), kao i prijenos iz SE u ERC. Razlog zastoja u izlasku iz SE, kao i iz ERCa je najvjerojatnije posljedica utjecaja MCMVa na razinu izražaja i aktivnost staničnih regulacijskih bjelančevina Rab i Arf obitelji (Arf6, Rab35 i Rab22A), što smo pokazali konfokalnom mikroskopijom i Western blot analizom. Zaključak: Za normalan razvoj infekcije potrebne su serinske proteaze (npr. furin). Infekcijom MCMVom dolazi do kočenja izlaska endocitiranog materijala iz SE i ERCa, što je najvjerojatnije prouzročeno utjecajem MCMVa na regulacijske bjelančevine Rab i Arf obitelji (Arf6, Rab35 i Rab22A.Aim of the study: This study aimed to elucidate mechanisms that rearrange endosomal system in the early phase of MCMV infection. Therefore, we investigated the function of cellular proteases and major endosomal routes in infected cells as well the MCMV influence in the intracellular expression levels of regulatory protein families Rab and Arf. Materials and methods: For our experiments, we used murine embryonic fibroblasts (MEF) and immortalized fibroblast-like Balb3T3 cells and two recombinant MCMV viruses: Δm138-MCMV and Δ9-MCMV. Intracellular distribution and expression of endosomal regulatory proteins, endosomal marker proteins, and viral proteins, was analyzed by immunofluorescence techniques and imaging by confocal microscopy. Cellular kinetic parameters were determined by flow cytometry (using in-house developed software, made in collaboration with Faculty of Engineering of the University of Rijeka). The intracellular expression levels of endosomal markers and viral proteins were quantified by Western blot. Molecules were followed with or without the application of different chemical inhibitors. Results: Functional system of cellular proteases that are active in the neutral pH environment (including furin), is required for the development of MCMV infection. Functional assays revealed alteration of the endosomal flow in the early phase of MCMV infection, as demonstrated by several cargo molecules, including trafficking of TfR, MHC-I molecules and NBD-sphingomyelin through the cellular endosomal system. Their recycling was delayed, and cargo molecules were retained in EPERC, the perinuclear endosomal compartment remodeled by MCMV. The kinetic modeling revealed that MCMV infection does not affect the rapid recycling route, but inhibits the egress from sorting endosomes (SE) and endosomal recycling compartment (ERC) as well as transfer form SE to ERC. The inhibition of the egress from SEs and the ERC was associated with altered levels and subcellular distribution of cellular regulatory proteins, most importantly Arf6, Rab35, and Rab22A (demonstrated by the immunofluorescence imaging and Western blot analysis). Conclusion: Serine proteases (i.e. furin) are necessary for the regular development of infection. MCMV infection causes retention of endocytosed cargo in SEs and the ERC which is associated by redistribution of the small GTPases from Rab and Arf families (Arf6, Rab35, and Rab22A