| Abstract | Cilj istraživanja: cilj ovoga rada bio je utvrditi izražaj, stupanj aktivacije te unutarstaničnu
lokalizaciju Arf proteina i njihovih regulatornih molekula tijekom citomegalovirusne (CMV)
infekcije te ispitati kako narušen izražaj ili funkcija Arf proteina utječe na tijek CMV infekcije.
Materijali i metode: u Balb 3T3 stanicama inficiranim mišjim CMV-om u različitim
vremenima infekcije izražaj Arf, Arf GAP i Arf GEF proteina praćen je Western blot metodom,
dok je konfokalnom mikroskopijom analizirana njihova kolokalizacija s biljezima
unutarstaničnih odjeljaka i virusnim proteinima. Stupanj aktivacije Arf proteina ispitan je
pomoću subcelularnog frakcioniranja i eseja za određivanje aktivacije Arf proteina. Tijek CMV
infekcije u uvjetima narušenog izražaja i funkcije Arf proteina ispitan je pomoću siRNA metode
te pomoću inhibitora Arf GEF i GAP proteina. Dinamike internalizacije i recikliranja
analizirane su protočnom citometrijom.
Rezultati: izražaj Arf1, Arf3, Arf4, Arf5, BRAG2, Big1, Big2, citohezin1/2, Smap1 i ASAP2
raste, dok izražaj Arf6, Gbf1, ACAP1, ACAP2, ARAP1 i Git1 ostaje gotovo nepromijenjen
tijekom MCMV infekcije. Izražaj ADAP1 i AGAP1 blago raste u kasnoj fazi, a izražaj Psd i
ArfGAP1 iako blago raste u ranoj fazi, ponovno pada u kasnoj fazi MCMV infekcije. Arf1,
Arf4, Arf5 i Arf6 proteini značajno se kolokaliziraju s markerima ranih endosoma,
reciklirajućih endosoma i trans-Golgi mreže, dok ne pokazuju značajniju kolokalizaciju s
markerima Golgijeva aparata i virusnim proteinima, za razliku od Arf3 proteina. BRAG2, Big1,
Big2, ArfGAP1, Git1 i ASAP2 se nakupljaju u jukstanukelarnom području, Citohezin1/2, Gbf1
i AGAP1 se mobiliziraju na staničnu membranu i u subkortikalno područje, dok se
unutarstanična lokalizacija Psd, ACAP1, ACAP2, ARAP1, ADAP1 i Smap1 ne mijenja
značajnije tijekom CMV infekcije. U uvjetima utišanog izražaja Arf1 i Arf6 proteina smanjen
je izražaj virusnih proteina, kao i u stanicama tretiranima s Golgicidom A, Brefeldinom A,
NAV-2729 i EXO2. U stanicama u kojima je utišan izražaj Arf3 i Arf4 proteina ne dolazi do
stvaranja odjeljka za sklapanje viriona. Tijekom CMV infekcije usporava se recikliranje
transferinskog receptora, CD44 i MHC-I molekula.
Zaključak: tijekom CMV infekcije mijenja se izražaj i unutarstanična lokalizacija Arf proteina,
kao i nekih od njihovih regulatornih molekula. Arf1 i Arf6 te Big1, Big2, Gbf1 i BRAG2
proteini su važni za uspostavu CMV infekcije, a Arf3 i Arf4 proteini te Big1, Big2, BRAG2 i
Gbf1 GEF proteini za formiranje odjeljka za sklapanje viriona. CMV infekcija narušava
endosomalno prometovanje transferinskog receptora, CD44 i MHC-I molekula. |
| Abstract (english) | Objective: the aim of this study was to determine the expression, degree of activation, and
intracellular localization of Arf proteins and their regulatory molecules during cytomegalovirus
(CMV) infection, and to examine how impaired expression or function of Arf proteins affects
course of CMV infection.
Materials and Methods: in Balb 3T3 cells infected with murine CMV at different times of
infection, the expression of Arfs, Arf GAPs, and Arf GEFs was determined by Western blot,
and their colocalization with intracellular compartment markers and viral proteins was analyzed
by confocal microscopy. The degree of Arf protein activation was examined by subcellular
fractionation and an Arf activation assay. The course of CMV infection under conditions of
impaired expression and function of Arf proteins was analyzed by siRNA and Arf GEF and
GAP inhibitors. Internalization and recycling were analyzed by flow cytometry.
Results: the expression of Arf1, Arf3, Arf4, Arf5, BRAG2, Big1, Big2, cytohesine1/2, Smap1,
and ASAP2 increases, while the expression of Arf6, Gbf1, ACAP1, ACAP2, ARAP1, and Git1
remains almost unchanged during MCMV infection. The expression of ADAP1, and AGAP1
slightly increases at a late stage, and the expression of Psd, and ArfGAP1 slightly increases at
an early stage and then decreases in the late stages of MCMV infection. Arf1, Arf4, Arf5, and
Arf6 proteins significantly colocalize with markers of early endosomes, recycling endosomes,
and the trans-Golgi network, while showing no significant colocalization with markers of the
Golgi apparatus and viral proteins, in contrast to Arf3. BRAG2, Big1, Big2, ArfGAP1, Git1,
and ASAP2 accumulate in the juxtanuclear region, cytohesin1/2, Gbf1, and AGAP1 mobilize
in the plasma membrane and subcortical region, while intracellular localization of Psd, ACAP1,
ACAP2, ARAP1, and ARAP1 remains unchanged during CMV infection. Under conditions of
silenced expression of Arf1 and Arf6, the expression of viral proteins was reduced, as well as
in cells treated with Golgicide A, Brefeldin A, NAV-2729, and EXO2. In cells where the
expression of Arf3 and Arf4 proteins is silenced, there is no formation of the virion assembly
compartment. During CMV infection, recycling of the transferrin receptor, CD44, and MHC-I
molecules is slowed down.
Conclusion: during CMV infection, the expression and intracellular localization of Arf proteins
and some of their regulatory molecules, change. Arf1, Arf6, Big1, Big2, Gbf1, and BRAG2 are
important for the establishment of CMV infection, while Arf3, Arf4, BRAG2, Big1, Big2, and
Gbf1 are important for the formation of the virion assembly compartment. CMV infection
disrupts the endosomal transport of the transferrin receptor, CD44 and MHC-I molecules. |
