engleski

The extensive remodeling – reorganization of the endosomal and secretory system during the early phase of cytomegalovirus infection

Cytomegalovirus (CMV) infection causes the remodeling of the cellular endosomal system that terminates with the formation of the viral assembly compartment (viral factory). However, the core mechanisms and the consequences that consider the cell function are poorly understood. We have shown that infection with murine cytomegalovirus (MCMV) induce the early rearrangement (6 hrs p.i.) of intracellular membranes and formation of the juxtanuclear EPERC (early-phase endosomal retention compartment). The process includes the restructuring of early endosomes (EE), endosomal recycling compartment (ERC), and trans-Golgi network (TGN). EPERC is also the place where molecules that are normally transported through early/sorting endosomes are retained (e.g. transferrin receptor (TfR), MHC class-I (MHC-I) molecules, mannoza-6-phosphate receptor (M6PR), epidermal growth factor receptor (EGFR)). Therefore, considering the involvement of EE and ERC in generation of this remodeled compartment, we have performed functional tests in order to determine kinetics of recycling from endosomal system to the plasma membrane. We have shown that the recycling of TfR, MHC-I and Rae-1 is significantly decreased, by using the flow cytometry and confocal microscopy. The same result was with NBD-sphyngomyelin (NBD-SM), a molecule that serves as the nonspecific marker of membrane labeling, and is the indicator of general block in cellular recycling. Furthermore, we have used the in-house software to perform mathematic modeling for additional dissection of the recycling network. Therefore, we confirmed that TfR recycling is inhibited from EE and ERC, but not its rapid recycling through the peripheral recycling endosomes. It is important to notice that that endosomal rearrangement is not only important for generation of viral factory. Namely, virus has also the direct interest due to by-side imunoevasion effects – the fast downloading of surface MHC-I and Rae-1 molecules disables the antigen presentation and recognition of infected cell from the side of cytotoxic T lymphocytes, or by NK cells, respectively. Finally, we have followed the influence of early infection on the expression of small GTPases from Rab and Arf family in order to investigate the mechanism that MCMV use to generate the formation of EPERC. Western blot analysis has shown to us that downregulation of Rab22a, Rab4 and Rab 11 takes place already 4-8 hrs p.i. It is well known that those molecules play important roles in cellular recycling. Furthermore, by using confocal microscopy, we have found that this remodeled compartment is Arf6, Rab5, Rab22a and Rab11 positive, but Rab35, Rab8 and Rab10 negative. Interestingly, the results from the literature indicate that the Rab8 and Rab10 molecules are expressed in the cascade fashion, following the Rab35 activation. The inhibition of recycling in infected cells could be partially explained from the fact that overactivation of Arf6 leads to the block in recycling processes. The same effect of recycling inhibition follows the depletion of Rab35, the molecule that work antagonistically with Arf6. MICAL-L1 and ACAP1/2 are effectors of Rab35, and are also absent from EPERC. It is also interest that inhibitor of furin inhibits the EPERC formation, that indicate the importance of proteases during this process. Finally, we can conclude that MCMV in the early phase of infection induce the endosomal remodeling that includes EE, ERC and TGN, and cause the intracellular retention of recycling molecules. The process includes the viral involvement in the expression of Rab proteins, and especially the regulation of the Arf6-Rab35 cascade. The whole process is played in order to prepare the formation of viral assembly compartment in the late phase of the infection.

Assembly compartment ; Endosomal recycling ; Murine cytomegalovirus ; Rab proteins ; Sorting endosomes ; Transferrin receptor

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