engleski

Impact of late phase infection with MCMV on markers of intracellular membrane system in Balb 3T3 fibroblasts

Introduction: Mouse cytomegalovirus (MCMV) is a member of Herpesviridiae family. It is a large DNA virus with highly developed immunoevasive strategy. Upon entering into the host cell, it alters its functions and reorganizes life cycle of the cell to replicate itself. Amongst other changes, MCMV reorganizes endosomal system of the host cell very early into infection, finally leading to production of new infectious virions. Endosomal system is a dynamic complex of intracellular membranes and vesicular organelles that is strictly regulated in course to transport endocytic cargo to its final cellular destination. In that way, the endosomal system is essential for normal cell function. Objectives: In this research we investigated the perturbations of endosomal system in Balb 3T3 fibroblasts during late phase of infection with murine cytomegalovirus in non-activated and IFN-γ activated cells. Materials and Methods: Balb 3T3 cells were infected for 16 hours with recombinant murine cytomegalovirus Δm138-MCMV (ΔMC95.15) that has deleted FcR. The activation with IFN-γ was achieved by presenting it to the cells for 48 hours prior to the infection. Fully conformed Dd, open forms of Ld, transferrin, transferrin receptor and other markers of endocytic pathways were followed by corresponding antibodies or fluorescent labelled ligands. The intracellular routes were determined by immunofluorescence and confocal microscopy analysis. Infection in each cell was verified by simultaneous staining od MCMV immediate early 1 (IE1) protein together with selected cellular and viral markers. Results: Cells positive for infection were selected and more closely investigated. Early endosomal marker 1 (EEA1) demonstrated signs of aggregation close to the nucleus of the cell, as opposed to its usual “starry night” pattern. Transferrin (receptor) and fully conformed Dd molecules were found accumulated in juxanucelar area, highly condensed to one location. Rab5 protein did not show any significant difference both in its placement in the cell as well as its expression level. Rab11 protein, however, showed reduced and more dispersed positive staining in cells positive for infection. Although CD63 (Late endosomal protein 3) was localized near before mentioned juxanuclear accumulation, it had still shown perinuclear localization. However, its peripheral vesicular staining almost completely vanished. Similar situation was found with Lamp1 (Late endosomal protein 1). Nevertheless, MLN64 and NPC1, another two markers of late endosomal vesicles, showed no alteration in their staining pattern between infected and non-infected cells. Viral m06 protein seemed to conglomerate also near the juxanuclear area, in the space free of CD63 or Lamp1. Finally, when the cells had been activated with IFN-γ, which prevented productive infection, and then infected, the fully conformed Dd molecules have shown the same pattern as in non-infected cells. Further, IE1 remained clearly visible in IFN-γ activated and MCMV infected cells. Conclusion: During the late phase of MCMV infection endosomal compartments are being remodelled. Some of the markers, and consequently their corresponding compartments, may be accumulated in the juxanuclear area (i.e. EEA1, full Dd, and Tf(R)). On the other hand, late endosomal markers are completely retreated from the juxanuclear and peri- juxanucelar part of the cell, clearly making room for the other compartments and they even envelop the aforementioned area. However, their trafficking could be changed as shown by the loss of their peripheral vesicles. The localization of m06 in juxanuclear area indicates viral involvement in the integrity of remodeled endosomes.

MCMV; intracellular membrane system; Balb 3T3 fibroblasts

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