Upon photobleaching, we observed rapid recovery from the nonbleached pool, with the original prebleached fraction of fluorescence clearly restored in less than 100 seconds (Fig. 4A,E). In contrast, in YFP-HBcAg and CFP-MxA cotransfected cells, the fluorescence recovery of YFP-HBcAg aggregated with CFP-MxA in the perinuclear region was dramatically slowed, with a great proportion of the initial YFP-HBcAg fluorescence in the region Decitabine molecular weight failing to recover, even a much longer time after photobleaching (Fig. 4B,E), suggesting this proportion of HBcAg was irreversibly bound to the compartment. The irreversible binding of HBcAg to the perinuclear pool was further confirmed

by fluorescence loss in photobleaching (FLIP). YFP-HBcAg fluorescence in a small cytoplasmic region of the cell expressing only YFP-HBcAg was bleached repetitively. After about 200 seconds, the fluorescence signals were completely lost in the areas outside the region, indicating that YFP-HBcAg diffused between the bleached and unbleached areas (Fig. 4C,F). In cells coexpressing YFP-HBcAg and CFP-MxA, repetitive

photobleaching of a similar cytoplasmic Saracatinib solubility dmso pool failed to cause the loss of YFP-HBcAg from the perinuclear area in which it colocalized with CFP-MxA, indicating fixation of YFP-HBcAg in that area (Fig. 4D,F). These results indicate that the formation of MxA-HBcAg complexes is able to immobilize HBcAg in the perinuclear area. Interaction of MxA and viral nucleocapsid protein to generate complexes in the perinuclear compartment has been reported in many types of viral infection.7, 19 However, so far, the compartment has not been well-characterized. In an attempt to determine the essence of the perinuclear find more compartment where the MxA-HBcAg complex forms, we tested whether the MxA-HBcAg aggregates overlapped with the Golgi apparatus, the endoplasmic reticulum (ER), or the ER-Golgi intermediates, using distinct organelle

markers. Vero cells expressing CFP-MxA and YFP-HBcAg were either transfected with RFP-tagged ssKDEL, or stained by GM130 or p58 antibody. We found that the perinuclear MxA-HBcAg complexes colocalized with neither the ssKDEL-RFP used as an ER marker, nor the GM130, a Golgi matrix protein, nor the p58 as a marker of ER-Golgi intermediates (Fig. 5A). We also treated the cells with brefeldin A (BFA), a drug that disassembles the Golgi structure by inhibition of early steps in ER-Golgi transport.20 In the presence of BFA, when RFP-tagged galactosyltransferase, a Golgi-resident enzyme, redistributed to the ER, and the endogenous GM130 disassociated from the Golgi membranes (Fig. 5B), the MxA-HBcAg aggregation remained in the perinuclear region without notable changes (Fig. 5B), suggesting that the Golgi and the ER-Golgi intermediates are not involved in the formation of the aggregation. To further characterize the perinuclear compartment, we disrupted microtubules by treating the cells with nocodazole21 or by putting the cells on ice.