In the absence of specific localization signals, GFP expression yielded a diffuse fluorescent staining in both cytoplasm and nucleus. Expression of g9 was strongly associated with a bright punctuate pattern. Fluorescent signals were completely excluded from nuclei of transfected cells, and widely distributed into the cytoplasm, in discrete puncta. The punctuate staining is typical of vesicles, as expected for a membrane protein involved in intracellular transport and as already reported. Puncta were detectable throughout the cytoplasm, and a weak signal was also associated with the plasma membrane. US9 accumulation in the central region of the cell has been associated with markers of the Trans Golgi Network, the putative site of virions assembly and sorting, and this region has been defined as the steadystate site of US9 localization. In all our experiments, this fluorescent pattern was always reproduced, with only the intensity of the fluorescent signal in the perinuclear region possibly diverging. However, not to interfere with physiological variability, we did not attempt to establish stably transfected cell clones, and always performed transient transfections. Nevertheless, labeled vesicles in distal regions were always detected, even in cells with a heavy perinuclear fluorescent signal. Cells transfected with g9-TM showed a comparable yet clearly distinguishable fluorescent pattern, comprising both intracellular punctuate staining and plasma membrane fluorescence. Indeed, g9-TM labeling was more pronounced at the plasma membrane, as compared to the full length protein, while intra-cellular punctuate signal was reduced. These data suggest that the 32 aa long trans-membrane domain of US9 is sufficient to drive the insertion of the GFP molecule into the same vesicles already described for the full length protein; however, in the absence of the cytosolic domain, the fusion protein accumulates at the plasma membrane. On the other hand, as expected, removing the US9 trans-membrane domain from the fusion protein dramatically changes the fluorescent pattern.