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The recombinant adenovirus mutants allowed to express and localize the mutant 10.4-14.5 proteins in primary fibroblasts, such as SeBu cells (Fig. 32A-D). In SeBu cells infected with wt Ad2/F14.5 the ER and Golgi stained positively for 10.4K and 14.5K, and 14.5K was additionally detected at the plasma membrane. While upon infection with mutant Ad2/F14.5Y74 14.5 surface staining was similar to infection with wt Ad2 (Fig. 32B), ER/Golgi staining appeared less pronounced. Upon infection with the Ad2/F14.5Y122 mutant, cellular extrusions were prominently stained, indicating a markedly increased surface staining of 10.4-14.5K (Fig. 32C), compatible with the proposed role of the Y122_{XXΦ motif in directing internalization of the 10.4-} 14.5K complex. By contrast primary fibroblasts infected with Ad2/10.4LL-F14.5 showed no surface staining. Even without Bafilomycin treatment a significant proportion of the cells exhibited a highly vesicular staining for both 10.4 and 14.5 (Fig. 32D), instead of the ER/Golgi staining seen for wt proteins (Fig. 32A). To characterize the nature of the vesicles SeBu cells were infected with Ad2/F14.5 or Ad2/10.4LL-F14.5 and processed for dual label immunofluorescence analysis to compare steady-state localization of 14.5K with cellular marker proteins. In the mutant (Fig. 33A) and the wt (Fig. 33B) situation the perinuclear compartment was costained with GM130, a cis- Golgi marker protein. Upon Bafilomycin treatment, the number of cells bearing 14.5+ vesicles was further increased in cells infected with mutant Ad2/10.4LL-F14.5, but not in cells infected with wt Ad2, in which ER/Golgi staining for 14.5K (Fig. 33D, F, H) remained unaltered. In Ad2/10.4LL- F14.5- infected primary fibroblasts the distribution of 14.5+ vesicles was distinct from the localization of EEA1, a marker for early endosomes (Fig. 33C). Similarly, vesicles did only partially overlap with LBPA which represents late endosomes (Fig. 33E). But both 14.5K (Fig. 33G) and 10.4LL (Fig. 34E) extensively colocalized with Lamp-2+ vesicles.

Remarkably, the reduction of ER staining and redistribution of 10.4K to Lamp-2+ vesicles occurred selectively in cells expressing the 10.4 dileucine mutant (Fig. 34A). In cells infected with Ad2/F14.5 (Fig. 34B) or virus mutants Ad2/10.4-F14.5Y74 (Fig. 34C), Ad2/10.4-F14.5Y122A (Fig. 34D) 10.4K was found in the ER and the Golgi, and did not colocalize with Lamp-2+ vesicles following Bafilomycin treatment (Fig. 34 F-H). Thus, disruption of the 10.4K dileucine motif leads to a profound redistribution of 10.4-14.5K into Lamp-2+ vesicles,

Fig. 32 Localization of 10.4-14.5 complexes in primary fibroblasts infected with Ad2 mutants

Primary fibroblasts (SeBu cells) were infected with 200 PFU/cell of wt (A) or mutant Ad2/F14.5 viruses (B- D) as indicated on the right. Cells were processed for immunofluorescence analysis at 21h p.i. and stained for wt 10.4 or 10.4LL with polyclonal serum Bur3 or R71, respectively (green), and mAb M1 directed to FLAG-14.5 (red). Upon infection with Ad2/F14.5 (A) or Ad2/F14.5Y74 (B) 10.4 and 14.5 colocalize extensively in the ER/Golgi and 14.5K can be detected at the cell surface. (C) Increased cell surface staining of both 10.4K and 14.5K upon infection with mutant Ad2/F14.5Y122. (D) Infection with Ad2/10.4LL- F14.5: 10.4LL and 14.5K colocalize in a perinuclear compartment and vesicular structures.

accompanied by a reduction of ER staining, which indicated an increased ER export rate and a faster delivery of the complex into lysosomes. In the absence of the dileucine motif 10.4-14.5 is deviated from its normal trafficking route and is primarily transported to lysosomes for degradation. Together with the drastically lowered steady-state surface expression levels of Flag- 14.5K, the data suggest that LL may contribute to recycling to the plasma membrane.

Fig. 33 Disruption of the dileucine motif in 10.4K redistributes the steady-state localization of 14.5K in infected primary fibroblasts (SeBu cells)

SeBu cells were infected with 200 PFU/cell of Ad2/10.4LL-F14.5 (A, C, E, G) or wt Ad2/F14.5 (B, D, F, H) viruses. Infected cells remained untreated (A, B) or were treated for 11 hours with 100nM Bafilomycin A1

(C-H) before processing for immunoflorescence at 21 h p.i. Intracellular localization of 14.5K (Bur4, green) was compared to that of marker proteins (red) for the cis-Golgi (A, B: GM130) and different endosomal compartments: EEA1 (early endosomes in C,D), lysobisphosphatidic acid (LBPA, late endosomes in E, F), Lamp-2 (late endosomes and lysosomes in G, H). The mAbs used are listed in Materials and Methods.

Fig. 34 In infected primary fibroblasts disruption of the 10.4K dileucine motif diverts the mutant protein to lysosomes resulting in increased degradation

Primary fibroblasts (SeBu cells) were infected with 200 PFU/cell of Ad2/10.4LL-F14.5 (A,E) Ad2/F14.5 (B, F), Ad2/F14.5Y74 (C, G), Ad2/F14.5Y122 (D, H) viruses and processed for immunofluorescence at 21 h p.i. Wt 10.4K was detected with polyclonal Ab Bur3 and the 10.4K dileucine mutant with R71 (D, H). Intracellular localization of 10.4K (green) was compared to localization of galactosyltransferase (GLT, red, mAb GTL2), a cellular Golgi-resident protein (A-D). Subsequent to treatment for 11 hours with 100nM Bafilomycin A1 (E-H) cells were costained for 10.4K (green) and Lamp-2 (red, mAb 2D5, late

Fig. 35 10.4-14.5K-induced internalization and degradation of Fas in late endosomes/lysosomes

SV80Fas cells overexpressing Fas were mock-infected (A) or infected with Ad2 (B-C) and processed for confocal laser scanning microscopy at 16 h p. i., subsequent to treatment with 100 nM Baf for 11h.

(A, B) Intracellular localization of 14.5K (green, Ra14.5) was compared to Fas (red), which was detected using a combination of different mAb against Fas (anti-Fas-Mix). (C) Costaining of Fas (green, polyclonal rabbit serum anti-Fas) and Lamp-2 (red, mAb 2D5). Magnification was 100x instead of 63x as in A, B.

5.6. 10.4-14.5K and Fas do not profoundly colocalize in Ad2-infected cells

In Ad2-infected cells 10.4-14.5K induce internalization of Fas and its degradation in late endosomes/lysosomes (Elsing and Burgert, 1998; Tollefson et al., 1998). But it remained unknown, whether or not 10.4-14.5K localizes to the same endosomal/lysosomal vesicles. Therefore, the intracellular distribution of 10.4-14.5K and Fas was analyzed in infected SV80Fas cells which had been treated with Bafilomycin to inhibit Fas degradation. Bafilomycin treatment did not affect distribution of Fas in mock-infected cells and Fas was detected at the cell surface (Fig. 35A). By contrast, in the infected cells Fas was no longer found at the plasma membrane, but exclusively in vesicles (Fig. 35B). Fas+ vesicles costained with Lamp-2 (Fig. 35C). As Bafilomycin treatment inhibits the acidification of late endocytic compartments Lamp-2+ late endosomes/lysosomes (van Weert et al., 1995; Yoshimori et al., 1991) were increased in size and anti-Lamp-2 antibody stained outer membranes which appeared as red circles (Fig. 35C). Fas was found in dense, dot-like

Fig. 36 In Ad2-infected SV80Fas cells 10.4-14.5K do not profoundly colocalize with Fas

SV80Fas cells overexpressing Fas were infected with Ad2, treated with 100 nM Baf for 11h and processed for confocal laser scanning microscopy at 16 h p. i. Intracellular localization of 10.4K (A, B) or 14.5K (C, D) was compared to Fas localization. 10.4K and 14.5K (green) were detected using polyclonal Ab Bur3 and Ra14.5), respectively, and Fas (red) was costained using a combination of different mAb (anti-Fas-Mix).

structures that localized at the outer rim, but also in the inner core of lamp-2+ vesicles (Fig. 35C). Thus, following 10.4-14.5K-induced down-regulation Fas becomes incorporated into the inner core of lysosomes. Despite Bafilomycin treatment the majority of 10.4K positive cells retained the ER/Golgi staining pattern (Fig. 36A). Fas could be detected in vesicles which did not colocalize with the 10.4K positive structures, even if 10.4K was detected in swollen vesicles (Fig. 36B). Like for Fas, Bafilomycin-treatment also increased the number of 14.5K vesicles, but strikingly these 14.5 vesicles only partially overlapped with Fas+ late endosomes/lysosomes (Fig. 36C, D). Thus, at steady state no extensive colocalization of 10.4-14.5 with Fas was observed, suggesting that the association of Fas with the viral proteins might be rather short-lived.

6. Analysis of homologous E3/10.4-14.5K proteins of adenovirus 4 (subgenus E)