Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes

Abstract

Semliki forest virus (SFV) is internalized by clathrin-mediated endocytosis, and transported via early endosomes to late endosomes and lysosomes. The intracellular pathway taken by individual fluorescently labeled SFV particles was followed using immunofluorescence in untransfected cells, and by video-enhanced, triple-color fluorescence microscopy in live cells transfected with GFP- and RFP-tagged Rab5, Rab7, Rab4, and Arf1. The viruses progressed from Rab5-positive early endosomes to a population of early endosomes (about 10% of total) that contained both Rab5 and Rab7. SFV were sequestered in the Rab7 domains, and they were sorted away from the early endosomes when these domains detached as separate transport carriers devoid of Rab5, Rab4, EEA1, Arf1, and transferrin. The process was independent of Arf1 and the acidic pH in early endosomes. Nocodazole treatment showed that the release of transport carriers was assisted by microtubules. Expression of constitutively inactive Rab7T22N resulted in accumulation of SFV in early endosomes. We concluded that Rab7 is recruited to early endosomes, where it forms distinct domains that mediate cargo sorting as well as the formation of late-endosome-targeted transport vesicles.

Figure 1. Fluorophores Label Glycoproteins E1 and E2 of SFV

(A) Analysis of SFV-FITC by non-reducing SDS-PAGE with Coomassie blue staining in the left lane and fluorography in the right lane. E1 (49 kDa) and E2 (52 kDa) are the only proteins that are fluorescently labeled; capsid protein C and E3 are not. (B) Confocal microscopy of Cy5-labeled SFV, showing individual spots of uniform size (approximately 0.4 μm). The relative fluorescence intensity distribution of two spots is shown. The distribution of the fluorescence fits to a Gaussian function. (Fit is calculated using the equation: y = e ^{−x2.) Scale bar represents 1 μm.M}

Figure 2. Internalization, Penetration, and Degradation

SFV-Cy5 (at a MOI of 20) was allowed to bind to Vero cells for 60 min at 4 °C, followed by transfer to 37 °C for different time periods. (A) The cells were fixed and permeabilized 10 min after warming, followed by immunostaining using antibodies to EEA1. SFV-Cy5 is present in EEA1-positive endosomes. (B) The cells were fixed 20 min after warming and immunostained with anti-Rab7 antibodies. SFV-Cy5 is present in Rab7-positive endosomes. (C) Non-labeled SFV was used, and the cells were fixed and permeabilized 30 min after warming and immunostained with anti-LAMP-1 and anti-E1/E2 antibodies. SFV is present in endosomes positive for LAMP-1. (D) A FACS-based infection assay was used to determine the time course of SFV penetration during entry. Vero cells were infected with a MOI of one. NH₄Cl was added at different time points to inhibit infection. Cells were further incubated at 37 °C for 5 h, then fixed and immunostained for newly synthesized glycoproteins (n = 3). (E) Analysis of E1/E2 degradation was determined using immunoblotting. Virus (MOI of 50) was bound to Vero cells in the cold, and unbound virus was washed away. Cells were incubated for indicated times, and surface-associated viruses were removed by Proteinase K treatment. Cells were lysed, and after SDS-PAGE, immunoblotting was performed with an antibody against E1/E2. Note that contrary to non-reduced samples (Figure 1A), E1 and E2 co-migrate in SDS-PAGE after reduction. Scale bars represent 5 μm.

Figure 3. Endosomes Labeled with Two Markers Show Presence of Rab7 in Early Endosomes

Vero cells were fixed and viewed by confocal microscopy. (A) Confocal microscopy of immunolabeled cells using anti-EEA1 (green) and anti-Rab7 (red) antibodies, (B–F) The cells were transfected with fluorescent-protein-labeled constructs as follows: (B) GFP-Rab5 and RFP-Rab7, (C) GFP-Rab4 and RFP-Rab5, (D) Arf1-GFP and RFP-Rab5, (E) GFP-Rab4 and RFP-Rab7, and (F) Arf1-GFP and RFP-Rab7. Arrowheads show individual endosomes positive for one of the two markers, and arrows indicate endosomes positive for two markers. Scale bars represent 10 μm.

Figure 4. Progression of SFV through Endosomes Labeled for EEA1 and Rab7

(A) Confocal microscopy of Vero cells. SFV-Cy5 was allowed to bind to Vero cells (at a MOI of 20) for 60 min at 4 °C, followed by transfer to 37 °C and fixed after different times (a, 0 min; b, 10 min; c, 20 min; d, 30 min) and immunolabeled with EEA1 and anti-Rab7 antibodies. Shown are three viruses for each time point. Scale bars represent 10 μm. In panel a, SFV do not co-localize with any endosomal marker; in panel b, SFV are localized in EEA1-positive endosomes; in panel c, SFV are localized in EEA1- and Rab7-positive endosomes; and in panel d, SFV are localized in Rab7-positive endosomes. (B) Quantification of SFV-Cy5 in endosomes that contain EEA1 but no Rab7 (green line), endosomes that contain both (blue line), and structures that contain only Rab7 (red line) over time. The values are normalized such that 100% represents the number of cell-associated viruses at each time point corrected for the degradation.

Figure 5. Sorting of Rab7 and SFV from Early Endosomes

(A–C) Selected images obtained from a time series starting 20 min after warming. Vero cells were transfected with (A) GFP-Rab5 and RFP-Rab7, (B) GFP-Rab4 and RFP-Rab5, or (C) GFP-Rab4 and RFP-Rab7; SFV-Cy5 was then added. SFV-Cy5 is sorted away from Rab5- and Rab4-positive endosomes in a Rab7-positive vesicle (arrowheads). Scale bars represent 2.5 μm. (D) Confocal microscopy of a Vero cell transfected with the dominant negative GFP-Rab7T22N and RFP-Rab5, followed by incubation with SFV-Cy5. Virus was internalized for 1 h at 37 °C. SFV-Cy5 is present in large Rab5-positive endosomes (arrowheads). Scale bar represents 10 μm. (E and F) Confocal microscopy of Vero cells transfected with the dominant negative GFP-Rab7T22N (insets), followed by incubation with SFV-Cy5. Virus was internalized for 1 h at 37 °C. Cells were fixed, permeabilized, and immunostained for (E) EEA1 or (F) LAMP-1. In all, 86.3% of SFV-Cy5 (red) is present in large EEA1-positive endosomes (green in [E]) and not in LAMP-1-positive structures (green in [F]) (arrowheads). Note that SFV-Cy5 is present in LAMP-1-positive structures in untransfected cells (arrows). Scale bars represent 10 μm.

Figure 6. Sorting of SFV into Late Endosomes is Arf1- and pH-Independent

(A and B) Selected images, obtained from time series recorded approximately 20 min after warming. Vero cells were transfected with (A) Arf1-GFP and RFP-Rab5, or (B) Arf1-GFP and RFP-Rab7, then SFV-Cy5 was added. Arf1 is present on Rab5-positive endosomes. SFV-Cy5 is sorted away from Arf1- and Rab5-positive endosomes in a Rab7-positive vesicle (arrowheads). Scale bars represent 2.5 μm. (C and D) Confocal microscopy of a Vero cell transfected with GFP-Rab7 (green in [E]) or GFP-Rab5 (green in [D]) and Arf1T31N-myc and incubated with SFV-Cy5 (red). Cells were fixed, permeabilized, and immunostained using an antibody against myc-tag (9E10) after 1 h of internalization. SFV-Cy5 is not present in Rab5 but is present in Rab7-positive endosomes (arrowheads), indicating that Arf1 is not needed for the sorting of SFV into Rab7-positive endosomes. Scale bars represent 10 μm. (E) Wide field fluorescence image of Bafilomycin A₁–treated (25 nM) Vero cells expressing YFP-Rab5 and CFP-Rab7, at 2 h after internalization of SFV-AF594. SFV is present in Rab7-positive endosomes (arrowheads), indicating that no acidic pH is needed for the sorting of SFV into Rab7-positive endosomes. Scale bar represents 5 μm. (F) NH₄Cl-treated (20 mM) Vero cells expressing YFP-Rab5 and CFP-Rab7 incubated with Dextran–Texas Red for 2 h. Dextran is present in Rab7-positive endosomes (arrowheads), indicating that no acidic pH is needed for the sorting of SFV and Dextran into Rab7-positive endosomes. Scale bar represents 5 μm.

Figure 7. Endosomes Move along Microtubules

(A) Confocal microscopy of a Vero cell transfected with GFP-Rab5 and RFP-Rab7 after treatment with 5 μM nocodazole. Endosomes are enlarged and many (approximately 75%) Rab5-positive endosomes are also positive for Rab7 (arrowheads). Scale bar represents 10 μm. (B) Cells treated as in (A) were incubated with SFV-Cy5, and fixed 1 h after warming. SFV-Cy5 is present in bigger endosomes positive for Rab5 and Rab7 (arrowheads). Scale bar represents 5 μm. (C) Confocal microscopy of a Vero cell treated with 5 μM nocodazole and incubated with SFV-Cy5. After 1 h of internalization at 37 °C, cells were fixed, permeabilized, and immunostained for EEA1 and Rab7. Endosomes positive for both markers can be seen (white rectangles) (41%), and SFV-Cy5 is captured in these structures (yellow rectangles). The protrusions of Rab5 shown in (A) are not seen in (C) and are probably caused by overexpression of the Rab proteins. Scale bar represents 10 μm.

Figure 8. Schematic Representation of the SFV Transport Pathway from the Plasma Membrane to Lysosomes

After uncoating of clathrin, the primary endocytic vesicles deliver the virus to Rab5-positive early endosomes, which contain Rab5 and Arf1 domains but do not contain Rab7. Rab7 is then recruited to these endosomes, and it forms additional domains (blue) that exclude the other early endosome-associated small GTPases. The virus moves into the Rab7 domain, and when this domain leaves the endosome as a Rab7-containing transport vesicle, the virus also leaves the Rab5-positive organelle. The Rab7-containing carrier moves along microtubules and fuses with other carriers or late endosomes. Also shown is an alternative route from the early to the late endosome, involving Arf1 and the ECV, which is not used by SFV. CCP, clathrin-coated pit; CCV, clathrin-coated vesicle; EE, early endosome; MT, microtubule; RE, recycling endosome; LE, late endosome; Lys, lysosome.