Abstract
Simian virus 40 (SV40) is unusual among animal viruses in that it enters cells through caveolae, and the internalized virus accumulates in a smooth endoplasmic reticulum (ER) compartment. Using video-enhanced, dual-colour, live fluorescence microscopy, we show the uptake of individual virus particles in CV-1 cells. After associating with caveolae, SV40 leaves the plasma membrane in small, caveolin-1-containing vesicles. It then enters larger, peripheral organelles with a non-acidic pH. Although rich in caveolin-1, these organelles do not contain markers for endosomes, lysosomes, ER or Golgi, nor do they acquire ligands of clathrin-coated vesicle endocytosis. After several hours in these organelles, SV40 is sorted into tubular, caveolin-free membrane vesicles that move rapidly along microtubules, and is deposited in perinuclear, syntaxin 17-positive, smooth ER organelles. The microtubule-disrupting agent nocodazole inhibits formation and transport of these tubular carriers, and blocks viral infection. Our results demonstrate the existence of a two-step transport pathway from plasma-membrane caveolae, through an intermediate organelle (termed the caveosome), to the ER. This pathway bypasses endosomes and the Golgi complex, and is part of the productive infectious route used by SV40.
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Acknowledgements
We thank all laboratory members for discussions and suggestions throughout this work, M. Kowarik, K. Breiner and M. Molinari for critical reading of the manuscript, and A. Mezzacasa for help with microscopes. We also thank U. Lathinen for cDNA encoding caveolin-1, D. Toomre for Ptk2 cells expressing YFP–α-tubulin, and M. Steegmaier for antibodies against syntaxin 17. This work was supported by the Swiss National Science Foundation and by the Eidgenossische Technische Hochschule Zürich.
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Movie 1
Dynamics of TRX-SV40 bound to the plasma membrane of CV-1 cells directly after shifting to 37 ºC recorded with wide field microscopy. A part of the membrane in which both mobile and stationary spots can be discerned (recorded at 0.33 Hz, shown at 20 Hz, 50 frames). The mobile spots (arrows) move randomly in the membrane. There is considerable bleaching due to the high exposure. Scale bar: 2 µm. (MOV 958 kb)
Movie 2
Dynamics of TRX-SV40 and CAV1–GFP on the plasma membrane of CV-1 cells directly after shifting to 37º C recorded with confocal laser scanning microscopy. The movie shows parts of the membrane with TRX-SV40 (red) virions and CAV1-GFP positive microdomains (green), and the resulting yellow spots on the membrane (recorded at 0.33 Hz, shown at 20 Hz, 80 frames). These spots are stationary in the membrane, but that three of them (indicated by arrowheads) disappear, whereas others remain in place. CAV1–GFP is present as a diffusive dynamic staining between the spots. Scale bar: 2 µm. (MOV 3056 kb)
Movie 3
Dynamics of TRX-SV40 being internalised in CV-1 cells, incubated for 1 h at 37 ºC after virus binding before recording with wide field microscopy (recorded at 0.33 Hz, shown at 10 Hz, 50 frames). Small vesicles gain in speed (arrow), by contrast with spots (of about the same size) on the membrane (asterisk), which have not been internalised yet. Some larger vesicles are already visible (arrowhead). The vesicles movements do not share a common orientation. Scale bar: 7 µm. (MOV 1134 kb)
Movie 4a
Dual-colour live-fluorescence laser scanning confocal microscopy recorded in CAV1–GFP transfected CV-1 cells 4 h after virus binding and shifting to 37 ºC (recorded at 0.33 Hz, shown at 10 Hz). The movie shows part of a cell (part of the nucleus is visible in upper left corner) in which virus has accumulated in CAV1–GFPcontaining organelles, but is also present in unlabelled vesicles. Arrowheads indicate the formation of multi-domain organelles and separation of virus from a CAV1–GFP containing vesicle. The vesicles are dynamic and they interact frequently in a 'kiss-and-run' fashion. High-speed travelling structures are only positive for virus (red). Scale bar: 2 µm. (MOV 1511 kb)
Movie 4b
Same as movie 4a, especially focussed on tubule formation from a virus-positive vesicle (lower arrowhead), on which some CAV1–GFP is still located (upper arrowhead). Double positive vesicles (yellow, left upper region) do not move. Scale bar: 2 µm. (MOV 1171 kb)
Movie 4c
Similar to movie 4a, but at 20 Hz. This enlargement shows the dynamics of sorting indicated by arrowheads. The separation of green and red domains on one organelle (upper arrowhead), and the fast sorting and detachment of virus-positive tubules from double positive vesicles are visible. Scale bar: 5 µm (MOV 294 kb)
Movie 4d
Similar to movie 4c.Upper arrow, sorting of virus; middle arrow, partial fusion of one double positive vesicle with another (upper arrow) and subsequent fusion. Right arrow, dynamics of several small organelles, as examples of fusion and fission reactions. Scale bar: 5 µm. (MOV 310 kb)
Movie 5a
Dynamics of TRX-SV40 targeted to a perinuclear accumulation 6 h after virus binding (recorded at 0.5 Hz, shown at 10 Hz, 50 frames). Large tubular carriers that can span a large part of the cell (arrows) and move both towards and away from the perinuclear accumulation site. In the periphery, dynamics of tubule formation (white box, shown in movie 7a). Scale bar: 5 µm. (MOV 1735 kb)
Movie 5b
White box in movie 5a, showing the dynamic formation of tubules from a vesicular structure (arrowhead). Note how tubules extend from the vesicular structure and 'search for their way out'. Scale bar: 2 µm. (MOV 472 kb)
Movie 5c
The same as movie 5a, from another part of the same cell. (MOV 293 kb)
Movie 6a
Dual-colour live-fluorescence microscopy recorded in PtK2 cells expressing YFP-atubulin, 6 h after virus binding and shifting to 37 ºC (recorded at 0.25 Hz, shown at 10 Hz). There is an intricate network of microtubules. Virus has started to accumulate near the nucleus. Tubules containing virus move along pre-assembled microtubule tracks (arrows). Vesicular structures in the periphery are not moving (asterisk, bottom left). There is considerable bleaching of YFP-atubulin. Scale bar: 10 µm. (MOV 2695 kb)
Movie 6b
Same as movie 6a, a selection of the upper left region from movie 6a. Scale bar: 10 µm. (MOV 1681 kb)
Movie 6c
Same as movie 6a, focussed on vesicular structures from which tubules come out (arrowheads). The tubules grow along the aligning microtubules. Scale bar: 5 µm. (MOV 630 kb)
Movie 6d
Same as movie 6a, focussed on vesicular structures from which tubules come out (arrowheads). The tubules grow along the aligning microtubules. Scale bar: 5 µm. (MOV 410 kb)
Movie 7a
Same as movie 6, but the cells have been treated with nocodazole throughout the whole experiment. Viruses have entered the cells and have accumulated in vesicular structures, but do not move towards the nucleus. Vesicular structures are dynamic, but show no net movement. There is a large amount of small vesicles. Scale bar: 10 µm. (MOV 2931 kb)
Movie 7b
Selection of movie 7a (see white box) indicating that fission of bigger vesicular structures results in the formation of multiple small vesicles (arrow), instead of larger tubules. Scale bar: 2 µm. (MOV 313 kb)
Movie 8
Fluorescence recovery after photobleaching (FRAP) experiment in CV-1 cells recorded with laser scanning confocal microscopy (recorded at 0.017 Hz, shown at 10 Hz) 6 h after virus binding and shifting to 37 ºC. The movie shows a cell which has accumulated a large amount of virus at the perinuclear site, but yet contains a lager amount of carriers. In the second frame, the prominent accumulation (indicated by yellow line) completely disappeared (owing to bleaching with a high power laser beam), and subsequently recovers with newly targeted virus. The structure before and after bleaching is similar. Scale bar: 5 µm. (MOV 1490 kb)
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Pelkmans, L., Kartenbeck, J. & Helenius, A. Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat Cell Biol 3, 473–483 (2001). https://doi.org/10.1038/35074539
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DOI: https://doi.org/10.1038/35074539
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