Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER

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.

A nucleic acid-hydrolyzing antibody penetrates into cells via caveolae-mediated endocytosis, localizes in the cytosol and exhibits cytotoxicity

“…However, unlike Ctx-B (Fig. 6B) and SV40 [32], 3D8 scFv-containing caveolae/caveosomes did not translocate to early endosomes, late endosome/lysosomes (Fig. 5), ER or Golgi (Fig.…”

“…The effect of nocodazole on the cytosolic release of 3D8 scFv was further investigated. Nocodazole, a microtubule-depolymerzing agent, inhibits transport of caveolae/caveosomes into caveolin-free carrier vesicles which move rapidly along microtubules to eventually reach other subcellular vesicular organelles [32]. In the pulse-chase experiment, where cells were pulsed with 3D8 scFv (10 mM) for 2 h, treated with nocodazole (20 mM) for 30 min, and then incubated for another 4 h, nocodazole did not block the divergence of the internalized 3D8 scFv into the cytosol (Fig.…”

“…Caveosomes are non-degradative endosomal compartments with neutral pH and contain a caveolae marker (cav-1), lacking classic endosomal markers [13,28]. SV40 virus and Ctx-B are well-known for their caveolar endocytosis and then merging with caveosome before further sorting to ER and/or Golgi [13,28,32]. 3D8 scFv internalized by caveolar endocytosis was colocalized with Ctx-B ( Fig.…”

“…6C). Previously nocodazole, a microtubule depolymerizing agent, was found to confine SV40 in caveolae/caveosome compartments, inhibiting its further trafficking into ER [32]. Escape of some CPPs or viruses from early/late endosomes into the cytosol before its merging with lysosomes depends on acidification of endosomes.…”

The Process of Wrapping Virus Revealed by a Force Tracing Technique and Simulations

“…By force tracing with constant piezoceramics position, the process of viral invagination via cell membranes was recorded until it reaches the balance between endocytosis force and pulling force of AFM cantilever. The force tracing technique occupies several advantages: (i) It allows the single virion investigation, which provides a potent approach to study viral behaviors at the single particle level; (ii) The force of viral invagination is obtained, which is unavailable by real‐time fluorescence microscopy,17, 22 (iii) It enables to extremely fast record the viral invagination at the microsecond level and provide detailed dynamic information, which is unachievable by other current techniques.…”