Jürgen Kartenbeck

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

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.

Clathrin- and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae

Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)–deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1–independent pathway. Internalization was rapid (t 1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.

GM1 structure determines SV40-induced membrane invagination and infection

Incoming simian virus 40 (SV40) particles enter tight-fitting plasma membrane invaginations after binding to the carbohydrate moiety of GM1 gangliosides in the host cell plasma membrane through pentameric VP1 capsid proteins. This is followed by activation of cellular signalling pathways, endocytic internalization and transport of the virus via the endoplasmic reticulum to the nucleus. Here we show that the association of SV40 (as well as isolated pentameric VP1) with GM1 is itself sufficient to induce dramatic membrane curvature that leads to the formation of deep invaginations and tubules not only in the plasma membrane of cells, but also in giant unilamellar vesicles (GUVs). Unlike native GM1 molecules with long acyl chains, GM1 molecular species with short hydrocarbon chains failed to support such invagination, and endocytosis and infection did not occur. To conceptualize the experimental data, a physical model was derived based on energetic considerations. Taken together, our analysis indicates that SV40, other polyoma viruses and some bacterial toxins (Shiga and cholera) use glycosphingolipids and a common pentameric protein scaffold to induce plasma membrane curvature, thus directly promoting their endocytic uptake into cells.

Membrane flow and interconversions among endomembranes

II1. Selective membrane flow as an integral aspect of endomembrane transformations . . . . . . 75 A. General manifestations of membrane dynamics . . . . . . . . . . . . . . . . . . . . . . . 76 B. Endocytosis, lysosomes and intercellular digestion . . . . . . . . . . . . . . . . . . . . . . 76 C. Membrane fluidity and vectorial migration of membrane constituents including endomembrane compartments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 D. Exocytosis, secretory vesicles, and membrane recycling . . . . . . . . . . . . . . . . . . . 82

Targeting of Hepatitis C Virus Core Protein to Mitochondria through a Novel C-Terminal Localization Motif

ABSTRACT The hepatitis C virus (HCV) core protein represents the first 191 amino acids of the viral precursor polyprotein and is cotranslationally inserted into the membrane of the endoplasmic reticulum (ER). Processing at position 179 by a recently identified intramembrane signal peptide peptidase leads to the generation and potential cytosolic release of a 179-amino-acid matured form of the core protein. Using confocal microscopy, we observed that a fraction of the mature core protein colocalized with mitochondrial markers in core-expressing HeLa cells and in Huh-7 cells containing the full-length HCV replicon. Subcellular fractionation confirmed this observation and showed that the core protein associates with purified mitochondrial fractions devoid of ER contaminants. The core protein also fractionated with mitochondrion-associated membranes, a site of physical contact between the ER and mitochondria. Using immunoelectron microscopy and in vitro mitochondrial import assays, we showed that the core protein is located on the mitochondrial outer membrane. A stretch of 10 amino acids within the hydrophobic C terminus of the processed core protein conferred mitochondrial localization when it was fused to green fluorescent protein. The location of the core protein in the outer mitochondrial membrane suggests that it could modulate apoptosis or lipid transfer, both of which are associated with this subcellular compartment, during HCV infection.

Host Cell Factors and Functions Involved in Vesicular Stomatitis Virus Entry

ABSTRACT Vesicular stomatitis virus (VSV) is an animal virus that based on electron microscopy and its dependence on acidic cellular compartments for infection is thought to enter its host cells in a clathrin-dependent manner. The exact cellular mechanism, however, is largely unknown. In this study, we characterized the entry kinetics of VSV and elucidated viral requirements for host cell factors during infection in HeLa cells. We found that endocytosis of VSV was a fast process with a half time of 2.5 to 3 min and that acid activation occurred within 1 to 2 min after internalization in early endosomes. The majority of viral particles were endocytosed in a clathrin-based, dynamin-2-dependent manner. Although associated with some of the surface-bound viruses, the classical adaptor protein complex AP-2 was not required for infection. Time-lapse microscopy revealed that the virus either entered preformed clathrin-coated pits or induced de novo formation of pits. Dynamin-2 was recruited to plasma membrane-confined virus particles. Thus, VSV can induce productive internalization by exploiting a specific combination of the clathrin-associated proteins and cellular functions.

Different modes of internalization of proteins associated with adhaerens junctions and desmosomes: experimental separation of lateral contacts induces endocytosis of desmosomal plaque material.

The distribution and fate of two junctional complexes, zonula adhaerens and desmosomes, after dissociation of cell‐cell contacts is described in MDBK cells. Junctions were split between adjacent cells by treatment with EGTA and proteins associated with the plaques of zonulae adhaerentes and desmosomes were localized by immunological methods. Splitting of these junctions is accompanied by the dislocation of desmosomal plaque protein from the cell periphery and its distribution in punctate arrays over the whole cytoplasm. By contrast, vinculin associated with zonulae adhaerentes is still seen at early times (0.5‐1 h) in a conspicuous belt‐like structure which, however, is displaced from the plasma membrane. Strong vinculin staining is maintained on leading edges of free cell surfaces. Electron microscopy of EGTA‐treated cells exposed to colloidal gold particles reveals the disappearance of junctional structures from the cell periphery and the concomitant appearance of a distinct class of gold particle‐containing vesicles which are coated by dense plaques. These vesicle plaques react with antibodies to desmosomal plaque proteins and are associated with filaments of the cytokeratin type. In the same cells, extended dense aggregates are seen which are most probably the membrane‐detached vinculin‐rich material from the zonula adhaerens . The experiments show that, upon release from their junction‐mediated connections with adjacent cells, major proteins associated with the cytoplasmic side of the junctions remain, for several hours, clustered within plaques displaced from the cell surface. While plaque material of adhaerens junctions containing vinculin is recovered in large belt‐like aggregates, desmosomal plaque protein remains attached to membrane structures and appears on distinct vesicles endocytotically formed from half‐desmosomal equivalents.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular Assembly and Secretion of Recombinant Subviral Particles from Tick-Borne Encephalitis Virus

ABSTRACT It is believed that flavivirus assembly occurs by intracellular budding of the nucleocapsid into the lumen of the endoplasmic reticulum (ER). Recombinant expression of tick-borne encephalitis (TBE) virus envelope proteins prM and E in mammalian cells leads to their incorporation into enveloped recombinant subviral particles (RSPs), which have been used as a model system for studying assembly and entry processes and are also promising vaccine candidates. In this study, we analyzed the formation and secretion of TBE virus RSPs and of a membrane anchor-free E homodimer in mammalian cells. Immunofluorescence microscopy showed that E was accumulated in the lumen of the ER. RSPs were observed by electron microscopy in the rough and smooth ER and in downstream compartments of the secretory pathway. About 75% of the particles appeared to be of the size expected for RSPs (about 30 nm in diameter), but a number of larger particles and tubular structures were also observed in these compartments. Secretion of membrane anchor-free E dimers was detected 30 min after synthesis of prM and E, and secretion of RSPs was detected 1 h after synthesis of prM and E. We also found that the presence of the single N-linked oligosaccharide side chain on the E protein and its trimming by glucosidases was necessary for secretion of RSPs and truncated E dimers. Our results suggest that incorporation of prM and E into RSPs occurs at the ER membrane without other viral elements being required, followed by rapid transport along the compartments of the secretory pathway and secretion. Moreover, the carbohydrate side chain of E is involved in at least one assembly or transport step.

Lymphocytic choriomeningitis virus uses a novel endocytic pathway for infectious entry via late endosomes

The endocytic entry of lymphocytic choriomeningitis virus (LCMV) into host cells was compared to the entry of viruses known to exploit clathrin or caveolae/raft-dependent pathways. Pharmacological inhibitors, expression of pathway-specific dominant-negative constructs, and siRNA silencing of clathrin together with electron and light microscopy provided evidence that although a minority population followed a classical clathrin-mediated mechanism of entry, the majority of these enveloped RNA viruses used a novel endocytic route to late endosomes. The pathway was clathrin, dynamin-2, actin, Arf6, flotillin-1, caveolae, and lipid raft independent but required membrane cholesterol. Unaffected by perturbation of Rab5 or Rab7 and apparently without passing through Rab5/EEA1-positive early endosomes, the viruses reached late endosomes and underwent acid-induced penetration. This membrane trafficking route between the plasma membrane and late endosomes may function in the turnover of a select group of surface glycoproteins such as the dystroglycan complex, which serves as the receptor of LCMV.

Cytoskeletons of retinal pigment epithelial cells: Interspecies differences of expression patterns indicate independence of cell function from the specific complement of cytoskeletal proteins

SummaryIn vertebrate tissue development a given cell differentiation pathway is usually associated with a pattern of expression of a specific set of cytoskeletal proteins, including different intermediate filament (IF) and junctional proteins, which is identical in diverse species. The retinal pigment epithelium (RPE) is a layer of polar cells that have very similar morphological features and practically identical functions in different vertebrate species. However, in biochemical and immunolocalization studies of the cytoskeletal proteins of these cells we have noted remarkable interspecies differences. While chicken RPE cells contain only IFs of the vimentin type and do not possess desmosomes and desmosomal proteins RPE cells of diverse amphibian (Rana ridibunda, Xenopus laevis) and mammalian (rat, guinea pig, rabbit, cow, human) species express cytokeratins 8 and 18 either as their sole IF proteins, or together with vimentin IFs as in guinea pig and a certain subpopulation of bovine RPE cells. Plakoglobin, a plaque protein common to desmosomes and the zonula adhaerens exists in RPE cells of all species, whereas desmoplakin and desmoglein have been identified only in RPE desmosomes of frogs and cows, including bovine RPE cell cultures in which cytokeratins have disappeared and vimentin IFs are the only IFs present. These challenging findings show that neither cytokeratin IFs nor desmosomes are necessary for the establishment and function of a polar epithelial cell layer and that the same basic cellular architecture can be achieved by different programs of expression of cytoskeletal proteins. The differences in the composition of the RPE cytoskeleton further indicate that, at least in this tissue, a specific program of expression of IF and desmosomal proteins is not related to the functions of the RPE cell, which are very similar in the various species.