Mammalian ortheoreoviruses are currently being investigated as novel cancer therapeutics, but the cellular mechanisms that regulate susceptibility to reovirus oncolysis remain poorly understood. In this study, we present evidence that virion disassembly is a key determinant of reovirus oncolysis. To penetrate cell membranes and initiate infection, the outermost capsid proteins of reovirus must be proteolyzed to generate a disassembled particle called an infectious subviral particle (ISVP). In fibroblasts, this process is mediated by the endo/lysosomal proteases cathepsins B and L. We have analyzed the early events of infection in reovirus-susceptible and -resistant cells. We find that, in contrast to susceptible glioma cells and Ras-transformed NIH3T3 cells, reovirus-resistant cancer cells and untransformed NIH3T3 cells restrict virion uncoating and subsequent gene expression. Disassembly-restrictive cells support reovirus infection, as in vitro-generated ISVPs establish productive infection, and pretreatment with poly(I:C) does not prevent infection in cancer cells. We find that the level of active cathepsin B and L is increased in tumors and that disassembly-restrictive glioma cells support reovirus oncolysis when grown as a tumor in vivo. Together, these results provide a model in which proteolytic disassembly of reovirus is a critical determinant of susceptibility to reovirus oncolysis.

The oligomeric state of the Na/Ca-K exchanger in the plasma membrane of bovine photoreceptors was investigated using chemical cross-linking techniques. In the natural membrane, virtually all Na/Ca-K exchanger could be cross-linked mainly to a complex having an apparent molecular mass of 490 kDa by cupric phenanthroline catalyzed disulfide bonding as evidenced by Western blotting. Stable cross-links of the exchanger were also obtained with the thiol-specific reagent N,N'-p-phenylidenedimaleimide. Neuraminidase treatment reduced the apparent molecular mass of the highly glycosylated Na/Ca-K exchanger and of the 490 kDa cross-link product by 50 and 85 kDa, respectively. DL-1,4-Bismaleimido-2,3-butanediol (BMBD), a novel cleavable dimaleimide, was synthesized in order to produce cross-links that were stable to reductive conditions. Purification of the BMBD cross-linked exchanger followed by two-dimensional SDS polyacrylamide electrophoresis identified the cross-linked homodimers of the exchanger. There was no indication of higher oligomers, suggesting that the exchanger exists as a dimer in the plasma membrane. Hydrodynamic properties of the detergent-solubilized exchanger were determined by velocity sedimentation and gel filtration chromatography. The Triton X-100-solubilized exchanger ran as a single species having a Stokes radius of 10.0 nm, a sedimentation coefficient of 5.4 S, and a partial specific volume of 0.74 mL/g in Triton X-100. Similar results were obtained for the CHAPS-solubilized exchanger. A molecular mass of 236 and 205 kDa was calculated for the exchanger-detergent complex and the detergent-free protein, respectively. Neuraminidase treatment further reduced the molecular mass of the exchanger indicating that glycosylation contributes significantly to the mass of the exchanger. Cross-links of the exchanger were not detected if cross-linking was attempted after solubilization in 10 mM CHAPS. However, after reconstitution of the purified exchanger into soybean phosphatidylcholine vesicles, chemical cross-linking yielded again dimers. On the basis of these cross-linking and hydrodynamic studies, we conclude that the exchanger exists as a homodimer in the rod outer segment plasma membrane but dissociates into a monomer when solubilized in detergent.

The structural and functional properties of the bovine rod photoreceptor Na/Ca-K exchanger and its distribution in vertebrate photoreceptor cells were studied using a panel of monoclonal antibodies. Antibodies that bind to distinct epitopes along the large hydrophilic N-terminal segment of the exchanger labeled the extracellular surface of the rod outer segment plasma membrane, whereas antibodies against a large hydrophilic loop between the two membrane domains labeled the intracellular side. Enzymatic deglycosylation studies indicated that the exchanger primarily contains O-linked sialo-oligosaccharides located within the N-terminal domain. Removal of the extracellular domain with trypsin or the large intracellular domain with kallikrein did not alter the Na ؉ -or K ؉ -dependent Ca 2؉ efflux activity of the exchanger when reconstituted into lipid vesicles. Anti-exchanger antibodies were also used to visualize the distribution of the exchanger in the retina by light and electron microscopy. The exchanger was localized to the plasma membrane of rod outer segments. No labeling was observed in the disk membranes, cone photoreceptor cells, or other retinal neurons, and only faint staining was seen in the rod inner segment. These results indicate that the O-linked glycosylated rod Na/ Ca-K exchanger is specifically targeted to the plasma membrane of rod photoreceptors and has a topological organization similar to that reported for the cardiac Na/Ca exchanger. The large intracellular and extracellular domains do not directly function in the transport of ions across the rod outer segment plasma membrane, but instead may play a role in protein-protein interactions that maintain the spatial organization of the exchanger in the plasma membrane or possibly regulate transport activity of the exchanger.