Adenovirus-dependent changes in cell membrane permeability: role of Na+, K+-ATPase

Abstract

Adenovirus-dependent release of choline phosphate from KB cells at pH 6.0 was partially blocked by ouabain. In K+-containing medium, maximum inhibition of release was obtained by i0-5 M ouabain and half-maximal inhibition was achieved by about 0.5 x 10-6 M ouabain. Ouabain did not block either the binding or the uptake of adenovirus by KB cells. Without K+, about 25% of cell-associated choline phosphate was released by adenovirus, whereas with 1 mM K+ about 50% was released. This activation by K+ was blocked by 0.1 mM ouabain. HeLa cells behaved like KB cells, but a mutant of HeLa cells resistant to ouabain (D98-OR) released much lower amounts of choline phosphate in response to human adenovirus type 2 (Ad2). Wild-type D98-OR cells bound nearly the same amount of adenovirus as did normal HeLa cells. Ad2 also increased the activity of Na+,K+-ATPase in KB cells, with maximum activation at 50 pg of Ad2 per ml. In D98-OR cells, Ad2 failed to activate Na+,K+ ATPase activity. Ad2-dependent lysis of endocytic vesicles (receptosomes) was assayed by measuring Ad2-dependent enhancement of epidermal growth factor-Pseudomonas exotoxin toxicity. This action of adenovirus was increased when K+ was present in the medium. Under the conditions used, K+ had no effect on the amount of Ad2 or epidermal growth factor taken up by the cells. On the basis of these results, it is suggested that Ad2-dependent cellular efflux of choline phosphate and adenovirus-dependent lysis of receptosomes may require Na+,K+-ATPase activity. grown in suspension culture. Ad2 and 35S-labeled Ad2 were prepared by the procedure described earlier (11,28). All of the experiments were conducted by using UV-lightinactivated virus as described earlier (23).Choline release assays. Ad2-dependent release of choline from cells was measured as described previously (26). (We have shown previously [26] that most of the choline released from the cells is in the form of choline phosphate.) In brief, KB, HeLa, or D98-OR cells were plated at a density of 0.5 x 106 cells per 35-mm (diameter) dish and were used 24 h later. Cells were labeled with [3H]choline (10 ,uCi/ml) for 30 min in Dulbecco modified Eagle medium containing 2 mg of bovine serum albumin per ml (medium A). Cells were washed four times with medium A and once with basal Eagle medium, without bicarbonate, containing 2 mg of bovine serum albumin per ml (medium B) adjusted to pH 7.0. Cells were exposed to Ad2 (1 to 50 jig/ml) at 4°C for 60 min; 1 ,ug/ml corresponds to an average of 9 x 103 particles added per cell. Choline release was monitored at 37°C for 60 min, usually in medium B adjusted to pH 6.0 with 25 mM HEPES (N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid). In certain experiments, isotonic sucrose-HEPES (225 mM sucrose, 5 mM glucose, 25 mM HEPES; pH 6.0) or isotonic saline (125 mM NaCl, 5 mM glucose, 25 mM HEPES; pH 6.0) was used. If required, various concentrations of other ions were also included in these buffers. The effect of ouabain was studied by adding various concentrations of ouabain (10-9...

Integrin alpha v beta 5 selectively promotes adenovirus mediated cell membrane permeabilization.

“…This attribute has facilitated the use of adenovirus as a vector for gene therapy (20,21) since this virus also promotes the entry of foreign DNA into the cytoplasm before it can be degraded in lysosomes (7,35), thus overcoming one of the major impediments to gene delivery. Although the precise mechanism by which adenovirus penetrates the cell endosome has yet to be elucidated, virus-induced enhancement of cell membrane permeability has been detected by the release of small molecules such as [3H]choline phosphate from intact cells (23,26). Enhancement of cell membrane permeability is thought to be indicative of events leading to virus penetration of the cell endosome since it occurs with increasing time of incubation at 37°C and also requires mildly acidic conditions (23).…”

“…Although the precise mechanism by which adenovirus penetrates the cell endosome has yet to be elucidated, virus-induced enhancement of cell membrane permeability has been detected by the release of small molecules such as [3H]choline phosphate from intact cells (23,26). Enhancement of cell membrane permeability is thought to be indicative of events leading to virus penetration of the cell endosome since it occurs with increasing time of incubation at 37°C and also requires mildly acidic conditions (23).…”

“…Ad2 also induces cell membrane permeabilization at pH 6.0 (2,23), an activity associated with virus disruption of the cell endosome (penetration). Ad2-mediated membrane permeabilization has been demonstrated by the release of fluorescent dyes from liposomes (2) or by release of 51chromium or pH]choline from human epithelial cells (23). A number of earlier studies indicated that Ad2 penton base has an important role in membrane permeabilization.…”

Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis

“…Adenovirus is a well-studied example of this model. It can be found in vesicles (12,16); inhibitors of endocytosis and weak bases reduce infectivity if they are applied early in infection (50); cell plasma membranes can be made permeable when they are exposed to adenovirus at acid pH, presumably mimicking the enhanced permeability of acidic endosomal vesicles with enclosed adenovirus (44); and finally, reversible, acid-dependent hydrophobicity of adenovirus surface proteins has been demonstrated, providing both a potential mechanism of membrane penetration arnd an analog to the acid-dependent fusion proteins of enveloped viruses (45). Similar sensitivity to weak bases has recently been described for members of two other families of nonenveloped viruses: Reoviridae (reovirus type 3) and Picornaviridae (poliovirus, rhinovirus, and footand-mouth disease virus) (7,9,30,31,39,54).…”

Virus-mediated release of endosomal content in vitro: different behavior of adenovirus and rhinovirus serotype 2.

“…4 A) and during active acidification (16%; see Fig. 5) is compared, it is evident that ~34% of the endosomes labeled with adenovirus can lower their pH below 6.0 in vitro, the pH threshold for membrane permeabilization (Seth et al, 1985).…”