Requirement for vacuolar H+ -ATPase activity and Ca2+ gradient during entry of rotavirus into MA104 cells

Abstract

The mechanism by which rotavirus and other nonenveloped viruses enter the cell is still not clear. We have proposed an endocytosis model where the critical step for virus uncoating and membrane permeabilization is the decrease in Ca(2+) concentration in the endosome. In this paper, we monitored rotavirus entry by measuring alpha-sarcin-rotavirus coentry and infectivity in MA104 cells. The participation of endocytosis, acidification, and endosomal Ca(2+) concentration on virus entry was studied by inhibiting the endosomal H(+)-ATPase with bafilomycin A1 and/or increasing the extracellular calcium reservoir by addition of 10 mM CaEGTA. Rotavirus-alpha-sarcin coentry was inhibited by bafilomycin A1 and by addition of 10 mM CaEGTA. These effects were additive. These substances induced a significant inhibition of infectivity without affecting virus binding and postentry steps. These results are compatible with the interpretation that bafilomycin A1 and CaEGTA block rotavirus penetration from the endosome into the cytoplasm and support our hypothesis of a Ca(2+)-dependent endocytosis model.

FIG. 1.

Effect of bafilomycin A1 or 10 mM CaEGTA in the extracellular medium on rotavirus-mediated entry of α-sarcin. (A) MA104 cells were preincubated in a methionine-free MEM for 1 h. During the last 30 min, bafilomycin A1 (500 nM) was added to a group of wells, while another group of wells were left untreated for controls. Then, monolayers were infected with OSU or SA11 rotavirus at a multiplicity of infection of 500 FFU/cell in the presence or absence of α-sarcin (100 mg/ml) and/or bafilomycin A1. After 15 min, inoculum was removed and replaced by methionine-free MEM supplemented with l-[³⁵S]methionine (0.1 μCi/well) without α-sarcin but in the continuous presence or absence of bafilomycin A1. (B) Preincubation, infection, and protein labeling were performed as described above for panel A, except that 10 mM CaEGTA was added only during infection and protein labeling periods. Each value is the mean ± standard error (error bar) (In panel A, n = 21 values from seven experiments with the OSU strain or n = 6 values from two experiments with the SA11 strain. In panel B, n = 9 values from three experiments with the OSU strain or n =6 values from two experiments with SA11 strain. The OSU and SA11 values were compared, and statistically significant different values are indicated by an asterisk [P < 0.001 in paired Student's t test]). (C) Different concentrations of bafilomycin A1 (0, 10, 100, and 500 nM) were used during preincubation, infection, and protein labeling periods with or without 10 mM CaEGTA during infection and labeling periods. Data are plotted as the means of four measurements from one experiment.

FIG. 2.

Inhibition of rotavirus-mediated entry of α-sarcin induced by bafilomycin A1 and/or CaEGTA as a function of the incorporation time of l-[³⁵S]methionine. Preincubation and infection with OSU strain of MA104 cell monolayers were performed as detailed in the legend to Fig. 1. The concentration of bafilomycin was constant (500 nM), and protein labeling periods were 15, 60, and 120 min. Data are plotted as the means plus standard errors (error bars) of nine measurements from three experiments.

FIG. 3.

Effects of 10 mM CaEGTA and bafilomycin A1 on viral synthesis. MA104 cells were preincubated in a medium containing bafilomycin A1 (500 nM) for 30 min. Then, they were infected by serial dilutions of OSU rotavirus in a medium supplemented or not supplemented with 10 mM CaEGTA and/or bafilomycin A1. After 15 min, the virus was removed and washed twice with a medium containing 5 mM EGTA without Ca²⁺ to remove bound virus. Infection was allowed to proceed for 18 h in the presence of CaEGTA or bafilomycin A1, and infectivity (infect.) was determined. Values are means plus the standard errors of the means (error bars) of six independent experiments performed in triplicate. Values that were statistically significantly different from the control value are indicated by an asterisk (P < 0.01 by paired Student's t test).

FIG. 4.

Hypothetical model of rotavirus entry by endocytosis. See text for details.