ROTAVIRUSES INDUCE AN EARLY MEMBRANE PERMEABILIZATION OF MA104 CELLS AND DO NOT REQUIRE A LOW INTRACELLULAR CA2+ CONCENTRATION TO INITIATE THEIR REPLICATION CYCLE

In this work, we found that rotavirus infection induces an early membr ane permeabilization of MA104 cells and promotes the coentry of toxins , such as alpha-sarcin, into the cell. This cell permeability was show n to depend on infectious virus and was also shown to be virus dose de pendent, with 10 infectious particles per cell being sufficient to ach ieve maximum permeability; transient, lasting no more than 15 min afte r virus entry and probably occurring concomitantly with virus penetrat ion; and specific, since cells that are poorly permissive for rotaviru s were not permeabilized. The rotavirus-mediated coentry of toxins was not blocked by the endocytosis inhibitors dansylcadaverine and cytoch alasin D or by the vacuolar proton-ATPase inhibitor bafilomycin A1, su ggesting that neither endocytocis nor an intraendosomal acidic pH or a proton gradient is required for permeabilization of the cells. Compou nds that raise the intracellular concentration of calcium ([Ca2+](i)) by different mechanisms, such as the calcium ionophores A23187 and ion omycin and the endoplasmic reticulum calcium-ATPase inhibitor thapsiga rgin, did not block the coentry of alpha-sarcin or affect the onset of viral protein synthesis, suggesting that a low [Ca2+](i) is not essen tial for the initial steps of the virus life cycle. Since the entry of alpha-sarcin correlates with virus penetration in all parameters test ed, the assay for permeabilization to toxins might be a useful tool fo r studying and characterizing the route of entry and the mechanism use d by rotaviruses to traverse the cell membrane and initiate a producti ve replication cycle.