SELECTIVE DEPLETION OF STORED CALCIUM BY THAPSIGARGIN BLOCKS ROTAVIRUS MATURATION BUT NOT THE CYTOPATHIC EFFECT

Rotavirus matures inside the endoplasmic reticulum (ER), a site of int racellular calcium storage. Total cell Ca2+ depletion has been shown t o impair virus maturation, arresting this process at the membrane enve loped intermediate form following its budding into the ER. On the othe r hand, rotavirus infection leads to an increase in the internal Ca2concentration ([Ca2+](i)) and sequestered Ca2+ pools. We have used tha psigargin, an inhibitor of the Ca2+-ATPase of the ER, to release store d Ca2+ and to study its role in rotavirus morphogenesis and cytopathic effect. Thapsigargin (0.1 to 1 mu M) released stored Ca2+ from MA-104 cells, as measured by chlorotetracycline fluorescence. The concentrat ion of cytoplasmic Ca2+, measured with fura 2, increased in infected c ells whether treated or not with thapsigargin. Infectivity was decreas ed dose dependently by thapsigargin (3 log units at 0.25 to 1 mu M). I n infected cells treated with thapsigargin, glycosylation of VP7 and N S28 was inhibited. Electron microscopy of infected cells treated with thapsigargin showed normal synthesis of viroplasm. However, only membr ane-enveloped, not double-shelled, particles could be observed within the ER. The conformation of VP7 in infected cells treated with thapsig argin appeared to be altered, as suggested by decreased immunofluoresc ence reactivity with monoclonal antibodies to highly conformation-depe ndent VP7 epitopes, The progression of cell death in infected cells, a s measured by penetration of ethidium bromide, was not affected by tha psigargin, These results indicate that rotavirus maturation depends on a high sequestered [Ca2+], specifically in the ER. Cell death is the result of the accumulation of a viral product and is not related to th e production of infective particles. This viral product(s) may be resp onsible for the increase in [Ca2+](i) which in turn leads to Cell deat h.