Selective depletion of stored calcium by thapsigargin blocks rotavirus maturation but not the cytopathic effect

Abstract

Rotavirus matures inside the endoplasmic reticulum (ER), a site of intracellular calcium storage. Total cell Ca 2؉ depletion has been shown to impair virus maturation, arresting this process at the membrane-enveloped intermediate form following its budding into the ER. On the other hand, rotavirus infection leads to an increase in the internal Ca 2؉ concentration ([Ca 2؉ ] i) and sequestered Ca 2؉ pools. We have used thapsigargin, an inhibitor of the Ca 2؉-ATPase of the ER, to release stored Ca 2؉ and to study its role in rotavirus morphogenesis and cytopathic effect. Thapsigargin (0.1 to 1 M) released stored Ca 2؉ from MA-104 cells, as measured by chlorotetracycline fluorescence. The concentration of cytoplasmic Ca 2؉ , measured with fura2, increased in infected cells whether treated or not with thapsigargin. Infectivity was decreased dose dependently by thapsigargin (3 log units at 0.25 to 1 M). In infected cells treated with thapsigargin, glycosylation of VP7 and NS28 was inhibited. Electron microscopy of infected cells treated with thapsigargin showed normal synthesis of viroplasm. However, only membrane-enveloped, not double-shelled, particles could be observed within the ER. The conformation of VP7 in infected cells treated with thapsigargin appeared to be altered, as suggested by decreased immunofluorescence reactivity with monoclonal antibodies to highly conformationdependent VP7 epitopes. The progression of cell death in infected cells, as measured by penetration of ethidium bromide, was not affected by thapsigargin. These results indicate that rotavirus maturation depends on a high sequestered [Ca 2؉ ], specifically in the ER. Cell death is the result of the accumulation of a viral product and is not related to the production of infective particles. This viral product(s) may be responsible for the increase in [Ca 2؉ ] i , which in turn leads to cell death.

Autophagy hijacked through viroporin-activated calcium/calmodulin-dependent kinase kinase-β signaling is required for rotavirus replication

“…NSP4 increases ER membrane permeability to calcium without blocking SERCAmediated calcium uptake (27,28). In contrast, Michelangeli et al (29) reported that TG treatment reduces virus yield due to improper VP7 assembly onto particles, which was attributed to TG blocking calcium uptake rather than the indirect elevation in cytosolic calcium. Our data show that NSP4 viroporin-mediated increases in cytoplasmic calcium activate a specific calcium-induced signaling pathway to initiate autophagy, which enables trafficking of NSP4 and VP7 to viroplasms for subsequent virus assembly.…”

Viral calciomics: Interplays between Ca2+ and virus

“…These lines of evidence suggest that rotavirus-induced external Ca 2+ entry pathway is characteristic of a Ca 2+ channel rather than nonselective impairment of the plasma membrane. Such early activation of Ca 2+ entry is speculated to maintain an elevated ER Ca 2+ pool that is required for virion maturation and stability [47,48].…”

I, 2. Physiology and pathophysiology of the gut in relation to viral diarrhea

“…Among these perturbances is a progressive increase in Ca 2+ membrane permeability which leads to an elevation of cytosolic Ca 2÷ concentration and sequestered pools, without an apparent failure of regulatory mechanisms in virus-infected MA104, HT29 and Caco-2 cells (Brunet et al, 2000a;Michelangeli et al, 1991;Perez et al, 1999). In parallel to the increase in plasma membrane permeability and cytosolic Ca 2+ concentration, there is an enhancement of ER sequestered Ca 2+ stores (Brunet et al, 2000a;Michelangeli et al, 1995;Perez et al, 1999). This effect is likely to be due to the activation of ER Ca 2÷ pumps.…”