Reovirus-induced apoptosis is preceded by increased cellular calpain activity and is blocked by calpain inhibitors

The cellular pathways of Apoptosis have not been fully characterized; however, Calpain, a cytosolic calcium-activated cysteine Protease, has been implicated in several forms of programmed cell death. Reoviruses induce Apoptosis both in vitro and in vivo and serve as a model for studying virus-induced cell death. We investigated the potential role of Calpain in reovirus-induced Apoptosis in vitro by measuring Calpain activity as well as evaluating the effects of Calpain inhibitors. L929 cells were infected with reovirus type 3 Abney (T3A), and Calpain activity, measured as cleavage of the fluorogenic Calpain substrate Suc-Leu-Leu-Val-Tyr-AMC, was monitored. There was a 1.6-fold increase in Calpain activity in T3A-infected cells compared to mock-infected cells; this increase was completely inhibited by preincubation with Calpain Inhibitor I (N-acetyl-leucyl-leucyl-norleucinal [aLLN]), an active-site inhibitor. Both aLLN and PD150606, a specific Calpain Inhibitor that interacts with the calcium-binding site, inhibited reovirus-induced Apoptosis in L929 cells by 54 to 93%. Apoptosis induced by UV-inactivated reovirus was also reduced 65 to 69% by aLLN, indicating that inhibition of Apoptosis by Calpain inhibitors is independent of effects on viral replication. We conclude that Calpain activation is a component of the regulatory cascade in reovirus-induced Apoptosis.