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; howev er, calpain, a cytosolic calcium-activated cysteine protease, has been impl icated in several forms of programmed cell death. Reoviruses induce apoptos is both in vitro and in vivo and serve as a model for studying virus-induce d cell death. We investigated the potential role of calpain in reovirus-ind uced 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 fluor ogenic 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 m ock-infected cells; this increase was completely inhibited by preincubation with calpain inhibitor I (N-acetyl-leucyl-leucyl-norleucinal [aLLN]), an a ctive site inhibitor. Both aLLN and PD150606, a specific calpain inhibitor that interacts with the calcium-binding site, inhibited reovirus-induced ap optosis in L929 cells by 54 to 93%. Apoptosis induced by UV-inactivated reo virus was also reduced 65 to 69% by aLLN, indicating that inhibition of apo ptosis by calpain inhibitors is independent of effects on viral replication . We conclude that calpain activation is a component of the regulatory casc ade in reovirus-induced apoptosis.