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.
Debiasi
R L
RL
Departments of Pediatric Infectious Diseases, and Denver Veterans Affairs Medical Center, Denver, Colorado 80262, USA.
Squier
M K
MK
Pike
B
B
Wynes
M
M
Dermody
T S
TS
Cohen
J J
JJ
Tyler
K L
KL
D000179:Acrylates / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D051379:Mice; D012087:Reoviridae / Q000502:physiology*; D015398:Signal Transduction; D014466:Ultraviolet Rays
D000179:Acrylates; D015853:Cysteine Proteinase Inhibitors; C100442:PD 150606; D002154:Calpain