ACTIVATION OF CASPASES IN PIG-KIDNEY CELLS INFECTED WITH WILD-TYPE AND CRMA SPI-2 MUTANTS OF COWPOX AND RABBITPOX VIRUSES/

The cowpox virus (CPV) CrmA and the equivalent rabbitpox virus (RPV) S PI-2 proteins have anti-inflammatory and antiapoptosis activity by vir tue of their ability to inhibit caspases, including the interleukin-1 beta-converting enzyme (ICE; caspase-1), Infection of LLC-PK1 pig kidn ey cells with a CPV CrmA mutant, but not with wild-type (wt) CPV, resu lts in the induction of many of the morphological features of apoptosi s (C. A. Ray and D. J. Pickup, Virology 217:384-391, 1996). In our stu dy, LLC-PK1 cells infected with CPV Delta crmA, but not those infected with wt CPV, showed induction of poly(ADP-ribose) polymerase (PARP)- and lamin A-cleaving activities and processing of the CPP32 (caspase-3 ) precursor to a mature 18-kDa form. Surprisingly, infection of LLC-PK 1 cells with either wt RPV (despite the presence of the SPI-2 protein) or RPV Delta SPI-2 resulted in cleavage activity against PARP and lam in A and the appearance of the mature subunit of CPP32/caspase-3. The biotinylated specific peptide inhibitor Ac-Tyr-Val-Lys (biotinyl)-Asp- 2,6 dimethylbenzoyloxymethylketone [AcYV(bio)KD-aomk] labeled active c aspase subunits of 18, 19, and 21 kDa in extracts from LLC-PK1 cells i nfected with CPV Delta crmA, wt RPV, or RPV Delta SPI-2 but not wt CPV . Mixed infection of LLC-PK1 cells with wt RPV and wt CPV gave no PARP -cleaving activity, and all PARP cleavage mediated by SPI-2 and CrmA m utants of RPV and CPV, respectively, could be eliminated by coinfectio n with wt CPV. These results suggest that the RPV SPI-2 and CPV CrmA p roteins are not functionally equivalent and that CrmA, but not SPI-2 p rotein, can completely prevent apoptosis in LLC-PK1 cells under these conditions.