title: Activation of caspases in pig kidney cells infected with wild-type and CrmA/SPI-2 mutants of cowpox and rabbitpox viruses.
abstract: The cowpox virus (CPV) CrmA and the equivalent rabbitpox virus (RPV) SPI-2 proteins have anti-inflammatory and antiapoptosis activity by virtue of their ability to inhibit caspases, including the interleukin-1beta-converting enzyme (ICE; caspase-1). Infection of LLC-PK1 pig kidney cells with a CPV CrmA mutant, but not with wild-type (wt) CPV, results in the induction of many of the morphological features of apoptosis (C. A. Ray and D. J. Pickup, Virology 217:384-391, 1996). In our study, 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-PK1 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 lamin 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 caspase subunits of 18, 19, and 21 kDa in extracts from LLC-PK1 cells infected 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 mutants of RPV and CPV, respectively, could be eliminated by coinfection with wt CPV. These results suggest that the RPV SPI-2 and CPV CrmA proteins are not functionally equivalent and that CrmA, but not SPI-2 protein, can completely prevent apoptosis in LLC-PK1 cells under these conditions.
authors.affiliation: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville 32610-0266, USA.
authors.forename: J
authors.initials: J
authors.lastname: Macen
authors.forename: A
authors.initials: A
authors.lastname: Takahashi
authors.forename: K B
authors.initials: KB
authors.lastname: Moon
authors.forename: R
authors.initials: R
authors.lastname: Nathaniel
authors.forename: P C
authors.initials: PC
authors.lastname: Turner
authors.forename: R W
authors.initials: RW
authors.lastname: Moyer
mesh_terms: D000818:Animals; D001710:Biotin; D020170:Caspase 1; D053148:Caspase 3; D020169:Caspases; D002457:Cell Extracts; D002460:Cell Line; D002522:Chlorocebus aethiops; D015608:Cowpox virus / Q000235:genetics / Q000502:physiology*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors; D004789:Enzyme Activation; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D018374:LLC-PK1 Cells; D034904:Lamin Type A; D034882:Lamins; D051379:Mice; D008807:Mice, Inbred BALB C; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D015843:Serpins / Q000235:genetics / Q000378:metabolism*; D013552:Swine; D014616:Vaccinia virus / Q000235:genetics / Q000502:physiology*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D002457:Cell Extracts; D015853:Cysteine Proteinase Inhibitors; D034904:Lamin Type A; D034882:Lamins; D009687:Nuclear Proteins; D015843:Serpins; D014764:Viral Proteins; D001710:Biotin; C074641:interleukin-1beta-converting enzyme inhibitor; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D020170:Caspase 1