Myxoma virus Serp2 is a weak inhibitor of granzyme B and interleukin-1beta-converting enzyme in vitro and unlike CrmA cannot block apoptosis in cowpox virus-infected cells.
The Serp2 protein encoded by the leporipoxvirus myxoma virus is essential for full virulence (F. Messud-Petit, J. Gelfi, M. Delverdier, M. F. Amardeilh, R. Py, G. Sutter, and S. Bertagnoli, J. Virol. 72:7830-7839, 1998) and, like crmA of cowpox virus (CPV), is reported to inhibit the interleukin-1beta-converting enzyme (ICE, caspase-1) (F. Petit, S. Bertagnoli, J. Gelfi, F. Fassy, C. Boucraut-Baralon, and A. Milon, J. Virol. 70:5860-5866, 1996). Serp2 and CrmA both contain Asp at the P1 position within the serpin reactive site loop and yet are only 35% identical overall. Serp2 protein was cleaved by ICE but, unlike CrmA, did not form a stable complex with ICE that was detectable by native gel electrophoresis. Attempts to covalently cross-link ICE-serpin inhibitory complexes were successful with CrmA, but no complex between ICE and Serp2 was visible after cross-linking. Purified His10-tagged Serp2 protein was a relatively poor inhibitor of ICE, with a Ki of 80 nM compared to 4 pM for CrmA. Serp2 protein resembled CrmA in that a stable complex with the serine proteinase granzyme B was detectable after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, Serp2 was less effective at inhibiting granzyme B activity (Ki = 420 nM) than CrmA (Ki = 100 nM). Finally, Serp2 was tested for the ability to replace CrmA and inhibit apoptosis in LLC-PK1 cells infected with a CPV recombinant deleted for CrmA but expressing Serp2. Unlike wild-type-CPV-infected cells, apoptosis was readily observed in cells infected with the recombinant virus, as indicated by the induction of both nuclear fragmentation and caspase-mediated cleavage of DEVD-AMC [acetyl-Asp-Glu-Val-Asp-(amino-4-methyl coumarin)]. These results indicate that Serp2 is unable to functionally substitute for CrmA within the context of CPV and that the inhibition spectra for Serp2 and CrmA are distinct.
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 32610-0266, USA.
P C
PC
Turner
M C
MC
Sancho
S R
SR
Thoennes
A
A
Caputo
R C
RC
Bleackley
R W
RW
Moyer
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D020170:Caspase 1 / Q000378:metabolism; D061945:Caspase Inhibitors; D015608:Cowpox virus / Q000472:pathogenicity*; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005814:Genes, Viral; D053804:Granzymes; D006801:Humans; D018374:LLC-PK1 Cells; D051379:Mice; D008969:Molecular Sequence Data; D009233:Myxoma virus / Q000235:genetics; D010455:Peptides / Q000378:metabolism; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000378:metabolism*; D015843:Serpins / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D010455:Peptides; D015842:Serine Proteinase Inhibitors; C120680:Serp2 protein, myxoma virus; D015843:Serpins; D014764:Viral Proteins; C074641:interleukin-1beta-converting enzyme inhibitor; C509213:GZMB protein, human; D053804:Granzymes; C509214:Gzmb protein, mouse; D012697:Serine Endopeptidases; D020170:Caspase 1