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.
Turner
P C
PC
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 32610-0266, USA.
Sancho
M C
MC
Thoennes
S R
SR
Caputo
A
A
Bleackley
R C
RC
Moyer
R W
RW
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