Myxoma virus Serp2 is a weak inhibitor of granzyme B and interleukin-1 beta-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 f or Full virulence (F. Messud-Petit, J. Gelfi, M. Delverdier, M. F. Amardeil h, 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-1 b eta-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 react ive site loop and yet are only 35% identical overall. Serp2 protein was cle aved by ICE but, unlike CrmA, did not form a stable complex with ICE that w as detectable by native gel electrophoresis. Attempts to covalently cross-l ink ICE-serpin inhibitory complexes were successful with CrmA, but no compl ex between ICE and Serp2, was visible after cross-linking. Purified His(10) -tagged Serp2 protein was a relatively poor inhibitor of ICE, with a K-i of 80 nM compared to 4 pM for CrmA. Serp2 protein resembled CrmA in that a st able complex with the serine proteinase granzyme B was detectable after sod ium dodecyl sulfate-pulyacrylamide gel electrophoresis. However, Serp2 was less effective at inhibiting granzyme B activity (K-i = 420 nM) than CrmA ( K-i = 100 nM). Finally, Serp2 was tested for the ability to replace CrmA an d inhibit apoptosis in LLC-PK1 cells infected with a CPV recombinant delete d for CrmA but expressing Serp2. Unlike wild-type-CPV-infected cells, apopt osis was readily observed in cells infected with the recombinant virus, as indicated by the induction of both nuclear fragmentation and caspase-mediat ed 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 a nd CrmA are distinct.