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Journal of Virology, August 1999, p. 6394-6404, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Peter C. Turner,1 M. Carmen Sancho,1,dagger S. R. Thoennes,1 A. Caputo,2 R. C. Bleackley,2 and Richard W. Moyer1,*

Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida 32610-0266,1 and Biochemistry Department, University of Alberta, Edmonton, Alberta T6G 2H7, Canada2

Received 19 January 1999/Accepted 30 April 1999

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.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Box 100266, Gainesville, FL 32610-0266. Phone: (352) 392-7077. Fax: (352) 846-2042. E-mail: rmoyer{at}medmicro.med.ufl.edu.

dagger Present address: Centro de Investigaciones Biológicas (CSIC), 28006 Madrid, Spain.

Journal of Virology, August 1999, p. 6394-6404, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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