CrmA serves to regulate inflammation by blocking the proteolytic activation of the precursor proIL-1β by inhibition of IL-1β converting enzyme (ICE caspase-1) ( 40). ICE is the prototypic member of the family of caspases that in mammals has at least thirteen members ( 3, 51). The prototypic poxvirus serpin is the cowpox virus CrmA protein ( 37), which has aspartic acid at the P1 position in the RSL, consistent with the ability of CrmA to inhibit caspases (cysteine proteinases which cleave after aspartic acid) and granzyme B. It is this residue which largely determines proteinase specificity ( 38). Typical of serpins, the P1 residue of a given serpin is located within the reactive site loop (RSL) close to the C terminus. ![]() Many genera of poxviruses also encode serpins (serine proteinase inhibitors), some of which inhibit inflammation by interfering both with the processing of cellular cytokines from precursors and with apoptosis ( 53). Orthopoxviruses such as cowpox virus (CPV) and vaccinia virus (VV) express a secreted receptor for interleukin-1β (IL-1β) (VV open reading frame B15R) and a complement control protein homolog (ORF C21L), in addition to other immune modulators, including a variety of soluble cytokine receptors (viroceptors) and a cytokine mimic (virokine) which belongs to the epidermal growth factor superfamily. Leporipoxviruses, such as MYX, encode secreted receptors for tumor necrosis factor alpha (TNF-α) (gene T2), gamma interferon (IFN-γ) (gene T7) ( 27), and for chemokines (gene T1 and gene T7 ). Examples of such genes include those which subvert the host immune response by interfering with cytokine action ( 27, 36, 42). Many of the genes involved in the virulence, pathogenesis, and host range of poxviruses are nonessential for growth in cell culture and typically reside at either end of the linear viral genome, outside the central conserved core of genes devoted to housekeeping functions ( 29). However, MYX exists in a nonpathogenic symbiotic relationship with its natural host, the South American rabbit ( Sylvilagus sp.), and other leporipoxviruses, such as the Shope fibroma virus, induce only a mild disease. This disease is almost invariably lethal and involves fulminating lesions and immunosuppression leading to severe gram-negative bacterial secondary infections of the respiratory tract ( 26). Myxoma virus (MYX) is the causative agent of myxomatosis in the European rabbit Oryctolagus cuniculus. 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. 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. 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. However, Serp2 was less effective at inhibiting granzyme B activity ( K i = 420 nM) than CrmA ( K i = 100 nM). Serp2 protein resembled CrmA in that a stable complex with the serine proteinase granzyme B was detectable after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ![]() 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. 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. Serp2 protein was cleaved by ICE but, unlike CrmA, did not form a stable complex with ICE that was detectable by native gel electrophoresis. Serp2 and CrmA both contain Asp at the P1 position within the serpin reactive site loop and yet are only 35% identical overall. 72:7830–7839, 1998) and, like crmA of cowpox virus (CPV), is reported to inhibit the interleukin-1β-converting enzyme (ICE, caspase-1) (F. The Serp2 protein encoded by the leporipoxvirus myxoma virus is essential for full virulence (F.
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