Cowpox virus CrmA, Myxoma virus SERP2 and baculovirus P35 are not functionally interchangeable caspase inhibitors in poxvirus infections

Abstract

Cowpox virus (CPV) expresses the serpin (serine proteinase inhibitor) CrmA, an anti-inflammatory, anti-apoptotic protein required for production of red pocks on chicken chorioallantoic membranes (CAMs). In vitro, CrmA inhibits several caspases and granzyme B. Altering the critical P1-aspartate in the CrmA reactive centre loop to alanine resulted in a virus (CPV-CrmA-D303A) that resembled CPV deleted for CrmA (CPVDCrmA : : lacZ); on CAMs it produced white, inflammatory pocks with activated caspase-3 and reduced virus yields, suggesting that CrmA activities are mediated via proteinase inhibition. CrmA in CPV was replaced with SERP2 from Myxoma virus (MYX) or baculovirus P35, which inhibit similar proteinases in vitro. SERP2 and P35 each blocked caspase-3-mediated apoptosis but were unable to control inflammation of CAMs. However, SERP2 and P35 restored virus yields, indicating that the decreased virus titres seen with CPVDCrmA : : lacZ resulted from apoptosis rather than inflammation. To compare the activities of CrmA and SERP2 further, rabbits were infected with MYX recombinant viruses. Intradermal infection of rabbits with MYX was uniformly lethal, generating raised primary lesions and many secondary lesions. In contrast, deletion of SERP2 from MYX (MYXDSERP2 : : lacZ) caused little mortality and produced flat primary lesions with few secondary lesions. Replacement of SERP2 with CrmA (MYXDSERP2 : : CrmA) resulted in partial complementation with flat primary lesions, many secondary lesions and death in 70 % of the rabbits. Therefore, CrmA and SERP2 were not functionally interchangeable during infection of CAMs or rabbits, implying that these serpins have activities that are not evident from biochemical studies with human caspases.

Poxvirus Proteomics and Virus-Host Protein Interactions

“…MYXV also encodes three serpins, designated SERP-1, SERP-2, and SERP-3. Although studies have shown the MYXV serpins exhibit functional similarly to those of orthopoxviruses, SERP-2 and CrmA are not interchangeable during viral infection and pathogenesis in vivo (116). These studies demonstrate the complexity and specificity of the interactions between viral pathogens and their host.…”

Viral Subversion of Apoptotic Enzymes: Escape from Death Row

“…For example, replacement of CrmA by SERP-2 enabled the resulting recombinant cowpox virus to inhibit apoptosis and reach wild-type virus yields in infected CAMs. However, SERP-2 did not block inflammation in this context (86), which is the original hallmark of CrmA function (89). Similarly, the reciprocal recombinant myxoma virus containing CrmA in place of SERP-2 was not fully virulent in its European rabbit host and did not cause the fulminant skin lesions characteristic of infection with wild-type virus (86).…”

“…However, SERP-2 did not block inflammation in this context (86), which is the original hallmark of CrmA function (89). Similarly, the reciprocal recombinant myxoma virus containing CrmA in place of SERP-2 was not fully virulent in its European rabbit host and did not cause the fulminant skin lesions characteristic of infection with wild-type virus (86). Both recombinant viruses could inhibit apoptosis of infected cells, suggesting that poxvirus serpins may have host-specific roles in control of inflammation.…”

Control of granzymes by serpins

“…100 Despite these similarities, Serp2 and crmA are not fully interchangeable in vivo. [99][100][101] This suggests either additional host-specific protease targets or virus-specific functions unrelated to protease inhibition that are beyond the scope of this review. The nematode Caenhorabditis elegans has also been shown to encode a potent gzmB inhibitor, Srp-2.…”