Co-Regulation of NF-κB and Inflammasome-Mediated Inflammatory Responses by Myxoma Virus Pyrin Domain-Containing Protein M013

Abstract

NF-κB and inflammasomes both play central roles in orchestrating anti-pathogen responses by rapidly inducing a variety of early-response cytokines and chemokines following infection. Myxoma virus (MYXV), a pathogenic poxvirus of rabbits, encodes a member of the cellular pyrin domain (PYD) superfamily, called M013. The viral M013 protein was previously shown to bind host ASC-1 protein and inhibit the cellular inflammasome complex that regulates the activation and secretion of caspase 1-regulated cytokines such as IL-1β and IL-18. Here, we report that human THP-1 monocytic cells infected with a MYXV construct deleted for the M013L gene (vMyxM013-KO), in stark contrast to the parental MYXV, rapidly induce high levels of secreted pro-inflammatory cytokines like TNF, IL-6, and MCP-1, all of which are regulated by NF-κB. The induction of these NF-κB regulated cytokines following infection with vMyxM013-KO was also confirmed in vivo using THP-1 derived xenografts in NOD-SCID mice. vMyxM013-KO virus infection specifically induced the rapid phosphorylation of IKK and degradation of IκBα, which was followed by nuclear translocation of NF-κB/p65. Even in the absence of virus infection, transiently expressed M013 protein alone inhibited cellular NF-κB-mediated reporter gene expression and nuclear translocation of NF-κB/p65. Using protein/protein interaction analysis, we show that M013 protein also binds directly with cellular NF-κB1, suggesting a direct physical and functional linkage between NF-κB1 and ASC-1. We further demonstrate that inhibition of the inflammasome with a caspase-1 inhibitor did not prevent the induction of NF-κB regulated cytokines following infection with vMyxM013-KO virus, but did block the activation of IL-1β. Thus, the poxviral M013 inhibitor exerts a dual immuno-subversive role in the simultaneous co-regulation of both the cellular inflammasome complex and NF-κB-mediated pro-inflammatory responses.

Orf Virus ORFV121 Encodes a Novel Inhibitor of NF-κB That Contributes to Virus Virulence

“…Some poxviral NF-B inhibitors have been shown to affect virus virulence and disease pathogenesis to some extent; however, the degree of virus attenuation resulting from single-gene deletions has been variable and, in most cases, very modest (1,8,10,11,21,48). With a few exceptions (CPXV CPXV006 and MYXV M013 and M150R), no singlegene deletion rendered marked virus attenuation in vivo, suggesting that at the host level multiple NF-B inhibitors encoded by individual poxviruses exert complementary functions to effectively suppress the NF-B signaling pathway during virus infection (4,24,30,42).…”

“…For example, CPXV006, a virulence determinant for CPXV in mice, binds to and prevents the degradation of the NF-B subunit NF-B-p105 (30). Similarly, MYXV M013, a significant virulence determinant for MYXV in rabbits, has been shown to interact with NF-B-p105 (24,42). MYXV M150R, an additional virulence determinant for MYXV in rabbits, was shown to colocalize with NF-B-p65 in the cell nucleus, although its actual function is unknown (4).…”

“…Pathogenesis studies using mutant viruses, with the deletion of selected NF-B inhibitors, have shown a wide range of phenotypes in vivo, with individual gene deletions resulting in phenotypes from no effect (1,8,21,48) to marked virus attenuation (4,24,30,42).…”

Innate immune activation of NFκB and its antagonism by poxviruses

“…CP77 might also target p65 for proteasomal degradation through these binding activities [30]. Other poxviral proteins demonstrated or suspected to directly interact with NFkB include: MYXV protein M150 which co-localises with NFkB and supresses inflammation [31]; VARV protein G1 which contains ankyrin repeats, is conserved in a number of orthopoxviruses (CPV, MYXV and ECTV) and has been shown to associate with p105 and block NFkB nuclear translocation [32]; MYXV protein M013 which also binds p105 to prevent its processing and subsequent NFkB activation [33].…”

“…The earliest reported inhibitor was CPV protein CrmA (and orthologs such as B13R in VACV) which inhibits IL1b processing by targeting Caspase 1 [93]. The MYXV protein M013, which was discussed earlier as a direct inhibitor of NFkB, is like many poxviral inhibitors multi-functional in that it also blocks caspase-1 activation and proIL1b processing by targeting ASC [33]. Another inflammasome, NLRP1, is also targeted by poxviruses: the VACV Bcl-2 family member, F1L was shown to bind NLRP1 thereby limiting pro-IL-1b processing though the precise mechanism by which poxvirus infection activates this type of inflammasome is not yet clear [94].…”