Abstract
Among the repertoire of immunoregulatory proteins encoded by myxoma virus, M013 is a viral homologue of the viral pyrin domain-only protein (vPOP) family. In myeloid cells, M013 protein has been shown to inhibit both the inflammasome and NF-κB signaling pathways by direct binding to ASC1 and NF-κB1, respectively. In this study, a three-dimensional homology model of the M013 pyrin domain (PYD) was built based on similarities to known PYD structures. A distinctive feature of the deduced surface electrostatic map of the M013 PYD is the presence of a negatively region consisting of numerous aspartate and glutamate residues in close proximity. Single-site mutations of aspartate and glutamate residues reveal their role in interactions with ASC-1. The biological significance of charge complementarity in the M013-ASC-1 interaction was further confirmed by functional assays of caspase-1 activation and subsequent secretion of cytokines. M013 also has a unique 33-residue C-terminal tail that follows the N-terminal PYD, and it is enriched in positively charged residues. Deletion of the tail of M013 significantly inhibited the interactions between M013 and NF-κB1, thus compromising the ability of the viral protein to suppress the secretion of pro-inflammatory cytokines. These results demonstrate that vPOP M013 exploits distinct structural motifs to regulate both the inflammasome and NF-κB pathways.
Highlights
Among the repertoire of immunoregulatory proteins encoded by myxoma virus, M013 is a viral homologue of the viral pyrin domain-only protein family
We found that mutation in selected charged residues of M013 pyrin domain diminished the interaction between M013 and ASC-1
At 6 h.p.i., the level of activated caspase-1 was significantly reduced for the cells transfected with WT M013 (WT-M013) compared with C1, LPS treatment as a positive control, or cells transfected with plasmids expressing mutants M013PYD, D29N, D57N, and D41A (Fig. 4A)
Summary
The amino acid sequence of M013_PYD was aligned to the known host cPOPs and selected other cellular pyrin domain– containing proteins using Clustal Omega [24]. At 6 h.p.i., the level of activated caspase-1 was significantly reduced for the cells transfected with WT-M013 compared with C1 (i.e. cells infected with vMyx-M013KO virus alone), LPS treatment as a positive control, or cells transfected with plasmids expressing mutants M013PYD, D29N, D57N, and D41A (Fig. 4A). This suggests that despite the low expression (Fig. 2A), M013 mutants were able to induce the secretion of signature cytokines of both the NLRP3 inflammasome and NF-B1 pathways at 6 h.p.i. A robust increase was observed for most of the cytokines in transfected THP-1 cell supernatants at, 24 h.p.i. In contrast to D41A and C2, levels of IL-6 were substantially higher for control C1, mutants M013PYD, E28Q, D29N, and D57N (Fig. 4G). These results suggest that weakened binding of M013 mutants to ASC-1 correlates with the increased activation of caspase-1 and the production of specific indicator cytokines
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