Poxviruses encode proteins that suppress host immune responses, including secreted decoy receptors for pro-inflammatory cytokines such as interleukin-1 (IL-1) and the vaccinia virus proteins A46R and A52R that inhibit intracellular signaling by members of the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) family. In vivo, the TLRs mediate the innate immune response by serving as pathogen recognition receptors, whose oligomerized intracellular Toll/IL-1 receptor (TIR) domains can initiate innate immune signaling. A family of TIR domaincontaining adapter molecules transduces signals from engaged receptors that ultimately activate NF-B and/or interferon regulatory factor 3 (IRF3) to induce proinflammatory cytokines. Data base searches detected a significant similarity between the N1L protein of vaccinia virus and A52R, a poxvirus inhibitor of TIR signaling. Compared with other poxvirus virulence factors, the poxvirus N1L protein strongly affects virulence in vivo; however, the precise target of N1L was previously unknown. Here we show that N1L suppresses NF-B activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-␣ and IKK--dependent signaling to NF-B. N1L associated with several components of the multisubunit I-B kinase complex, most strongly associating with the kinase, TANK-binding kinase 1 (TBK1). Together these findings are consistent with the hypothesis that N1L disrupts signaling to NF-B by Toll/IL-1Rs and TNF superfamily receptors by targeting the IKK complex for inhibition. Furthermore, N1L inhibited IRF3 signaling, which is also regulated by TBK1. These studies define a role for N1L as an immunomodulator of innate immunity by targeting components of NF-B and IRF3 signaling pathways.
The outbreak of monkeypox in the Unites States in the summer of 2003 was the first occurrence of this smallpox-like disease outside of Africa. This limited human epidemic resulted from cross-infection of prairie dogs by imported African rodents. Although there were no human fatalities, this outbreak illustrates that monkeypox is an emerging natural infection and a potential biological weapon. We characterized a virulence factor expressed by monkeypox (monkeypox inhibitor of complement enzymes or MOPICE). We also compared its structure and regulatory function to homologous complement regulatory proteins of variola (SPICE) and vaccinia (VCP). In multiple expression systems, 5–30% of MOPICE, SPICE, and VCP consisted of function-enhancing disulfide-linked homodimers. Mammalian cells infected with vaccinia virus also expressed VCP dimers. MOPICE bound human C3b/C4b intermediate to that of SPICE and VCP. Cofactor activity of MOPICE was similar to VCP, but both were ∼100-fold less efficient than SPICE. SPICE and VCP, but not MOPICE, possessed decay-accelerating activity for the C3 and C5 convertases of the classical pathway. Additionally, all three regulators possessed heparin-binding capability. These studies demonstrate that MOPICE regulates human complement and suggest that dimerization is a prominent feature of these virulence factors. Thus, our data add novel information relative to the functional repertoire of these poxviral virulence factors. Furthermore, targeting and neutralizing these complement regulatory active sites via mAbs is a therapeutic approach that may enhance protection against smallpox.
The complement system contributes to host defenses against invasion by infectious agents. A 35-kilodalton protein, encoded by vaccinia virus and secreted from infected cells, has sequence similarities to members of a gene family that includes complement control proteins. Biochemical and genetic studies showed that the viral protein binds to derivatives of the fourth component of complement and inhibits the classical complement cascade, suggesting that it serves as a defense molecule to help the virus evade the consequences of complement activation.
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