The MC159 Protein from the Molluscum Contagiosum Poxvirus Inhibits NF-κB Activation by Interacting with the IκB Kinase Complex

Abstract

Molluscum contagiosum virus (MCV) causes persistent neoplasms in healthy and immunocompromised people. Its ability to persist likely is due to its arsenal of viral immune evasion proteins. For example, the MCV MC159 protein inhibits TNFR1-induced NF-κB activation and apoptosis. The MC159 protein is a viral FLIP and, as such, possesses two tandem death effector domains (DEDs). We show here that, in HEK293T cells, the expression of wild-type MC159 or a mutant MC159 protein containing the first DED (MC159 A) inhibited TNF-induced NF-κB, or NF-κB activated by PMA or MyD88 over-expression, whereas a mutant protein lacking the first DED (MC159 B) did not. We hypothesized that the MC159 protein targeted the IKK complex to inhibit these diverse signaling events. Indeed, the MC159 protein, but not MC159 B, co-immunoprecipitated with IKKγ. MC159 co-immunoprecipitated with IKKγ when using MEFs lacking either IKKα or IKKβ, suggesting that the MC159 protein interacted directly with IKKγ. MC159-IKKγ co-immunoprecipitations were detected during infection of cells with either MCV isolated from human lesions or with a recombinant MC159-expressing vaccinia virus. MC159 also interacts with TRAF2, a signaling molecule involved in NF-κB activation. However, mutational analysis of MC159 failed to reveal a correlation between MC159-TRAF2 interactions and MC159’s inhibitory function. We propose that MC159-IKK interactions, but not MC159-TRAF2 interactions, are responsible for inhibiting NF-κB activation.

Inhibition of interferon gene activation by death-effector domain-containing proteins from the molluscum contagiosum virus

“…For example, within the context of a single FLIP, both DED1 and DED2 must be present for antiapoptosis function of MC159 (53,54,71). In contrast, MC159 DED1 is sufficient to inhibit NF-κB activation (41). Here, either DED1 or DED2 of MC159 is sufficient to inhibit IRF3 activation.…”

“…All luciferase activities were measured as relative light units using a BioTek luminometer and the Dual Luciferase Reporter Assay System (Promega). Relative luciferase activity ratios were calculated as described previously (41). In some instances, a portion of each cellular lysate from luciferase assays was analyzed for protein expression by immunoblotting, using the antiserum indicated under each immunoblot in the figures.…”

“…For both assays, 24 h later, cells were lysed in cytoplasmic extraction buffer. The protein concentration of each clarified cellular lysate was determined using bicinchoninic acid assay (Pierce), and 20 μg of protein from each lysate was prepared for immunoblotting as previously described (41). Anti-serum used to probe immunoblots for this assay was as follows: polyclonal rabbit antiphospho-TBK1 (Ser-172; Millipore), monoclonal rabbit anti-TBK1 (Cell Signaling), and polyclonal rabbit anti-actin (Calbiochem), rabbit anti-phospho-IRF7 (Cell Signaling), monoclonal anti-IRF7 (Cell Signaling), polyclonal rabbit anti-MC159 (31), polyclonal rabbit anti-MC160 (31), and monoclonal mouse anti-FLAG (Sigma-Aldrich).…”

“…TNF-induced NF-κB activation (38)(39)(40)(41)(42). Together, these publications highlight that although they are evolutionarily related and similar in amino acid sequence, each FLIP is distinct enough to possess unique biological properties, and that these properties may have evolved out of the different requirements a virus has to survive and replicate in its host cell environment.…”

Functional Comparison of Molluscum Contagiosum Virus vFLIP MC159 with Murine Cytomegalovirus M36/vICA and M45/vIRA Proteins

“…It has been shown that overexpression of MC159 inhibits NF-B activation by several stimuli, including death receptor stimulation or MyD88 overexpression (16)(17)(18), and that MC159 acts at the level of the inhibitor of B kinase (IKK) complex by interacting with the IKK␥ subunit (also called NF-B essential modulator [NEMO]) (18). However, it has also been shown that MC159 can activate NF-B: transient expression of MC159 in human Jurkat or 293 cells led to a significantly increased NF-B activity, and the ambivalent effect appeared to depend on MC159 and RIP1 expression levels (19).…”