Poxviruses express proteins that limit host immune responses to infection. For example, the molluscum contagiosum virus MC160 protein inhibits tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activation. This event correlates with MC160-induced IKK1 protein degradation, suggesting a mechanism for the above-mentioned phenotype. IKK1 is stabilized when it associates with the cellular heat shock protein 90 (Hsp90). Here, Hsp90 overexpression restored IKK1 levels in MC160-expressing cells, suggesting that MC160 competitively interacted with Hsp90. In support of this, further investigation showed that a mutant MC160 protein comprising only the C-terminal region (C protein) immunoprecipitated with Hsp90. In contrast, Hsp90 IP with a mutant MC160 protein consisting of only the N-terminal tandem death effector domains (DEDs) (N protein) was dramatically decreased. Since cells expressing either the N or C mutant MC160 protein remained similarly resistant to TNF-alpha-induced NF-kappaB activation, the N mutant protein probably utilized a different mechanism for inhibiting NF-kappaB. One likely mechanism for the N protein lies in its association with the DED-containing procaspase-8 protein, a cellular apoptosis precursor protein that regulates NF-kappaB activation. Here, IPs revealed that this association relied on the presence of the DED-containing N terminus of the MC160 protein but not the C-terminal portion. These interactions appear to have relevance with NF-kappaB activation, since the expression of the viral DEDs strongly inhibited procaspase-8-mediated NF-kappaB activation, an event not substantially altered by the C protein. Thus, the MC160 protein utilizes at least two distinct mechanisms for impeding NF-kappaB activation, association with Hsp90 to result in IKK1 protein degradation or interaction with procaspase-8.