Poxvirus Host Range Protein CP77 Contains an F-Box-Like Domain That Is Necessary To Suppress NF-κB Activation by Tumor Necrosis Factor Alpha but Is Independent of Its Host Range Function

Tumor necrosis factor alpha (TNF-alpha) activates the nuclear factor kappa B (NF-kappa B) signaling pathway that regulates expression of many cellular factors playing important roles in innate immune responses and inflammation in infected hosts. Poxviruses employ many strategies to inhibit NF-kappa B activation in cells. In this report, we describe a poxvirus host range protein, CP77, which blocked NF-kappa B activation by TNF-alpha. Immunofluorescence analyses revealed that nuclear translocation of NF-kappa B subunit p65 protein in TNF-alpha-treated HeLa cells was blocked by CP77. CP77 did so without blocking I kappa B alpha phosphorylation, suggesting that upstream kinase activation was not affected by CP77. Using GST pull-down, we showed that CP77 bound to the NF-kappa B subunit p65 through the N-terminal six-ankyrin-repeat region in vitro. CP77 also bound to Cullin-1 and Skp1 of the SCF complex through a C-terminal 13-amino-acid F-box-like sequence. Both regions of CP77 are required to block NF-kappa B activation. We thus propose a model in which poxvirus CP77 suppresses NF-kappa B activation by two interactions: the C-terminal F-box of CP77 binding to the SCF complex and the N-terminal six ankyrins binding to the NF-kappa B subunit p65. In this way, CP77 attenuates innate immune response signaling in cells. Finally, we expressed CP77 or a CP77 F-box deletion protein from a vaccinia virus host range mutant (VV-hr-GFP) and showed that either protein was able to rescue the host range defect, illustrating that the F-box region, which is important for NF-kappa B modulation and binding to SCF complex, is not required for CP77' s host range function. Consistently, knocking down the protein level of NF-kappa B did not relieve the growth restriction of VV-hr-GFP in HeLa cells.