Poxvirus host range protein CP77 contains an F-box-like domain that is necessary to suppress NF-kappaB activation by tumor necrosis factor alpha but is independent of its host range function.
Tumor necrosis factor alpha (TNF-alpha) activates the nuclear factor kappaB (NF-kappaB) 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-kappaB activation in cells. In this report, we describe a poxvirus host range protein, CP77, which blocked NF-kappaB activation by TNF-alpha. Immunofluorescence analyses revealed that nuclear translocation of NF-kappaB subunit p65 protein in TNF-alpha-treated HeLa cells was blocked by CP77. CP77 did so without blocking IkappaBalpha phosphorylation, suggesting that upstream kinase activation was not affected by CP77. Using GST pull-down, we showed that CP77 bound to the NF-kappaB 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-kappaB activation. We thus propose a model in which poxvirus CP77 suppresses NF-kappaB 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-kappaB 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-kappaB modulation and binding to SCF complex, is not required for CP77's host range function. Consistently, knocking down the protein level of NF-kappaB did not relieve the growth restriction of VV-hr-GFP in HeLa cells.
Chang
Shu-Jung
SJ
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
Hsiao
Jye-Chian
JC
Sonnberg
Stephanie
S
Chiang
Cheng-Ting
CT
Yang
Min-Hsiang
MH
Tzou
Der-Lii
DL
Mercer
Andrew A
AA
Chang
Wen
W
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D016207:Cytokines / Q000378:metabolism; D044783:F-Box Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D010766:Phosphorylation; D011212:Poxviridae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D016207:Cytokines; D044783:F-Box Proteins; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D014764:Viral Proteins; D044767:Ubiquitin-Protein Ligases; D051550:I-kappa B Kinase