SHU-JUNG CHANGHsiao, Jye-ChianJye-ChianHsiaoSonnberg, StephanieStephanieSonnbergChiang, Cheng-TingCheng-TingChiangYang, Min-HsiangMin-HsiangYangTzou, Der-LiiDer-LiiTzouMercer, Andrew AAndrew AMercerChang, WenWenChang2024-01-102024-01-102009-050022538Xhttps://scholars.lib.ntu.edu.tw/handle/123456789/638339Tumor 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.en[SDGs]SDG3journal article10.1128/JVI.01835-08192117462-s2.0-66149101255https://api.elsevier.com/content/abstract/scopus_id/66149101255