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Journal of Virology, May 2009, p. 4140-4152, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.01835-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Shu-Jung Chang,^1,4 Jye-Chian Hsiao,¹ Stephanie Sonnberg,² Cheng-Ting Chiang,¹ Min-Hsiang Yang,³ Der-Lii Tzou,³ Andrew A. Mercer,² and Wen Chang^1*

Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China,¹ Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand,² Institute of Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China,³ Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan, Republic of China⁴

Received 1 September 2008/ Accepted 1 February 2009

Tumor necrosis factor alpha (TNF-) activates the nuclear factor B (NF-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-B activation in cells. In this report, we describe a poxvirus host range protein, CP77, which blocked NF-B activation by TNF-. Immunofluorescence analyses revealed that nuclear translocation of NF-B subunit p65 protein in TNF--treated HeLa cells was blocked by CP77. CP77 did so without blocking IB phosphorylation, suggesting that upstream kinase activation was not affected by CP77. Using GST pull-down, we showed that CP77 bound to the NF-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-B activation. We thus propose a model in which poxvirus CP77 suppresses NF-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-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-B modulation and binding to SCF complex, is not required for CP77's host range function. Consistently, knocking down the protein level of NF-B did not relieve the growth restriction of VV-hr-GFP in HeLa cells.

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* Corresponding author. Mailing address: Institute of Molecular Biology, Academia Sinica, 128, Sect. 2, Academic Road, Taipei, Taiwan, Republic of China. Phone: 886-2-2789-9230. Fax: 886-2-2782-6085. E-mail: mbwen{at}ccvax.sinica.edu.tw

Published ahead of print on 11 February 2009.

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Journal of Virology, May 2009, p. 4140-4152, Vol. 83, No. 9
0022-538X/09/$08.00+0     doi:10.1128/JVI.01835-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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