Control of TANK-binding Kinase 1-mediated Signaling by the γ134.5 Protein of Herpes Simplex Virus 1

Dustin Verpooten,Yijie Ma,Songwang Hou,Zhipeng Yan,Bin He

doi:10.1074/jbc.m805905200

Dustin Verpooten, Yijie Ma + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m805905200

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Abstract

TANK-binding kinase 1 (TBK1) is a key component of Toll-like receptor-dependent and -independent signaling pathways. In response to microbial components, TBK1 activates interferon regulatory factor 3 (IRF3) and cytokine expression. Here we show that TBK1 is a novel target of the gamma(1)34.5 protein, a virulence factor whose expression is regulated in a temporal fashion. Remarkably, the gamma(1)34.5 protein is required to inhibit IRF3 phosphorylation, nuclear translocation, and the induction of antiviral genes in infected cells. When expressed in mammalian cells, the gamma(1)34.5 protein forms complexes with TBK1 and disrupts the interaction of TBK1 and IRF3, which prevents the induction of interferon and interferon-stimulated gene promoters. Down-regulation of TBK1 requires the amino-terminal domain. In addition, unlike wild type virus, a herpes simplex virus mutant lacking gamma(1)34.5 replicates efficiently in TBK1(-/-) cells but not in TBK1(+/+) cells. Addition of exogenous interferon restores the antiviral activity in both TBK1(-/-) and TBK(+/+) cells. Hence, control of TBK1-mediated cell signaling by the gamma(1)34.5 protein contributes to herpes simplex virus infection. These results reveal that TBK1 plays a pivotal role in limiting replication of a DNA virus.

Highlights

The IKK-related kinases function as essential components that phosphorylate interferon regulatory factor 3 (IRF3), as well as the closely related IRF7, which translocates to the nucleus and induces antiviral genes, such as interferon-␣/␤ and ISG56 [7, 8]
These results indicate that early expression of ␥134.5 is required to suppress phosphorylation and nuclear translocation of IRF3 in HSV infection. ␥134.5 Null Mutant Replicates More Efficiently in TBK1Ϫ/Ϫ
As TANKbinding kinase 1 (TBK1) activates the expression of ISG56 and IFN-␤, insight into ␥134.5 function, we examined whether the ␥134.5 protein directly disrupted this process

Summary

EXPERIMENTAL PROCEDURES

Cells and Viruses—Vero, HEL, and 293T cells were from the American Type Culture Collection. 293T cells were transfected with the indicated amounts of pcDNA3, FLAG-TBK1, HA-␥13.45, FLAG-dN200, and IRF3. At 40 h after transfection, cells were harvested and lysed in 50 mM Tris-HCl (pH 7.4) buffer containing 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 1 ␮g/ml aprotinin/leupeptin/pepstatin, mM Na3VO4, 1 mM NaF. With ice-cold methanol and acetone for 5 min Following this step, cells were washed with phosphate-buffered saline and stained with 4Ј,6-diamidino-2-phenylindole (1.5 ␮g/ml) in the VECTASHIELD mounting medium. Cell Fractionation Assays—Infected or transfected cells were lysed in phosphate-buffered saline containing 0.4% Nonidet P-40 and protease inhibitor mixtures (Sigma) and kept on ice with gentle inversion. Samples were subjected to electrophoresis and Western blot analysis with antibodies against IRF3 (Santa Cruz Biotechnology), GRP78 (glucose-regulated protein 78) (BD Transduction Laboratories), and histone H3 (Cell Signaling), respectively

RESULTS

Findings

DISCUSSION