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Herpes simplex virus 1 DNA polymerase processivity factor UL42 inhibits TNF-α-induced NF-κB activation by interacting with p65/RelA and p50/NF-κB1

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Abstract

Herpes simplex virus 1 (HSV-1) is the archetypal member of the alphaherpesvirus with a large genome encoding over 80 viral proteins, many of which are involved in virus–host interactions and show immune modulatory capabilities. In this study, we demonstrated that the HSV-1 UL42 protein, a DNA polymerase processivity factor, was a novel antagonism of the canonical NF-κB signaling pathway. UL42 was shown to significantly suppress TNF-α mediated NF-κB activation. Co-immunoprecipitation experiment revealed that UL42 bound to the NF-κB subunits p65 and p50. Fluorescence microscopy demonstrated that UL42 abolished nuclear translocation of p65 and p50 upon TNF-α-stimulation. But the inhibiting capacity of UL42 2R/2A (R279A, R280A) and UL42 3R/3A (R113A, R279A and R280A) mutants were less than wild type UL42. Also UL42 bound to the Rel homology domain of the NF-κB subunit p65 and p50. Notably, the N-terminal of UL42 was sufficient to interact with p65 and p50 and abolished NF-κB reporter gene activity. Thus, it was first time we demonstrated that HSV-1 UL42 appeared to prevent NF-κB-dependent gene expression by retaining p65 and p50 in the cytoplasm, and UL42-dependent transcriptional activation were inherently coupled to promote HSV-1 lytic replication, which also may contribute to immune evasion and pathogenesis of HSV-1.

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Acknowledgments

This work was supported by grants from the Major State Basic Research Development Program of China (973 Program) (2011CB504800 and 2010CB530100); the Start-up Fund of the Hundred Talents Program of the Chinese Academy of Sciences (20071010-141); the National Natural Science Foundation of China (81171584). We thank Dr. Rao Anjana (Immune Disease Institute, Harvard Medical School), and Dr. Gangmin Hur (Chungnam National University) for providing plasmids.

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Authors and Affiliations

  1. State Key Laboratory of Virology, Molecular Virology and Viral Immunology Research Group, Wuhan Institute of Virology, Chinese Academy of Sciences, 44 Xiaohongshan Wuchang, Wuhan, 430071, China

    Jie Zhang, Shuai Wang, Kezhen Wang & Chunfu Zheng

  2. Institutes of Biology and Medical Sciences, Soochow University, Suzhou, 215123, Jiangsu, China

    Shuai Wang, Kezhen Wang & Chunfu Zheng

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  1. Jie Zhang
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Correspondence to Chunfu Zheng.

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430_2013_295_MOESM1_ESM.tif

Fig S1. UL42 inhibits TRAF2, RIP1, NIK, IKKα-induced NF-κB Activity. The HEK 293T cells were co-transfected with NF-κB-Luc, pRL-TK and TRAF2 (A), RIP1 (B), NIK (C), IKKα and (D) expression plasmids along with indicated amount of UL42 expression plasmid. Luciferase activity was analyzed as described in Fig. 1A. Cell lysates were analyzed by immunoblotting (IB) with tag-specific Abs (bottom panels) to detect the expression of Flag-TRAF2, Flag-TAK1 and HA-IKKα. The UL42 expression was detected by anti-HA mAb. IB of β-actin was used to verify equal loading of protein in each lane. The data represent means plus standard deviations for three replicates. Statistical analysis was performed using Student’s t test. **, P < 0.01. (TIFF 1338 kb)

430_2013_295_MOESM2_ESM.tif

Fig S2. UL42(1-390aa) interacts with p65 and p50. The HEK 293T cells were transfected with UL42(1-390aa)-Flag expression plasmid. At 24 h after transfection, cells harvested and lysed, the samples were then subjected to immunoprecipitation assays using anti-p65 pAb (IP: p65), anti-p50 pAb (IP: p50) or control antibody IgG. Immunoprecipitated samples (IP) were separated by 10 % SDS-PAGE, and proteins were transferred onto a PVDF membrane. IB was probed with the indicated Ab. (TIFF 497 kb)

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Zhang, J., Wang, S., Wang, K. et al. Herpes simplex virus 1 DNA polymerase processivity factor UL42 inhibits TNF-α-induced NF-κB activation by interacting with p65/RelA and p50/NF-κB1. Med Microbiol Immunol 202, 313–325 (2013). https://doi.org/10.1007/s00430-013-0295-0

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