Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 9, 1446
eCollection

Latent Membrane Protein 1 of Epstein-Barr Virus Promotes RIG-I Degradation Mediated by Proteasome Pathway

Affiliations

Latent Membrane Protein 1 of Epstein-Barr Virus Promotes RIG-I Degradation Mediated by Proteasome Pathway

Chongfeng Xu et al. Front Immunol.

Abstract

RIG-I signaling is critical to host innate immune response against RNA virus infection, and also can be activated against many kinds of cancer. Oncogene LMP1 of Epstein-Barr virus (EBV) contributes to various tumors progress. In this study, we have provided strong evidence that LMP1 inhibits Sendai virus mediated type I interferon production and downregulates RIG-I signaling pathway by promotion RIG-I degradation dependent on proteasome. Nineteen kinds of E3 ligase are identified by IP-MS as LMP1-interactors, they are candidate E3s, which are possibly recruited by LMP1 to mediate RIG-I degradation. CHIP is among these E3s, which has been reported to lead RIG-I degradation. Notably, we find C666-1, an EBV-positive nasopharyngeal carcinoma cell line, expresses low level of RIG-I, even treated with IFN-α, RIG-I expression could not be induced. This evidence indicates that EBV employs a unique strategy to evade RIG-I mediated immune responses.

Keywords: Epstein–Barr virus cancer; LMP1; RIG-I; immunotherapy of cancer; nasopharygeal carcinoma.

Figures

Figure 1
Figure 1
LMP1-inhibits SeV and RIG-I-mediated transactivation of IFN-β. (A) 293T cells were transfected with 0.1 µg IFN-β, ISRE, or NF-κB luciferase reporter together with 0.1 µg pCMV-β-gal (as internal control), and 0.5 µg pCMV-Myc-LMP1 or empty vector. 24 h after transfection, 293T cells were challenged with SeV for 12 h. Luciferase activity was analyzed at 12 h post-infection and relative luciferase activity (RLU) (relative to the basal level of reporter gene in the presensence of pCMV-Myc vector after nomalization with co-transfected β-gal activity) was determined compared to uninfection control. The data are shown as the mean ± SD and are representative of one of three independent experiments (n = 3). Results are representative of at least three experiments run in triplicates. (B) CNE2 cells were transfected with pCMV-LMP1, after 24 h transfection, infected by SeV for 4 and 8 h, phosphorylated IRF3 of CNE2 cells was detected by Western blot. (C) 293T cells were transfected with 0.5 µg pCMV-LMP1 (vector as control), 0.25 µg signal molecules 0.1 µg IFN-β and 0.1 µg β-gal. Luciferase activity was analyzed after 24 h post transfection. (D) 293T cells were transfected with 0.25 µg signal molecules, pEGFP-IRF3, and pCMV-LMP1 (vector as control). 24 h post transfection, phosphorylated IRF3 was detected by Western blot. (E) 293T cells were transfected with 0.25 µg signaling molecules and pCMV-LMP1 (vector as control). 24 h post transfection, endogenously induction of ISG15 was detected by Western blot. (F) 293T cells were transfected with 1 µg pCMV-LMP1 (vector as control) per well in 6-well plates, stimulated with SeV (MOI = 1) for 6 and 12 h, supernatants were collected to test IFNs antivirus titers. IFNs activity was determined by IFN assays: calculating the protection of the WISH cells against the cytopathic effects caused by VSV.
Figure 2
Figure 2
LMP1-promotes degradation of RIG-I and MG132 inhibited RIG-I degradation. (A) Immunoblot analysis of endogenously induced RIG-I and phosphorylated IRF3 in CNE2 cells. CNE2 were transfected with pCMV-LMP1 (empty vector as control) for 24 h, then infected with Sendai virus for 4 h, after infection, CNE2 was treated with CHX (100 mg/ml) for 3 h, RIG-I and pIRF3 of cell lysates were analyzed with Western blot. (B) Immunoblot analysis of FLAG tagged RIG-I in 293T cell lysates, which were transfected with pCMV-LMP1 (empty vector as control) and pcDNA-FLAG-RIG-I for 16 h, then treated with MG132 or NH4CL for 3 h. (C) FLAG tag was probed for immunoblot analysis of RIG-I and its N terminal and C terminal truncates in 293T cell lysates, which were co-transfected with FLAG-RIG-I/RIG-IN/RIG-IC and pCMV-LMP1 (vector as control) for 16 h, then treated with MG132 for 3 h. The Myc-LMP1 line was from two images, which were pictured from the same polyvinylidene difluoride film with different exposure time, the box area exposure time was longer than the right part.
Figure 3
Figure 3
LMP1 interacts with E3 ligases CHIP and TRAFD1. The 293T cells were transfected with plasmids E3 ligases FLAG-Myc-CHIP (MW: 28 kD) or FLAG-Myc-TRAFD1(MW: 65 kD) with Myc-LMP1 (MW: 55kD). After transfection of 24 h, Co-IP was performed with cell lysates with antibodies to FLAG tags. Immunoblot analysis of Myc-LMP1-immunoprecipitated by FLAG-tagged E3 with antibodies to c-Myc. Upper panel shows the IP results performed with anti-FLAG monoclonal antibody, and immunoblot analysis with anti Myc. The lower panel shows the input, immunoblot with anti-FLAG and anti-Myc monoclonal antibodies. * represents immunoprecipitated Myc-LMP1.
Figure 4
Figure 4
Expression of RIG-I is low in C666-1 cells and could not be induced by IFN-α. Nasopharyngeal epithelial cell line NP69 and nasopharyngeal carcinoma cell lines were cultured in the absence or presence of 100 U/ml of IFN-α for 12 h. Expression of RIG-I was determined by Western blot analysis.

Similar articles

See all similar articles

Cited by 2 articles

References

    1. Young LS, Rickinson AB. Epstein-Barr virus: 40 years on. Nat Rev Cancer (2004) 4(10):757–68.10.1038/nrc1452 - DOI - PubMed
    1. Sung WW, Chu YC, Chen PR, Liao MH, Lee JW. Positive regulation of HIF-1A expression by EBV oncoprotein LMP1 in nasopharyngeal carcinoma cells. Cancer Lett (2016) 382(1):21–31.10.1016/j.canlet.2016.08.021 - DOI - PubMed
    1. Lawson JS, Salmons B, Glenn WK. Oncogenic viruses and breast cancer: Mouse Mammary Tumor Virus (MMTV), Bovine Leukemia Virus (BLV), Human Papilloma Virus (HPV), and Epstein-Barr Virus (EBV). Front Oncol (2018) 8:1.10.3389/fonc.2018.00001 - DOI - PMC - PubMed
    1. Zhang J, Huang T, Zhou Y, Cheng ASL, Yu J, To KF, et al. The oncogenic role of Epstein-Barr virus-encoded microRNAs in Epstein-Barr virus-associated gastric carcinoma. J Cell Mol Med (2018) 22(1):38–45.10.1111/jcmm.13354 - DOI - PMC - PubMed
    1. Xu XX, Wan H, Nie L, Shao T, Xiang LX, Shao JZ. RIG-I: a multifunctional protein beyond a pattern recognition receptor. Protein Cell (2018) 9(3):246–53.10.1007/s13238-017-0431-5 - DOI - PMC - PubMed
Feedback