The anti-mouse Alexa Fluor 555(A21422, 1:750) and anti-rabbit Alexa Fluro 488 (A110081, 1:500) were purchased from Invitrogene
The anti-mouse Alexa Fluor 555(A21422, 1:750) and anti-rabbit Alexa Fluro 488 (A110081, 1:500) were purchased from Invitrogene. Plasmid Plasmids expressing MLL5 (tagged with 3Flag or HA) in mammalian cells were constructed by in-frame 2-MPPA insertion of a human MLL5 cDNA into pcDEF3 expression vector. of MLL5 that is specifically expressed in HPV16/18-positive cervical cancer cells is essential for transcriptional activation of the E6/E7 oncogene20. Another isoform of MLL5 protein, NKp44L, is a ligand for natural killer (NK) cell NKp44 receptor that mediates natural cytotoxicity toward tumor cells21. Nevertheless, whether MLL5 plays a role in HRAS innate antiviral immunity is largely unknown. In the present study, we show that MLL5 acts as a negative regulator in host antiviral immune responses. A fraction of MLL5 that was located in the cytoplasm and mediated interaction between RIG-I and its E3 ubiquitin ligase STUB1, leads to K48-linked polyubiquitination and proteasomal degradation of RIG-I. Ablation of MLL5 attenuated interaction between RIG-I and STUB1, and reduced K48-linked polyubiquitination and accumulation of RIG-I protein in cells. MLL5 deficiency potentiates 2-MPPA the production of type I IFN, proinflammatory cytokines and innate antiviral immune responses to RNA virus both in vitro and in vivo. Moreover, upon viral infection, MLL5 protein translocates from the nucleus to the cytoplasm to induce STUB1-mediated RIG-I degradation. Here we show an unexpected role for MLL5 in host antiviral immune responses, highlighting a mechanism of epigenetic modifiers in controlling viral infection. Results MLL5 suppresses RLR-mediated innate immune responses To explore the function of MLL5 in the antiviral immune response, we generated deficient (mice, and challenged them with diverse pathogen-associated molecular pattern (PAMP) ligands. The mRNA expression of type I IFN and proinflammatory cytokines were detected using quantitative reverse transcription PCR (qRT-PCR). We found that BMDMs expressed upregulated mRNA compared with those from wild-type BMDMs after synthetic RNA duplex poly(I:C) (polyinosinic:polycytidylic acid) or 5-pppRNA transfection, but not stimulation with other PAMP ligands, such as lipopolysaccharide (LPS) (TLR4 ligand), CpG-B (TLR9 ligand), R848 (TLR7/8 ligand), Pam3 (TLR1/2 ligand), poly(I:C)(TLR3 ligand), or intracellular IFN stimulatory DNA (ISD) (Fig.?1a). To test this further, we prepared primary peritoneal macrophages (PMs) or mouse embryonic fibroblasts (MEFs) from wild-type or mice, and transfected them with poly(I:C) or 5-pppRNA. In line with that, the levels of and or mRNA and the production of IFN- and TNF- or IL-6 cytokines were significantly higher in PMs (Fig.?1b, c) than in wild-type 2-MPPA cells when transfected with 2-MPPA poly(I:C) or 5-pppRNA, but not intracellular ISD. Open in a separate window Fig. 1 MLL5 selectively suppresses RLR-mediated antiviral immune response. a Expression of mRNA in BMDMs from wild-type (WT) or mice stimulated with poly(I:C) (100?g/ml), CpG-B (1?g/ml), R848 (1?g/ml), Pam3 (1?g/ml) and LPS (0.2?g/ml) for 4?h, or stimulated with intracellular poly(I:C) (1?g/ml), intracellular 5ppp-RNA (0.4?g/ml) and intracellular ISD (1?g/ml) for 6?h. served as control. b Expression of and mRNA in PMs from WT or mice stimulated with intracellular poly(I:C) (1?g/ml), intracellular 5ppp-RNA (0.4?g/ml) and intracellular ISD (1?g/ml) for 6?h, or infected with VSV-GFP (MOI:1), SeV (10 HA/ml) and HSV-1 (MOI:1) for 6?h. served as control. c ELISA quantification of IFN-, TNF- and IL6 secretion in PMs treated as in b. Data were from three independent experiments and were analyzed by Students PMs with vesicular stomatitis virus (VSV) or Sendai virus (SeV), then measured mRNA expression and cytokine production of IFN- and TNF- or IL-6. The DNA virus herpes simplex virus type 1 (HSV-1) was used as a negative control. We found that PMs had higher gene expression and protein secretion of IFN-, TNF-, and IL-6 than their wild-type counterparts had in response to infection with VSV or SeV, but not HSV-1 (Fig.?1b, c). Similar results were observed in MEF cells treated with poly(I:C) transfection (Supplementary Fig.?2a, b) or VSV infection (Supplementary Fig.?2c, d). We next generated HEK293T human embryonic kidney cells using a CRISPR-Cas9-based approach, and detected the role of MLL5 in antiviral immune responses in human cells (Supplementary Fig.?3a). Similarly, HEK293T cells increased intracellular poly(I:C)-induced expression of IFN- and TNF- (Supplementary Fig.?3b), indicating that the function of MLL5 in the antiviral immune response is conserved in mice and humans. Therefore, these results demonstrated that MLL5 selectively suppresses RLR-mediated production of type I IFN and proinflammatory cytokines. MLL5 suppresses RLR-mediated antiviral signaling To investigate further the effect of MLL5 in RLR-mediated immune signaling, we detected activation of transcription factors IRF3.