Identification of new type I interferon-stimulated genes and investigation of their involvement in IFN-β activation
Virus infection induces the production of type I interferons (IFNs). IFNs bind to their heterodimeric receptors to initiate downstream cascade of signaling, leading to the up-regulation of interferon-stimulated genes (ISGs). ISGs play very important roles in innate immunity through a variety of mechanisms. Although hundreds of ISGs have been identified, it is commonly recognized that more ISGs await to be discovered. The aim of this study was to identify new ISGs and to probe their roles in regulating virus-induced type I IFN production. We used consensus interferon (Con-IFN), an artificial alpha IFN that was shown to be more potent than naturally existing type I IFN, to treat three human immune cell lines, CEM, U937 and Daudi cells. Microarray analysis was employed to identify those genes whose expressions were up-regulated. Six hundred and seventeen genes were up-regulated more than 3-fold. Out of these 617 genes, 138 were not previously reported as ISGs and thus were further pursued. Validation of these 138 genes using quantitative reverse transcription PCR (qRT-PCR) confirmed 91 genes. We screened 89 genes for those involved in Sendai virus (SeV)-induced IFN-β promoter activation, and PIM1 was identified as one whose expression inhibited SeV-mediated IFN-β activation. We provide evidence indicating that PIM1 specifically inhibits RIG-I- and MDA5-mediated IFN-β signaling. Our results expand the ISG library and identify PIM1 as an ISG that participates in the regulation of virus-induced type I interferon production.
Keywordsinterferon-stimulated genes IFN-β signaling PIM1 RIG-I MDA5
Viral infection activates host innate immune response (Schneider et al., 2014). The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), RIG-I and melanoma differentiation-associated gene 5 (MDA5), are important to initiate innate immune in response to RNA virus invasion (Wilkins and Gale, 2010). Following recognition of viral RNAs, RLRs are recruited to an adaptor protein VISA (also known as MAVS, IPS-1 and Cardif), which further triggers TBK1/IKKε and IKKα/β kinases-mediated activation of IRF3 and NF-κB (Xu et al., 2005; Seth et al., 2005; Meylan et al., 2005; Kawai et al., 2005). These events ultimately lead to the induction of the expressions of type I IFNs and pro-inflammatory cytokines.
Interferons (IFNs) are a group of pleiotropic cytokines that are made and released by host cells in response to pathogen infections and tumorgenesis (Pestka, 2007). Based on their receptors, IFNs are divided into three classes (Uze et al., 2007; de Weerd et al., 2007). Type I IFNs comprise IFN-α, IFN-β, IFN-ε, IFN-κ and IFN-ω, which bind to the type I IFN heterodimeric receptor complex of IFN-α receptor 1 (IFNAR1) and receptor 2 (IFNAR2) (Chen et al., 2004). Type II IFN, IFN-γ, signals through the IFN-γ receptor complex (IFNGR) (Pestka et al., 1997) and type III IFNs signal through IFN-λ receptor 1 or IL-10R2 (Kotenko et al., 2003).
Type I IFN binding to IFNAR activates a signaling cascade through the Janus kinase and signal transducer and activator of transcription (JAK-STAT) pathway (Aaronson and Horvath, 2002). This leads to the assembly of the IFN-stimulated gene factor 3 (ISGF3) complex, which is composed of STAT1-STAT2 dimers and IFN-regulatory factor 9 (IRF9) (Fu et al., 1992). The complex translocates to the nucleus and binds to the IFN-stimulated response elements (ISREs) present in the promoters of IFN-stimulated genes (ISGs), thereby initiating the transcription of those genes.
Systematic identification of ISGs revealed that there may be more than a thousand ISGs (Martensen and Justesen, 2004; Schoggins and Rice, 2011). In these studies, the naturally existing IFN-α was commonly used as the stimulator. However, there are 13 IFN-α subtypes in addition to the other type I IFNs. Although these type I IFNs use the same receptor, their downstream effects are different to some extents. Based on the sequence alignment, Alton et al. designed a consensus alpha IFN (Con-IFN) (Alton, 1983). Compared with the naturally existing recombinant type I IFN (IFN-α2a and IFN-α2b), Con-IFN displayed remarkable enhanced natural killer cell activation, antiviral, antiproliferative, and gene-induction activities (Klein et al., 1988; Blatt et al., 1996). Here we used Con-IFN to stimulate three immune cell lines and identified dozens of new ISGs.
Although innate immune responses provide one of the first lines of defense against viral infection (Schneider et al., 2014), uncontrolled immune activation could be harmful to the host (Yap and Lai, 2010); the IFN signaling needs to be tightly controlled. A number of ISGs have been reported to play roles in the maintenance of the homeostasis. For example, several components in the RLR pathway are IFN responsive, such as RIG-I, IRF3 and IRF7 (Schneider et al., 2014; Schoggins et al., 2011). The induction of these proteins in turn reinforces IFN production. The expression of some TRIM proteins, such as TRIM13 and TRIM25, is up-regulated by type I IFNs (Rajsbaum et al., 2008). These proteins also play important roles in the regulation of immune responses (Narayan et al., 2014; Gack et al., 2007; Versteeg et al., 2013).
We screened the newly identified ISGs for those participating in the modulation of virus-induced innate immune activation. PIM1 was found to negatively regulate Sendai virus (SeV)-triggered IFN-β promoter activation.
Identification of ISG candidates by microarray analysis
Identification of new ISGs by qRT-PCR
Screen for the ISGs that modulate SeV-triggered IFN-β activation
PIM1 negatively regulates SeV-triggered activation of IFN-β promoter
PIM1 inhibits RIG-I- and MDA5-mediated activation of IFN-β promoter
Given their important roles in the innate immune defenses, ISGs have been extensively studied. The effects of type I IFNs on the transcriptome of several cell types have been investigated in previous studies (Liu et al., 2012; Der et al., 1998; de Veer et al., 2001; Hilkens et al., 2003; Calcaterra et al., 2006; Indraccolo et al., 2007), and the effects of IFNs on B cells were only partially studied (Salamon et al., 2012; Pfeffer et al., 1991). Unlike previous studies, here we used Con-IFN, a bio-optimized highly potent type I interferon alpha (Blatt et al., 1996), to treat three human immune cell types, T lymphoblast-derived CEM, B lymphoblast-derived Daudi and monocyte-derived U937 cells. The three cell types displayed different gene expression profiles in response to Con-IFN (Fig. 1B). In addition, even for a particular cell type, some genes were induced upon Con-IFN treatment for 4 h but not for 12 h and vice versa (Fig. 1). Our strategy used here helped to increase the coverage of ISGs.
The IFN signaling mediated by RLRs functions as an effective mechanism against RNA virus infection. A few ISGs have been reported to modulate this pathway. We thus tested whether the ISGs identified here were involved in the regulation of the SeV-triggered IFN-β activation. Among the 89 genes tested, 15 affected the pathway (Fig. 4). Only two genes, MS4A12 and MS4A13, enhanced the SeV-triggered IFN-β activation, while the other 13 genes negatively regulated the activation (Fig. 4). These results further suggest that the immune response to viral infection is regulated by multiple mechanisms. Whether these ISGs directly regulate the IFN-β activation or indirectly by interfering with the viral replication needs to be further investigated.
Among the above 13 ISGs that negatively regulated the SeV-triggered IFN activation, the serine/threonine kinase PIM1 displayed strong inhibitory activity (Fig. 4). We focused on this protein for further investigation for two reasons. First, PIM1 is a phosphokinase and phosphorylation of components in the RLR pathways is a common mechanism to modulate the pathway (Bachmann and Moroy, 2005; Quicke et al., 2017; Ivashkiv and Donlin, 2014). Second, it has been reported that inhibition of PIM1 with an inhibitor suppressed viral infection, postulating the possibility that PIM1 is involved in innate immune response (de Vries et al., 2015). Our results showed that PIM1 inhibited both MDA5- and RIG-I-mediated IFN-β promoter activation (Fig. 6). De-phosphorylation and the following poly-ubiquitination of RIG-I and MDA5 are required for their activation. Upon activation, the RLRs translocate to mitochondria and mitochondria-associated membranes where they interact with VISA, then trigger the downstream signaling (Quicke et al., 2017; Wies et al., 2013). PIM1 may interfere with the conformational changes of RIG-I and MDA5 via phosphorylation and disturb their interactions with the downstream adapter VISA. Protein kinase C-α (PKC-α), PKC-β and casein kinase II (CK2) are responsible for phosphorylation of RIG-I (Maharaj et al., 2012; Sun et al., 2011). However, proteins involved in the phosphorylation of MDA5 were not reported. It would be intriguing to investigate whether PIM1 is the undiscovered kinase to phosphorylate MDA5 in future studies.
In summary, our results here expand the ISG library and provide additional evidence that ISGs can negatively regulate the virus-induced type I IFN production. The antiviral activities of the new ISGs and their biological functions in vivo await further investigation.
Materials and methods
CEM, U937 and Daudi cells were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated (56°C, 30 min) fetal bovine serum (Gibco). HEK293T cells were maintained in DMEM supplemented with 10% fetal bovine serum (Gibco).
Plasmids and viruses
Plasmids expressing ISGs were cloned by standard molecular biology techniques. IFN-β-luc reporter, pTK-renilla, Flag-tagged RIG-I, MDA5, VISA, TBK1 and IKKε were generous gifts from Dr. Hongbing Shu (Wuhan University, China). The plasmids expressing shRNAs were generated by annealing pairs of oligonucleotides and cloning into pSuper-Retro (OligoEngine). The target sequences are as follows: Ctrli: 5′-GCGCGCTTTGTAGGATTCG-3′; shPIM1: 5′-CCATCCATGGATGCAAGAT-3′. SeV was kindly provided by Zhengfan Jiang (Peking University, China).
CEM, U937 and Daudi cells were treated with consensus interferon (1000 IU/mL; Interferon Alfacon-1; Amgen) for 0 h, 4 h or 12 h. Total RNA was extracted with the TRIzol reagent (Invitrogen, USA) following the manufacturer’s instructions. Whole genome transcript analysis was performed by Phalanx Biotech Group.
Quantitative reverse transcription-PCR
Total RNA was reverse transcribed using random primer in a 20 μL reaction mixture. Relative mRNA levels of candidate ISGs were measured by SYBR Green real-time PCR (RealmasterMix; SYBR Green; Tiangen) in Rotor-gene 6000 (Corbett Life Science) using the following program: (i) 95°C 10 min, 1 cycle; (ii) 95°C 15 s; 60°C 30 s; 72°C 30 s, 40 cycles. All data are shown as mean value for at least two independent measurements. GAPDH mRNA levels served as internal control. Primers (Table 1) used for the PCR assays are designed using the Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) or acquired from Primer Bank (https://pga.mgh.harvard.edu/primerbank/). Amplification efficiency was assessed for all primer sets, and primers with efficiencies 90%–110% were used.
Luciferase reporter assays
HEK293T cells were transfected with reporters IFN-β-luc and pTK-renilla, together with a plasmid expressing an ISG using Neofectin (NeoBiolab) for 24 h, or with an shRNA-expressing plasmid for 36 h. The cells were then infected with SeV for additional 12 h. Samples were lysed in passive lysis buffer (Promega). Firefly and renilla luciferase activities were measured using the dual-luciferase reporter assay system (Promega). Firefly luciferase activity was normalized with the renilla luciferase activity.
We thank Dr. Hongbing Shu for providing IFN-β-luc reporter, pTK-renilla, Flag-tagged RIG-I, MDA5, VISA, TBK1 and IKKε vectors, Dr. Zhengfan Jiang for Sendai virus (SeV). We thank Zhimin Wang, Xudong Zhao and Xiaofei Guo of the core facility of the Institute of Biophysics, CAS, for technical assistance. This work was supported by grants to Guangxia Gao from Chinese Academy of Sciences (KFZD-SW-209) and National Natural Science Foundation of China (Grant No. 81530066).
CK2, casein kinase II; Con-IFN, consensus interferon; IFNs, interferons; IRF9, IFN-regulatory factor 9; ISGF3, IFN-stimulated gene factor 3; ISGs, interferon-stimulated genes; ISREs, IFN-stimulated response elements; JAK-STAT, Janus kinase and signal transducer and activator of transcription; MDA5, melanoma differentiation-associated gene 5; PKC-α, protein kinase C-α; RIG-I, retinoic acid-inducible gene I; RLRs, RIG-I-like receptors; SeV, Sendai virus
Compliance with ethics guidelines
Xiaolin Zhang, Wei Yang, Xinlu Wang, Xuyuan Zhang, Huabin Tian, Hongyu Deng, Liguo Zhang and Guangxia Gao declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by the any of the authors.
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