Introduction
Host immune responses are divided into innate and adaptive immune responses, with the former acting as the first line of defense against infection [
1‐
3]. During virus infection, the conserved components called pathogen associated molecular patterns are recognized by host pathogen recognition receptors, leading to the activation of innate immune signaling and production of a series of cytokines and inflammatory factors [
4,
5]. The induction and action of type I interferon are important in human immune defense activation and intracellular antimicrobial progress by influencing the development of innate and adaptive immune responses, up-regulating antiviral responses, and restricting viruses replication [
6,
7]. Influenza A virus is a single-strand negative-sense RNA virus contagious pathogen that is responsible for severe respiratory infection in humans and animals worldwide and has received attention in the current COVID-19 pandemic [
3,
8]. The virus also induces type I interferon production primarily through the activation of RIG-I pathway, during which multiple host factors are involved [
1,
9‐
12].
Retinoic acid-inducible gene-I (RIG-I) is an important member of RIG-I-like receptors that detect viral nucleic acids in the cytosol [
13‐
15]. Once activated, RIG-I triggers the expression of downstream MAVS, TRAF3, and TBK-1/IKKε and ultimately induces IRF3 phosphorylation, nuclear localization, and type I interferon expression [
5,
16]. The expression of RIG-I and its polyubiquitination of N-terminal CARD domain (RIG-I-N) are critical for its function and are regulated by a variety of host factors [
13,
15,
17].
Ariadne-1 homolog (ARIH1) is a member of the Ariadne family of E3 ubiquitin ligases with many biological functions. ARIH1 is involved in tumorigenesis, cell development, and metabolism; however, its role in antiviral innate immunity is not fully known [
18,
19]. In this study, we found that influenza A virus, wild-type H1N1 virus A/ PR8 (H1N1/PR8), could up-regulate the expression of ARIH1 as a novel positive regulator of RIG-I signaling. ARIH1 also promotes the RIG-I induced production of type I interferon by interacting with SQSTM1/p62. Our findings reveal a new host factor of defense influenza A virus evasion by regulating antiviral immune response.
Materials and methods
Cell culture and transfection
Human type II alveolar epithelial (A549) cells cultured in Ham’s F12K medium (F-12, BasalMedia, China) with 10% fetal bovine serum (FBS, Gibco, Brazil) and 5% CO2 at 37 °C. Human embryonic kidney (HEK293T) cells cultured in RPMI-1640 (BasalMedia, China) and Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco's minimal essential medium (DMEM, BasalMedia, China) with 10% FBS and 5% CO2 at 37 ℃.
For the transient overexpression of specific proteins, cells were transfected with plasmid using Lipofectamine™2000 or Lipofectamine™3000 (Invitrogen). For gene silencing, ARIH1 siRNA (siARIH1) and control siRNA (siNC) were obtained from GenePharma (China). Cells were transfected with siRNAs using Lipofectamine™2000 or Chemi-Trans™ RNAiMAX Transfection Reagent (GeneCodex) following the manufacturer's protocol at a final concentration of 100 nM.
Antibodies and plasmids
Mouse monoclonal Flag tag antibody was from Sigma (USA); rabbit polyclonal HA tag antibody, rabbit polyclonal ARIH1 antibody and rabbit polyclonal IRF3 antibody were from ABclonal Biotechnology (China); rabbit polyclonal phosphorylated IRF3 (p-IRF3) antibody was from Abcam (USA); mouse monoclonal Myc tag antibody, rabbit polyclonal NP and M1 of H1N1/PR8 antibody and rabbit polyclonal RIG-I antibody were from Proteintech (Wuhan, China); mouse polyclonal GAPDH antibody were from BioPM (Wuhan, China); horseradish peroxidase (HRP) conjugated secondary antibodies were from Beyotime Biotechnology (China).
ARIH1, RIG-I, RIG-I-N (The N-terminal CARD domain of RIG-I), VISA, TBK1, and SQSTM1 expression plasimds; a luciferase reporter plasmid for the IFN-β promoter (pIFN-β-Luc); and a luciferase reporter plasmid pRNP-Luc with a pol I transcription unit were constructed by our laboratory. A Renilla control plasmid (pGL4.75 hRluc/CMV) was purchased from Promega. The PHW-PR8-PB2, PB1, PA, and NP plasmids were constructed from H1N1/PR8 as previous described [
20]. Myc-tag ubiquitin plasmid (pMyc-UbWT), Myc-tag ubiquitin mutant in which all lysine residues except K63 were mutated to arginine plasmid (pMyc-UbK63), and Myc-tag mutant in which only the K63 residue was mutated to arginine plasmid (pMyc-UbK63R) were get from Hedgehogbio (Shanghai, China).
Virus preparation and infection of cells
H1N1/PR8 and Sendai virus (SeV) were grown in 10-day-old fertilized eggs and stored at − 80 °C. The viral titer of H1N1/PR8 was measured using the Reed-Muench method. All H1N1/PR8 experiments were performed in Biosafety Level 2 facilities at the Key Laboratory of Infectious Disease & Biosafety, Provincial Department of Education, Guizhou, Zunyi Medical University, China.
For H1N1/PR8 infection, A549 cells were washed three times in phosphate buffer saline (PBS) to remove FBS and then incubated with the virus diluted in F-12 with 0.5 μg/ml TPCK treated trypsin for 1 h at 37 °C. After 1 h, the cells were washed and maintained in F-12 with 1% FBS, 1% penicillomycin and 0.5 μg/ml TPCK treated trypsin for the indicated times. For IFN-β expression, HEK293T cells and A549 cells were stimulated with SeV.
Western blot assay
Cells were lysed in RIPA lysis buffer (CST, USA) containing protease inhibitors (Calbiochem) for 30 min on ice and were then centrifuged at 10,000 rpm for 10 min. The supernatant was quantified by Detergent Compatible Bradford Protein Assay Kit (Beyotime Biotechnology, China), added the 1 × SDS Loading buffer and boiled for 5 min. The sample was separated on 10% SDS-PAGE, transferred to nitrocellulose membranes (PALL, Japan), and blocked with 5% bovine serum albumin (BSA), followed by immunoblotting with the indicated antibodies. Immunoreactive bands detected using ECL reagents (Advansta, USA) were developed by Image Lab system (Bio-Rad, USA).
Polymerase activity assay
HEK293T cells in 12-well plates was transfected with 0.5 μg of PHW-PR8-PB2, PHW-PR8-PB1, PHW-PR8-PA, PHW-PR8-NP, and pRNP-Luc; 0.01 μg of pGL4.75 hRluc/CMV; and 0.5 μg of ARIH1 expression plasmid or control plasmid using 6 μl of LipofectamineTM2000. The cells were incubated for 24 h and then lysed in 200 μl of passive lysis buffer. Luciferase and Renilla activities were assessed using a Dual-Luciferase Assay Kit (Promega).
TCID50 assay
Cell supernatants containing the virus were serially diluted tenfold with DMEM and applied in quadruplicate to 2 × 104 MDCK cells /well in a 96-well plate. On the fifth day post infection, the viral titer was determined by observing the cytopathogenic effect and was confirmed by hemagglutination. The TCID50 was determined based on the Reed-Muench method.
Quantitative RT-PCR assay
Total cellular RNA was extracted using TransZol Up Kit, reversed by TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix, and analyzed using PerfectStart™ Green qPCR SuperMixt following the instruction. All the reagents were purchased from Transgen Biotech, Beijing, China. All the primers were synthesized from Sangon Biotech, Shanghai, China. The primers used were: 5′-CTCTCCTGTTGTGCTTCTCC-3′ and 5′-GTCAAAGTTCATCCTGTCCTTG-3′ for IFN-β detection; 5′-GCGCTGGGTATGCGATCTC-3′ and 5′-CAGCCTGCCTTAGGGGAAG-3′ for ISG56 detection; 5′-GAAAGCAGTTAGCAAGGAAAGGT-3′ and 5′-GACATATACTCCATGTAGGGAAGTGA-3′ for CXCL10 detection; 5′-GACAACTTTGGTATCGTGGAA-3′ and 5′-CCAGGAAATGAGCTTGACA-3′ for GAPDH detection.
Luciferase reporter assay
HEK293T cells in 12-well plates were transfected with 0.5 μg of pIFN-β-Luc; 0.01 μg of pGL4.75 hRluc/CMV; 0.5 μg of RIG-I, RIG-I-N, VISA, TBK1, or IRF3; and 2 μg of the ARIH1 expressing plasmid or control plasmid using 6 μl of Lipofectamine™2000. The cells were incubated for 24 h and then lysed in 200 μl of passive lysis buffer. Luciferase and Renilla activities were assessed using a Dual-Luciferase Assay Kit (Promega).
Immunoprecipitation assay
Cells were lysed in RIPA lysis buffer (CST, USA) containing protease inhibitors (Calbiochem) for 30 min on ice and were then centrifuged at 12,000 rpm for 15 min. The supernatant was incubated with antibody for 1 h at 4 °C and the lysate-antibody complexes were incubated with Protein A/G Magnetic Beads (MedChemExpress) for 6 h at 4 °C. The precipitated agarose was washed four times with lysis buffer to remove nonspecific binding. The immune complex was eluted with 2 × SDS Loading buffer and boiled, separated on SDS-PAGE and analyzed by western blot assay.
Statistical analysis
The data are presented as means standard deviations (SD) from three independent experiments. Statistical significance was determined using two-tailed Student’s t test. A P value of less than 0.05 was considered statistically significant, and a P value of less than 0.01 was considered highly significant (*, P < 0.05 and **, P < 0.01). For western blot assay, the band intensities were analyzed using the software ImageJ.
Discussion
ARIH1 belongs to the E3 ubiquitin-protein ligase family, which participates in multiple biological functions. In cancer, ARIH1 can inhibit the function of drugs on tumor cells by regulating autophagy [
18,
33] and can act as a suppressor to inhibit tumor development [
19]. It also regulates blood glucose, vascular smooth muscle, and iron transport [
37‐
39]. ARIH1 can inhibit the bacterial proliferation and germ cell development of
C. elegans [
40,
41]. However, the antiviral activity of ARIH1 has not yet been sufficiently studied. Our research provides important evidence for this function of ARIH1.
The effect of ARIH1 on the proliferation of influenza A virus was first investigated. The results showed that ARIH1 overexpression inhibited H1N1/PR8 replication, and its silence resulted in the opposite phenomenon (Fig.
1A–D). This study also found that viral infection can up-regulate the expression of ARIH1 (Fig.
2 and Additional file
1: Fig. S2). The effects of ARIH1 on influenza A virus internalization and polymerase activity were examined, but no remarkable influence was found (Fig.
3A, B). This finding suggested that ARIH1 does not affect the proliferation of the virus by affecting its life cycle and implied that viruses may up-regulate ARIH1-induced host antiviral mechanisms. The main antiviral signaling pathway activated by influenza virus in the host is RIG-I signaling pathway, which is commonly stimulated by SeV [
13,
26]. Therefore, this study next examined the effect of ARIH1 on SeV-induced phosphorylation of IRF3 in host cells by Western blot assay, which can reflect the activation of the signaling pathways. The results showed that ARIH1 was overexpressed and phosphorylated IRF3 was up-regulated (Fig.
4A, C). Quantitative RT-PCR assay also indicated that ARH1 overexpression increased the mRNA levels of SeV-induced endogenous IFN-β and downstream ISG15 and CXCL10 (Fig.
5A and Additional file
1: Fig. S3A). Opposite results were obtained after the silencing of ARIH1, and the phenomenon was the same in A549 cells and HEK293T cells (Figs.
4C, D,
5A, and Additional file
1: Fig. S3B). The targets of ARIH1 in RIG-I signaling pathway in HEK293T cells were explored using double fluorescence reporting system. Only the overexpression of RIG-I or RIG-I-N enhanced the activation of IFN-β promoter (Fig.
6A). These results indicated that ARIH1 targets at RIG-I and positively regulats the activation of IFN-β signaling.
The activation of RIG-I is the first step for the induction of IFN-β signaling [
12,
28,
42]. The interaction between ARIH1 and RIG-I was examined by immunoprecipitation assay, but no relationship was observed (Fig.
6B, C). RIG-I activation involves K63-linked polyubiquitination and oligomerization and various host proteins, such as E3 ubiquitin ligase [
27,
29,
43]. As an E3 ubiquitin ligase, the effects of ARIH1 on the K63-linked polyubiquitination and oligomerization of RIG-I were analyzed. The results proved that ARIH1 did not promote the K63-linked polyubiquitination and oligomerization of RIG-I, indicating that ARIH1 does not influence the activation of RIG-I (Fig.
7A, B).
SQSTM1/p62-mediated autophagic degradation is an important mechanism of the negative regulation of type I IFN signaling [
32,
44]. ARIH1 is also involved in autophagy [
33]. Given that ARIH1 did not affect activation, endogenous RIG-I expression was analyzed by Western blot assay. The results showed that ARIH1 facilitated RIG-I expression (Fig.
8A). Therefore, the effect of ARIH1 on RIG-I regulated by SQSTM1 was then examined. ARIH1 was found to promote RIG-I signaling by inhibiting the degradation of RIG-I through its interaction with SQSTM1 (Fig.
8B, C). Finally, we tested the effect of ARIH1 as E3 ubiquitin ligase on SQSTM1, and discovered that ARIH1 did not affect the ubiquitination of SQSTM1 but could block its binding with RIG-I (Fig.
9A, B). This study provides insights into the underlying mechanism by which ARIH1 promotes IFN-β.
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