1. Introduction
Autophagy mediates the clearance of damaged or redundant cellular components, such as protein aggregates, damaged organelles, or invading pathogens. Initiation of autophagy is mediated by the VPS34-Beclin-1 complex and the MTOR signaling pathway [
1]. Autophagy can be either nonselective or selective. Nonselective autophagy is a cellular response to nutritional deficiencies, in which the cytoplasm is nonspecifically engulfed into autophagosomes, while selective autophagy is responsible for removing components specifically through cargo receptor-mediated recognition [
2,
3]. SQSTM1 is one of the most well-known cargo receptors and plays an important role in recognizing and removing aggregates, mitochondria, and pathogens [
4]. In addition, SQSTM1 was found to interact with a vault RNA directly, and the interaction prevented the oligomerization of SQSTM1 and subsequent selective autophagy [
5]. However, whether SQSTM1 facilitates the degradation of dsRNA is still unknown.
The RNA metabolism involved in RNA synthesis, processing, folding, modification, and degradation ensures proper RNA functions. RNA molecules are monitored for quality control. Faulty and excessive RNA molecules are eliminated by RNA decay enzymes [
6,
7], such as ribonuclease, RNA helicase, and other RNA binding proteins. However, a portion of cellular components, such as rRNA and tRNA, are not accessible to the RNA decay enzymes due to their highly structured and extensively restricted localization within ribonucleoprotein complexes and are difficult to degrade by canonical RNA degradation mechanisms. Autophagy is effective at removing these types of RNA [
8]. For example, cytoplasmic RNA granules, retrotransposons [
9], RNA-protein aggregates, and viral RNA could be degraded through autophagy [
10,
11]. However, whether long dsRNA from invading pathogens is subjected to autophagic degradation is largely unknown.
IBDV is a nonenveloped dsRNA virus that causes immunosuppressive and highly contagious diseases in young chickens. IBDV contains two bisegmented double-stranded RNA genomes called segments A (3.2 kb) and B (2.8 kb) [
12]. Genomic dsRNA binds to the cellular pattern recognition receptor MDA5 to initiate type I interferon (IFN) production [
13]. However, whether and how autophagy recognizes and removes cytoplasmic IBDV dsRNA is still unclear.
Here, our study shows that SQSTM1 directly interacts with IBDV dsRNA and mediates its degradation through autophagy. Our study provides evidence that the pathogen genome could be removed by SQSTM1-mediated selective autophagy, highlighting the antiviral role of autophagy during virus infection.
2. Materials and Methods
2.1. Cells, Virus, and Reagents
HEK293T cells (CRL-11268, ATCC, Rockefeller, MD, USA) and the chicken fibroblast cell line DF-1 (CRL-12203, ATCC, Rockefeller, MD, USA) were routinely grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (CCS30010.02; MRC, Lytton, QLD, Australia). IBDV strain NB (NB virus) isolated by the Key Laboratory of Animal Virology (Hangzhou, China) was adapted to growth in DF-1 cells [
14].
VPS34 KO cell lines were derived from the MOA Key Laboratory of Animal Virology (Hangzhou, China) [
15].
SQSTM1 KO cell lines were stored in the MOA Key Laboratory of Animal Virology (Hangzhou, China).
2.2. Antibodies
Rabbit polyclonal antibody against GAPDH (glyceraldehyde-3-phosphate dehydrogenase; ABPR001) was purchased from Xianzhi Biological Technology (Hangzhou, China), and antibodies against Myc (R1208-1) and GST (EM80701) were purchased from Huaan Biological Technology (Hangzhou, China). Anti-SQSTM1 antibody (ab109012) was purchased from Abcam (Cambridge, UK). Mouse monoclonal antibodies to the viral protein VP2 of IBDV were provided by the Key Laboratory of Animal Virology [
16,
17]. Horseradish peroxidase (HRP)-conjugated anti-mouse (074-1806) and anti-rabbit IgG (074-1506) were obtained from KPL (Milford, MA, USA). Alexa Fluor 546-conjugated anti-rabbit (A21085) and anti-mouse IgG (A10036) were purchased from Invitrogen (Carlsbad, CA, USA). Fluorescein isothiocyanate (FITC)-conjugated anti-mouse (172-1806) and anti-rabbit IgG (172-1506) were purchased from KPL (Milford, MA, USA). NP-40 lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40; P0013F) was purchased from Beyotime (Shanghai, China). Anti-GST resin (L00206) was obtained from Genescript (Nanjing, China).
2.3. Plasmids and Transfection
The full-length SQSTM1 gene was amplified by PCR from the cDNA of 293T cells and inserted into the plasmids pCMV-Myc-N (Clontech, Mountain View, CA, USA), pGEX-4T-1 (GE, Boston, MA, USA), and pCDH-CMV-MCS-EF1-Puro (SBI, Palo Alto, CA, USA), thereby designated as Myc-SQSTM1, GST-SQSTM1, and pCDH-SQSTM1, respectively. All constructs were confirmed by sequencing (Zhejiang Sunya Biotechnology Co, Hangzhou, China). All plasmids and RNA were transfected into cells using lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
2.4. Construction of SQSTM1 Knockout Cell Lines
293T cells were cultured in DMEM supplemented with 10% fetal bovine serum. The gRNA was designed online (
http:crispr.mit.edu/ accessed on 18 June 2018), the sgRNA sequence against SQSTM1 (sense: TAACTTACCATAGACATCTG antisense: CAGATGTCTATGGTAAGTTA) was inserted into the CRISPR/Cas9 plasmid PX459, which contains puromycin resistance. The reconstructed plasmids were transfected into 293T cells and then screened with puromycin at a concentration of 2 µg/mL. The survived cells were diluted into a 96-well plate to screen monoclonal cells. After that, knockout effects were validated by Western blot and sequence. The sequencing result shows the amplified fragment that covered the deletion region was totally removed.
2.5. Construction of STable 293T Cells Expressing SQSTM1
PCDH-SQSTM1 was cotransfected with the ViraPowerTM lentiviral packaging mix (K497500; Invitrogen, Carlsbad, CA, USA) into 293T cells to generate a lentiviral stock according to the manufacturer’s protocol, and an empty vector was used as control. After 72 h of transfection, viral particles were acquired from the medium by ultracentrifugation. After lentiviral preparation, 293T cells were seeded in the 6-wells plate and grown to 80% confluence overnight. 293T cells were separately infected with 2 mL pCDH-SQSTM1 or pCDH-CMV-MCS-EF1 virus for 6 h. After cells were cultured for another 24 h with complete medium, puromycin (4 µg/mL) was added to screen positive cells. Western blot and indirect immunofluorescence assay were used to detect the expression of SQSTM1 in the cell lines.
2.6. Cytotoxicity Assay
The cytotoxicity assay was performed using an enhanced CCK8 kit (C0014, Beyotime, Shanghai, China) according to the standard protocol. The cells seeded in 96-well plates were treated with the drug for 4 h or transfected with vectors for 8 h. After treatments or transfection, 10 µL CCK-8 reagent was added into the culture medium. After incubation for 1 h, the absorbance value was measured at 450 nm with a spectrophotometer. The cell viability was calculated as (A450treated/A450control).
2.7. IBDV Genomic dsRNA Extraction
DF-1 cells reached about 80% confluence, and culture medium was replaced with medium containing 2% FBS. The cells were infected with IBDV (MOI = 0.1) and cultured for 48 h. Then, the cells were frozen and thawed 3 times and centrifuged for 10 min. The supernatant was transferred to clean tubes and centrifuged at 50,000× g for 4 h. The pellet was resuspended in PBS (1% of the medium volume), and then 10% SDS (5% of the PBS volume) and proteinase K (1 mg/mL) were added to digest the viral proteins at 50 °C for 2 h. The dsRNA was purified by phenol/chloroform/isoamyl alcohol (25:24:1) extraction and precipitated with isopropanol. Finally, the dsRNA was dissolved in diethyl pyrocarbonate (DEPC)-treated water. Purified dsRNA was separated in 1% agarose gel and detected by a gel imaging analysis system (GenoSens 1880, Clinx, Shanghai, China).
2.8. IBDV dsRNA Degradation Assay
The dsRNA of IBDV was transfected into wild-type, VPS34 KO or SQSTM1 KO 293T cells at approximately 80% confluence by using lipofectamine 3000 (L3000015, Invitrogen, Carlsbad, CA, USA). The cells were harvested at 8 h post transfection. Then, the cells were lysed with Trizol, and total RNA was extracted with chloroform. Reverse transcription of total RNA was carried out using a first-strand synthesis system, and cDNA was obtained. The amount of dsRNA was measured by qRT-PCR and PCR.
2.9. Detection of IFN-β and IFN-Stimulated Gene
Myc-SQSTM1 or its variant Myc-SQSTM1RK/A and dsRNA were cotransfected into SQSTM1 KO cell lines. The cells were harvested at 8 h post transfection. Then cells were lysed with Trizol, and total RNA was extracted with chloroform. Reverse transcription of total RNA and cDNA was obtained. The relative levels of IFN-β mRNA, IFN-stimulated gene mRNA, and β-Actin mRNA from SQSTM1 KO cell lines were analyzed by qRT-PCR.
2.10. Primer Pairs for qRT-PCR and PCR
Primer pairs were as follows: for dsRNA (sense: CCTCTGGGAGTCACGAATTAAC; antisense: ACTCATGGTGGCAGAATCATC), for β-actin in 293T cell lines (sense: TCTGGCACCACACCTTCTAC, antisense: ATCTGGGTCATCTTCTCGC) and GAPDH in DF-1 cell lines (sense: CCCAGCAACATCAAATGGGCAGAT, antisense: TGATAACACGCTTAGCACCACCCT), for IFN-β in 293T cell lines (sense: TTGTTGAGAACCTCCTGGCT, antisense: TGACTATGGTCCAGGCACAG), for ISG56 in 293T cell lines (sense: TCATCAGGTCAAGGATAGTC, antisense: CCACACTGTATTTGGTGTCTAGG), for ISG15 in 293T cell lines (sense: AGGACAGGGTCCCCCTTGCC, antisense: CCTCCAGCCCGCTCACTTGC), for dsRNA analog 1 (sense: ACTACCAGCAGAACACCCCCATCGG, antisense: GCAGGACCATGTGATCGCGCTTCTC), for dsRNA analog 2 (sense: GAATCAGGGGATAACGCAGGAAA, antisense: GTAAGCGGCAGGGTCGGAACA), for IBDV A segment (sense: CACCAGAATGGGTAGCA, antisense: ATCGCAGTCAAGAGCAGA), for IBDV segment B (sense: CCTCTGGGAGTCACGAATTAAC; antisense: ACTCATGGTGGCAGAATCATC).
2.11. Indirect Immunofluorescence Assay
To observe the transfection efficiency of SQSTM1 and VPS34, 293T cells were transfected with Myc-SQSTM1 or Myc-VPS34 for 8 h, while DF-1 cells were transfected with Myc-chSQSTM1 for 8 h. Then, the cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.2% Triton X-100 for 10 min. Cells were incubated with an anti-Myc-tag antibody at 37 °C for 2 h. After three washes with PBST, cells were incubated with FITC-conjugated goat anti-rabbit IgG at 37 °C for 1 h. After three washes with PBST, the cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI) to stain the nuclei. Then, the cells were observed under a fluorescence microscope (Ti-E, Nikon, Minato-ku, Tokyo, Japan).
2.12. Confocal Microscopy and Structured Illumination Microscopy
To observe the colocalization of IBDV dsRNA and SQSTM1, DF-1 cells were transfected with Myc-SQSTM1 for 24 h and then infected with IBDV (MOI = 0.1) for 24 h. The cells were then fixed with 4% paraformaldehyde for 30 min and then permeabilized with 0.2% Triton X-100 for 10 min. The fixed cells were incubated with mouse anti-dsRNA monoclonal antibody and rabbit anti-Myc monoclonal at 37 °C for 2 h. After three washes with PBST, the cells were incubated with FITC-conjugated goat anti-rabbit IgG- and A546-conjugated donkey anti-mouse IgG at 37 °C for 1 h. After three washes with PBST, the cells were incubated with DAPI. The cells were observed under a Nikon laser confocal microscope (Ti-E + A1, Nikon, Minato-ku, Tokyo, Japan) and N-SIM microscope (Ti-E + SIM, Nikon, Minato-ku, Tokyo, Japan).
2.13. Western Blot
The cells were resuspended in PBS and centrifuged at 1000× g for 10 min at 4 °C. The cell pellets were lysed in NP-40 buffer. Then, SDS-PAGE loading buffer (P1016, Solarbio, Beijing, China) was added to the cell lysates to prepare the samples. Equivalent amounts of samples were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and then incubated with 5% skimmed milk dissolved well in PBST at 37 °C for 40 min. After three washes with PBST, the membrane was incubated with primary antibodies at 4 °C for 8 h. After four washes with PBST, the membrane was incubated with HRP-conjugated anti-mouse or anti-rabbit IgG at 37 °C for 40 min. After three washes with PBST, the membrane was visualized using a SuperSignal West Femto Maximum Sensitivity Substrate (34094, Thermo Fisher Scientific, Waltham, MA, USA) and imaged using a chemiluminescence imaging system(GE Amersham Imager680, Boston, MA, USA). In addition, densitometric analysis was performed.
2.14. RNA Binding Protein Immunoprecipitation Assay
Purified IBDV dsRNA was incubated with anti-GST resin at 4 °C for 1 h, and the dsRNA with nonspecific binding beads were removed by centrifugation at 1000× g for 5 min. Then, the supernatant was mixed with prokaryotically expressed recombinant GST-SQSTM1 protein and rotated at 4 °C for 2 h. The anti-GST resin was then added to the mixture and rotated at 4 °C for 2 h. After four washes with NP-40 lysis buffer, the samples were divided into two parts. One part was used to detect protein expression by Western blot; the other part was used to detect the bound RNA by PCR.
2.15. RNA Pull-Down
The IBDV genomic dsRNA extraction assay was conducted using a Pierce™ Magnetic RNA-Protein Pull-Down Kit (20164, Thermo Fisher Scientific, Waltham, MA, USA) according to standard protocols. Firstly, the IBDV dsRNA was labeled by using the Thermo Scientific Pierce RNA 3′ Desthiobiotinylation Kit, and then the labeled RNA was bound to streptavidin magnetic beads. After that, GST-SQSTM1 protein was added to the mixture. After two washes with wash buffer, the targeted protein was eluted with elution buffer. The samples were processed and detected by Western blot.
2.16. DsRNA Transcription
Both strands of 1000 bp DNA segments from pEGFP-C3 vector (primer pairs for dsRNA analog 1: sense: TAATACGACTCACTATAGGGTCCTACTTGGCAGTACATCT, antisense: TAATACGACTCACTATAGGGGTCCATGCCGAGAGTGATCC), 2810 bp DNA segments from pGL3-Basic vector (primer pairs for dsRNA analog 2: sense: TAATACGACTCACTATAGGGGGTACCGAGCTCTTACGCGT, antisense: TAATACGACTCACTATAGGGTTTTCCGAAGGTAACTGGCT), and DNAs encoding IBDV A and B segments (primer pairs for IBDV A: sense: TAATACGACTCACTATAGGGGGATACGATCGGTCTGACCC, antisense: TAATACGACTCACTATAGGGGGGGACCCGCGAACGGATCC, primer pairs for IBDV B: sense: TAATACGACTCACTATAGGGGGATACGATGGGTCTGACCC, antisense: TAATACGACTCACTATAGGGGGGGGCCCCCGCAGGCGAAG) were transcribed by T7 High-Yield RNA Transcription Kit (TR102, Vazyme, Nanjing, China). Transcriptional RNA was annealed to form double strands. RNase T1 was used to digest single-stranded RNA. DsRNA analogs were separated in 1% agarose gel and detected by a gel imaging analysis system (GenoSens 1880, Clinx, Shanghai, China).
2.17. Statistical Analysis
The protein bands were relatively quantified from Western blot analysis. Briefly, the mean gray value of protein bands within the linear range and background was measured by using Image J software (National Institutes of Health, Bethesda, MD, USA), and the quantification reflects the relative amounts as a ratio of each net band value relative to the net loading control. Data statistical significance was calculated by Student’s t-test. All data from three independent biological experiments are presented as mean ± SD (*, p < 0.05; **, p < 0.01).
4. Discussion
The degradation of RNA through a lysosome-dependent pathway was first demonstrated by Sameshima M. et al. in human fibroblasts [
20]. As expected, autophagy was then confirmed as a critical mechanism for the degradation of RNA [
21,
22,
23]. Recently, Fujiwara et al. characterized a novel RNA degradation mechanism named “RNautophagy” [
24]. Clearance of redundant intracellular RNA is physiologically important. Retrotransposon RNA, such as long interspersed element 1 (LINE 1) and short interspersed nucleotide elements, leads to translocations, inversions, deletions, and amplifications. Mutations in the genome by retrotransposon RNA have been linked to tumorigenesis [
25,
26]. Guo et al. showed that autophagy plays a critical role in removing retrotransposon RNA and consequently protecting the genome from mutations [
9]. Autophagy also plays a significant role in clearing major cellular RNA, namely, rRNA, which is assembled into ribosomes. The autophagic degradation of ribosomes, as well as rRNA, is critical in maintaining cell viability under nutritional stress [
27]. Similarly, clearance of stress granules and processing bodies is required because of its physiological importance for cellular viability [
28,
29]. Our study showed that autophagy is responsible for removing dsRNA of IBDV, confirming the antiviral role of autophagy. However, we could not exclude the possibility that IBDV might employ autophagy to digest naked dsRNA in order to avoid recognition by host immune receptors, thereby preventing the immune response.
Invading viruses and bacteria can be captured by autophagosomes and degraded by lysosomes. Spencer Shelly et al. found that autophagy decreased the replication of VSV, and repression of autophagy led to increased viral replication and pathogenesis in cells and animals [
10]. During Sindbis virus (SINV) infection, autophagy is activated and leads to the degradation of viral capsids [
11]. Hu et al. found that IBDV could induce autophagy at a very early stage of IBDV infection [
30]. Our data indicate that not only viral proteins but also the viral genome can be degraded by autophagy.
Cargo receptors, such as OPTN, NDP52, SQSTM1, and NBR1, mediate selective autophagy. Some of these proteins have been shown to regulate the degradation of nucleic acid. LAMP2C (lysosomal-associated membrane protein 2C) delivers RNA into lysosomes directly for degradation [
24]. NDP52 recognizes M. tuberculosis DNA and regulates its autophagic degradation [
31]. The SQSTM1 and NDP52 receptors were found to play a role in removing cytoplasmic RNA granules, such as stress granules (SG) and processing bodies (PB) [
9]. However, the authors of the study above did not prove the direct binding of RNA to the cargo receptors. In our study, we showed that SQSTM1 was able to bind to dsRNA directly, suggesting that this autophagic cargo receptor might be a cytoplasmic RNA sensor that regulates cytoplasmic RNA degradation. In addition, it is possible that IBDV might employ autophagy to digest naked dsRNA in order to avoid recognition by host immune receptors, thereby blunting the immune response.
In conclusion, we show that SQSTM1 directly binds to IBDV dsRNA and mediates its autophagic degradation, thereby suppressing IBDV replication, highlighting the antiviral role of selective autophagy in IBDV infection.