Background
Japanese encephalitis virus (JEV) is a mosquito-borne neurotropic virus of the family Flaviviridae. Epidemic encephalitis B is a mosquito-borne zoonosis caused by JEV, occurring mainly in Asia and the Pacific Rim. Japanese encephalitis is a major health hazard in China, where it is considered as a serious infectious disease. Pigs are the main amplifier and wintering host of JEV, which exhibits a pig–mosquito–human transmission pathway that is independent of pig breed and gender. In China, JEV genotypes I and III mainly infect pigs [
1]. JEV infection incidence is highest from July to September each year, at a rate of 20–30%. JEV infection can lead to miscarriage, stillbirths, and weak or mummified fetuses among pregnant sows, and can cause orchitis, testicular shrinkage, and hardening or loss of spermatogenic function in infected boars, eventually leading to infertility; moreover, piglets may die from JEV-induced encephalitis. Together, these effects limit herd expansion, causing huge economic losses in the pig industry [
2,
3]. Since large-scale application of vaccines and traditional veterinary drugs can have adverse effects on disease resistance and the environment, it is of great theoretical and practical significance to identify antiviral molecular agents and alternative means to prevent and control JEV.
Long non-coding RNA (lncRNA) is a type of non-coding RNA with a length greater than 200 nucleotides. LncRNA was originally thought to be merely genomic noise, with no biological function. However, recent studies have shown that lncRNA plays an important role in cellular processes, such as transcriptional regulation, chromosome modification, epigenetic regulation, and intranuclear transport [
4‐
9]. As multi-function non-coding RNAs, lncRNAs have received increasing attention in antiviral-related research. LncRNA MEG3 has been reported to be inhibited by RSV infection, whereas MEG3 inhibits RSV infection of respiratory epithelial cells by inhibiting the TLR4-dependent p38 MAPK and NF-κB signaling pathways [
7]. LncRNA also plays an important role in the natural immunity of pigs to blue ear virus [
10], and can be used as a diagnostic marker and therapeutic target for liver damage caused by dengue virus infection [
11,
12]. Recently, certain viruses have been shown to inhibit cell metabolism-related enzymes, such as GOT2 (mainly enriched in mitochondria), by activating the NF-κB signaling pathway and thereby promoting viral replication and proliferation [
13,
14].
JEV typically invades the central nervous system after infection, and can trigger a wide range of natural immune responses via substantial viral replication, leading to nerve cell necrosis [
15]. The neuroinflammation caused by JEV is mainly related to the loss of control of microglia, which release inflammation-related cytokines and chemokines such as IL-1β, IL-6, TNFα, and MCP1, causing an irreversible inflammatory response and leading to neuronal necrosis. Several studies have found that microglia can also serve as long-term JEV containers [
16]. Although many studies have investigated the molecular mechanisms by which micro RNAs (miRNAs) regulate JEV replication and proliferation [
17‐
20], the molecular mechanism of the effect of lncRNA on JEV proliferation remains to be explored. Recent studies have shown that lncRNA Malat1 was significantly upregulated in JEV-infected mouse Neuro2a cells via the PERK endoplasmic reticulum stress signaling pathway [
21], and that silencing lncRNA E52329 and N54010 can regulate the inflammatory response in JEV-infected mouse microglia cells by reducing the phosphorylation levels of JNK and MKK4 [
22].
In this study, we aimed to explore the role of lncRNA-SUSAJ1 (NONCODE-ID: NONSUST006715.1) in the proliferation of JEV. To this end, PK-15 cells were transduced with overexpression vector and antisense oligonucleotides (ASO) of lncRNA-SUSAJ1 to evaluate its potential role in the proliferation of JEV. We found that lncRNA-SUSAJ1 could inhibit the proliferation of JEV, and CCR1 as a key regulator of JEV proliferation was involved in the expression regulation of lncRNA-SUSAJ1 via transcript factor SP1.
Discussion
After porcine is naturally infected by mosquitoes carrying JEV, the virus first propagates in skin epithelial cells and lymph nodes, infects peripheral organs such as kidney, liver and spleen, and then invades, and then causes transient viremia. After that, the neurotropic virus spread to the central nervous system. Porcine kidney epithelial cell line, PK-15 cells, has a similar susceptibility and function as skin epithelial cells. In addition, scientists have conducted a large number of studies on JEV in PK-15 cells. Therefore, PK-15 cells are a good model to evaluate the role of lncRNAs in host response to JEV infection.
LncRNAs regulate many biological processes including gene imprinting, cell growth, cell differentiation, apoptosis, immune responses, the p53 pathway, stem cell self-renewal, and DNA damage response [
24‐
29]. LncRNA expression is usually tissue-specific or affects specific developmental stages [
30‐
32]. SARS coronavirus-infected mice were found to contain 500 annotated lncRNAs and 1,000 non-annotated genomic regions [
33]. LncRNA GAS5 has been found to suppress hepatitis C virus (HCV) replication via interaction with viral NS protein [
34]. LncRNA NEAT1 is crucial for the nucleocytoplasmic transport of mRNA in response to stimuli [
35]. Recent studies have also shown that virus lncRNA, or lncRNA produced during the viral life cycle, can regulate the host’s antiviral immune response, thus playing an important role in promoting the replication and proliferation of the virus and packaging of the genome into the virions [
36,
37]. Cellular lncRNA and virus-encoded lncRNA can form chimeric lncRNA, which impacts virus infection [
38,
39]. Some studies have shown that lncRNAs regulate the host’s innate immune response, including pathogen recognition receptor-related signaling and the production of interferons and cytokines [
40,
41].
In-depth study of lncRNAs has shown that they act as a medium for molecular scaffolds, guides, decoys, or signals in chromatin remodeling, transcription, post-transcription, or post-translational regulation [
42,
43]. lncRNAs exhibit both negative and positive functions for host’s innate immunity and virus replication [
44,
45]. Different forms of miRNAs lead to mRNA degradation through base pairing to mRNA sequence motifs; thus, lncRNAs utilize specific sequences or structural motifs to bind with DNA, RNA, or proteins, to modulate gene expression and protein activity including cis (impacting neighboring genes) and trans (impacting gene expression via chromosome conformation) functions [
42,
46].
In this study, we found that upregulation of lncRNA-SUSAJ1 transcription levels inhibited the expression of JEV nonstructural protein NS3 and JEV mRNA levels (Fig.
1c, d), and knockdown of lncRNA-SUSAJ1 promoted JEV proliferation (Fig.
2c, d). JEV-NS3 is a multifunctional protein consisting of 619 amino acid residues, one-third of which are n-terminal. The protein also has a catalytic domain of helicases, the activity of serine protease, nucleoside 5′ -triphosphatase, and RNA triphosphatase active [
47‐
49]. NS3 also play crucial roles in the replication and assembly of viruses, that has been confirmed in the Flaviviridae, such as Japanese encephalitis virus, dengue fever virus, yellow fever virus; Hepacivirus, such as hepatitis C [
50‐
52]. We speculate that lncRNA-SUSAJ1 could suppress JEV proliferation by inhibiting NS3, but it is still unclear that the lncRNA-SUSAJ1 interacts with protein JEV- NS3 to inhibit the activity directly or affecting the NS3 activity via other process. In this study, we investigated the effects of lncRNA on anti-virus in PK-15 cells, but the neuroinflammation caused by JEV is mainly related to the loss of control of microglia cells [
16], Furthermore, we will study the function of lncRNAs in microglial cell of swine.
CCR1, also called CD191, is a G protein-coupled receptor that can serve a therapeutic target for the treatment of inflammatory diseases. Mouse homolog studies have suggested that this gene plays roles in host protection, including the inflammatory response and susceptibility to viruses and parasites [
53]. CCR1 also directs leukocytes to inflammation sites [
54]. CCR1 is mainly expressed in lymphocytes, neutrophils, and monocytes [
55,
56]; its known ligands include CCL3, CCL5, CCL7, and CCL23 [
57]. In humans, CCR1 is highly expressed on monocytes, whereas in rodents, it is primarily expressed on neutrophils [
54,
58]. CCR1 recruits monocytes and type-1 T helper cells to activate inflammation after chronic HCV infection [
59]. In rheumatoid arthritis, CCR1 regulates the expression of TNFα and IL-10, and is therefore an efficient therapeutic target [
60]. Since lncRNA-SUSAJ1 suppresses JEV proliferation, CCR1 may play a positive role in promoting JEV proliferation post-infection. Downregulation of CCR1 expression has been reported after infection with
Leishmania infantum or coronavirus [
61,
62]. In the present study, we found that CCR1 expression was negatively correlated with lncRNA-SUSAJ1 expression after JEV infection, CCR1 expression was downregulated at 36 h after JEV infection, but recovered at 48 h (Fig.
3d). By contrast, lncRNA-SUSAJ1 expression was very high at 36 h after JEV infection, but decreased sharply at 48 h (Fig.
1a); therefore, we concluded that the regulation of lncRNA-SUSAJ1 expression by CCR1 is crucial for JEV proliferation. In this study, we found the transcript factor SP1 could regulate the expression of the lncRNA-SUSAJ1 (Fig.
4c, d), and found that CCR1 inhibited lncRNA-SUSAJ1 expression via the transcript factor SP1 (Figs.
4a, b,
5a–c); however, the mechanism by which CCR1 regulates SP1 remains unclear. Furthermore, the mechanism by which lncRNA-SUSAJ1 suppresses JEV proliferation requires further study.
Materials and methods
Cell culture, transfection and viral infection
PK-15 cells were cultured in Dulbecco’s Modified EagleMedium DMEM-F12 (GIBCO) containing 10% (v/v) foetal cattle Serum, 100 μg/ml penicillin /streptomycin mixtures at 37 °C with 5% CO2.
The lncRNA smart silencers were synthesized by Ribobio (Guangzhou, China) and siRNA (siCCR1) were synthesized by GenePharma (Shanghai, China). The sequences of siCCR1s were as follow: siCCR1A 5′-UCAUUGGCCUGAUCGGCAATT-3′, the sequences of siCCR1B: 5′-GGCUCUAUUUCAUUGGCUUTT-3′, the sequences of siCCR1C: 5′-GCAAGUAUCUACGGCAGUUTT-3′, the sequences of siSP1: 5′-GCAACAUCAUUGCUGCUAUTT-3′, the sequences of NC (Negative control): 5′-UUCUCCGAACGUGUCACGUTT-3′. PK-15 cells were seeded in 6-well or 12-well plates and grown to approximately 50–60% confluence for transfection. The cells were transfected with 50 nmol siRNA or 100 nmol lncRNA smart silencers using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. The cells were harvested at the indicated times.
The inhibitors were synthesized by Beyotime (China). AG490 (S1509, JAK inhibitor), LY294002 (S1737, PI3K inhibitor), SP600125 (S1876, JNK inhibitor), SB203580 (S1863, p38MAPK inhibitor), U0126 (S1901, MEK1/2 inhibitor), and ZK811752 (SD3699, CCR1 inhibitor).
The JEV strain SA14-14–2 (GenBank accession: AF315119.1) was propagated in BHK-21 cells according to the protocol of Yang (S. Yang et al., 2013). All infections were carried out by incubating the cells with virus at the MOI = 1, then the inoculum was removed, the cells were washed three times with PBS and fresh mediuma was added. The infection was performed and the infected PK-15 cells were maintained in DMEM supplemented with 2% FBS without penicillin /streptomycin mixtures.
Plasmid
Full-length pig lncRNA-SUSAJ1 oligos was synthesis by Nucleic acid synthesizer, Full-length pig SP1 CDS was inserted into the NheI and XhoI sites of the pcDNA3.1( +) vector (Invitrogen).
Real-time quantitative PCR analysis
Primers for lncRNA-SUSAJ1, CCR1, SP1, NF1, GATA1 and CEBP-alpha were designed using the Primer 5 software; Primers for glyceraldehyde 3- phosphate dehydrogenase (GAPDH) were used as an internal control. Total RNA was extracted from cells using TRIzol® Reagent (Invitrogen) according to the manufacturer's protocol. The reverse transcription of total RNA (1 μg) was performed using a RevertAid™ RT Reagent Kit (RR036A, Takara) in a 20 μl reaction volume according to the manufacturer. Primer information for the Real-time quantitative PCR is also available in the Supplemental information (Additional file
1: Table S1).
Western blot analysis
Cells were lysed with RIPA lysis buffer (P0013B, Beyotime, China) and 1 mM PMSF (ST506, Beyotime, China). Protein concentration of cell lysate was determined by the BCA method (Pierce, Rockford, USA). Ten micrograms of total protein per sample was loaded onto sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 3–4 h and transferred to PVDF membrane at 350 mA for 90 min (Version8, Roche, USA) using an electro-blotting method. After incubating in blocking buffer (PBST with 1% (w/v) BSA (A7030, Sigma)) for 1 h, membranes were incubated with rabbit polyclonal antibody for NS3 (GTX125868, Genetex, USA), rabbit polyclonal antibody for SP1 (ab13370, Abcam, USA) at 4 °C for 12 h. After primary antibodies were used, the membranes were washed before Horseradish Peroxidase (HRP)-conjugated Goat anti-rabbit IgG second-antibody (sc-2030, Santa Cruz, USA) was added for 1 h at room temperature and washed again. The membranes were visualized with an ECL Western blot detection kit (NC15080, Thermo). The β-actin (#4970, Cell Signalling Technology, USA) protein level was also examined as an internal control. The chemiluminescence intensity of each protein band was quantified using the Image J software, and then protein levels were normalized by the amount of β-actin protein.
Chromatin immunoprecipitation assay
Formaldehyde was added at a final concentration of 1% directly to media of PK-15 cells. Fixation proceeded at room temperature for 10 min and was stopped by the addition of glycine to a final concentration of 0.125 M for 15 min. Cells were centrifuged and rinsed 3 times in cold PBS with 1mMPMSF. Then, cell nuclei were collected according to the manufacturer's protocol, SimpleChIP Enzymatic CHIP Kit (#9002, Cell Signalling Technology, USA). Samples were sonicated on ice with an Ultrasonics sonicator at setting 5 for six 10 s pulses to an average chromatin length of approximately 400 to 800 bp. For the immunoprecipitation, 2 μg rabbit polyclonal antibody for SP1 (ab13370, Abcam, USA) in a final volume of 500 μl immunoprecipitation (IP) buffer were added in combination to the nuclear sonicate. After the immunoprecipitation, the IP was eluted and the DNA was recovered. DNA obtained from IP samples were quantified by real-time PCR and normalized to input DNA control samples. Primer information for the ChIP assay is available in the Supplemental information (Additional file
1: Table S1).
Statistics
Data are presented as means ± SEM. Significant differences were analyzed by Mann–Whitney test or one-way analysis of variance (ANOVA) using SPSS software (ver, 20.0, SPAA Inc, USA). P-values < 0.05 were considered to be statistically significant.
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