Background
Japanese encephalitis virus (JEV) is a mosquito-borne neurotropic virus that belongs to the family flaviviridae. JEV is mostly prevalent in eastern, southeastern and southern Asian countries where three billion people are at risk of contracting the disease [
1]. A 2011 review estimated that the annual incidence was 1.8/100,000 and 5.4/100,000 for children 0-14 years old in 24 JEV endemic countries [
2]. JEV is transmitted in an enzootic cycle between mosquito vectors and vertebrate hosts; humans contract JEV when bitten by an infected mosquito. Pigs act as amplifying hosts of JEV; therefore, the domestic pig was considered to be a risk factor in the transmission of the disease to humans [
3,
4]. JEV is also one of the main causes of infectious reproductive failure in swine, and this has resulted in significant economic losses for the swine industry. The primary symptoms of pigs infected with JEV are fetal abortion and stillbirth in infected sows and aspermia in boars [
5,
6]. In order to elucidate the pathogenesis of JEV in pigs, numerous studies on JEV have been conducted in PK-15 cells, a porcine kidney epithelial cell line, which have a similar susceptibility as skin epithelial cells to JEV [
7‐
9]. Therefore, PK-15 cells are a good model in which to evaluate the host response to JEV infection.
MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nucleotides in length, which play a crucial role in the post-transcriptional regulation of genes involved in fundamental biological processes, including cell differentiation, proliferation, apoptosis and cell signaling [
10,
11]. miRNAs regulate gene expression by guiding the RNA-induced silencing complex (RISC) to partially complementary sites within the 3’ UTR of target mRNAs via their seed region [
12]. miRNAs have been shown to regulate the host antiviral response by altering the expression of host genes required for virus replication and antiviral response [
13‐
16] or by directly targeting viral genomes and/or transcripts. [
17‐
20]
Recent studies suggest that miRNAs play a notable role in cellular immune response during Japanese encephalitis virus infection [
21‐
24]. For example, miR-15b and miR-155 were shown to induce an inflammatory response in JEV-infected microglial cells via suppression of RNF125 [
21] and SHIP1 [
23], respectively, whereas miR-146a was shown to inhibit the immune response against JEV by suppressing the JAK/STAT pathway in mouse monocytes [
22]. In addition, miR-29b regulated JEV-induced microglia activation, and miR-155 suppressed JEV replication and negatively modulated innate immune responses in microglial cells [
24,
25].
In this study, we investigated the role of host miRNAs in JEV infection of porcine cells. We show that expression of miR-124 is upregulated in response to JEV infection and that this results in the suppression of the target gene, DNM2, which is required for virus replication. Thus, we conclude that upregulation of miR-124 and the subsequent suppression of DNM2 represents a host response aimed at limiting JEV infection. Our study reveals an example of a miRNA that modulates JEV replication and also highlights a host factor that could be used for RNAi-mediated antiviral therapeutic strategies.
Discussion
MicroRNAs serve as multifunctional regulators, and the significance of miRNAs in virus-host interactions is becoming evident. A growing number of studies show that host miRNAs can drastically influence virus replication, either by facilitating enhanced replication [
40] or by mediating the host antiviral response [
17]. Therefore, many viruses, including JEV, modulate the expression of specific cellular miRNAs [
22‐
25]. JEV is a significant pathogen of acute CNS inflammatory disease in humans. In addition, pigs act as amplifying hosts of JEV. However, the role of host miRNAs in JEV-host interactions and pathogenesis has not been extensively studied.
miR-124 is highly expressed in neurons, and it plays a notable role in neuronal differentiation [
28]. In addition, miR-124 was attenuated in several tumors, and the overexpression of miR-124 inhibited the metastasis of breast cancer and hepatocellular carcinoma [
41,
42]. Interestingly, previous studies showed that miR-124 could play an important role in virus entry by disturbing receptor-mediated endocytosis, and miR-124 was upregulated in JEV-infected porcine cells [
30,
43]. These results suggest that miR-124 may be involved in the regulation of JEV infection. In order to determine the biological role of miR-124 in the host response to JEV infection, miR-124 was overexpressed through miRNA mimic transfection in order to assess its effect on JEV replication. The flow cytometry and Western blot experiments revealed that miR-124 exhibited a significant antiviral effect (Fig.
1). Thus, the key new finding of this study is that miR-124 is an anti-JEV miRNA.
miRNAs regulate many biological processes by interacting with their target genes. Host miRNAs were previously shown to directly target viral genomic RNAs; for example, miR-122 facilitates hepatitis C virus replication [
44], while miR-130b and miR-181 suppress the porcine reproductive and respiratory syndrome virus (PRRSV) [
19,
45]. According to the online software analysis, there were two potential miR-124 binding sites in the JEV genomic RNA. However, the luciferase reporter assay showed that miR-124 does not directly interact with the 2 predicted sites in the JEV RNA (Fig.
2). These data indicate that the antiviral effect of miR-124 is mediated by its interaction with the host genes. In order to test this hypothesis, we screened for miR-124 targets using TargetScan and ultimately focused on the DNM2 gene. The luciferase reporter assay showed that miR-124 directly interacts with the 3’ UTR of DNM2 and that DNM2 mRNA and protein levels were reduced in cells overexpressing miR-124 (Figs.
3 and
4).
Dynamin-2, a GTPase responsible for vesicle scission, forms helical polymers around the membrane neck of nascent endocytic buds and, upon GTP hydrolysis, mediates the fission of vesicles from the plasma membrane [
46]. Thus, the research showed that other factors may assist with the action of dynamin-2 in vivo because dynamin-2 is sufficient to mediate membrane fission [
47]. In addition, dynamin-2 was required for the caveolae and clathrin-mediated endocytosis pathways [
47,
48]. We previously found that JEV infected PK15 cells via clathrin-dependent endocytosis [
8], and miR-124 reduced caveolar density in porcine PK15 cells [
43]. Therefore, dynamin-2 may play an important role in JEV infection. Indeed, siRNA knockdown was performed in order to assess its effect on JEV replication in DNM2 depleted cells. The knockdown experiment demonstrated that dynamin-2 was required for JEV replication (Fig.
5). However, the step at which the inhibition of JEV infection by dynamin-2 occurs, perhaps at entry, still needs to be demonstrated.
Previous studies using high-throughput sequencing technology found that miR-124 was downregulated by JEV in swine testis cells [
30]. In this study, quantitative methods were used to determine expression levels of miR-124 in PK15 cells before and after JEV infection. The results confirmed the previous reports and further showed that miR-124 was upregulated immediately upon infection (4 hpi) and remained upregulated throughout the infection. In contrast, DNM2 expression levels increased during early JEV infection (first 12 hpi) and then sharply declined (24 hpi) in mRNA level, however, DNM2 protein level always remained downregulated throughout the infection (Fig.
6). Considering the important role of DNM2 in the regulation of virus entry, it is possible that miR-124-mediated suppression of DNM2 expression may block JEV-induced vesicle scission, thereby contributing to the antiviral effect of miR-124. However, miR-124 may also target other host regulators, and their roles in JEV replication may have yet to be evaluated.
Methods
Cell culture, transfection and viral infection
PK15 and baby hamster kidney (BHK-21) cell lines (obtained from the American Type Culture Collection, Manassas, VA) were grown in Dulbecco’s Modified Eagle Medium (DMEM, High glucose, Thermo Scientific Hyclone, Beijing, China) supplemented with 10 % fetal bovine serum (Gibco, Life Technologies, Austin, TX) and maintained in a humidified incubator at 37 °C and 5 % CO2.
The miRNA mimics and siRNA (siDNM2) were synthesized by GenePharma (Shanghai, China). The sequences of siDNM2 and mimics were as follows: siDNM2 5’-GGACAUGAUCCUGCAGUUTT-3’ [
49], miR-124 mimics: 5’- UAAGGCACGCGGUGAAUGCCA-3’, NC (Negative control): 5’-UUCUCCGAACGUGUCACGUTT-3’. PK15 cells were seeded in 24- or 6-well plates and grown to approximately 50 % confluence for transfection. The cells were transfected with 50 pmol miRNA mimics or siDNM2 using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The cells were cultured at the indicated times before harvesting.
The JEV attenuated strain SA14-14-2 (GenBank accession: AF315119.1) was propagated in BHK-21 cells as described previously [
8]. All infections were carried out by incubating the cells with a small volume of medium (300 μL in per well of 6-well cell culture plate) containing virus at the MOI = 1, then the inoculum was removed, the cells were washed three times with Phosphate Buffered Saline (PBS, Thermo Scientific Hyclone, Beijing, China) and fresh media was added. Infections were performed and the infected cells were maintained in DMEM supplemented with 2 % FBS.
Total RNA was isolated using TRIzol reagent according to the manufacturer’s instructions. Genomic DNA was removed from RNA samples by incubation with DNase I (Promega). The synthesis of cDNA was conducted with 1 μg of total RNA using an RT-PCR reagent kit (PrimeScript™ 1st Strand cDNA Synthesis Kit, TaKaRa Biotechnology, DaLian, China) with an oligo-dT primer for mRNA and a special stem-loop primer for miRNAs [
50]. Real-time quantitative PCR was performed on an Stratagene Mx3000P instrument (Agilent), using SYBR Green detection chemistry (SYBR® Premix Ex Taq™ II, TaKaRa). Relative expression levels were calculated by applying the 2
-ΔΔCt method [
51] using reference gene RPL32 for mRNA and U6 snRNA for miRNA relative to the control samples. The PCR program was: 95 °C for 2 min followed by 40 cycles of 95 °C for 10 s, 60°Cfor 20 s, and 72 °C for 15 s followed by melting curve analysis. The sequences of the qPCR primers are listed in Table
1.
Table 1
Primers used for reverse transcription, real-time quantification PCR
miR-124-RT | CTCAACTGGTGTCGTGGAGTCGGCAA | | |
| TTCAGTTGAGTGGCATTC | | |
miR-124-S | TCGGCAGGTAAGGCACGCGGTG | 62 | 64 |
miR-124-A | TCAACTGGTGTCGTGGAGTCGGC | | |
U6_S | GTGCTCGCTTCGGCAGCACATAT | 62 | 106 |
U6_A | AAAATATGGAACGCTTCACGAA | | |
DNM2- S | CCCACTCGCAAGACCAAA | 60 | 132 |
DNM2-A | GATGCGATTGATTCGGGC | | |
RPL32-S | CGGAAGTTTCTGGTACACAATGTAA | 60 | 96 |
RPL32-A | TGGAAGAGACGTTGTGAGCAA | | |
Protein extraction and western blot
Cells were harvested 48 h after transfection, and lysates were prepared using RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS, 1 mM phenylmethylsulfonyl fluoride [PMSF], Beyotime, China). The protein concentration was determined with the BCA Protein Assay kit (Solarbio, China). Equal amounts of protein lysate were separated on 12 % SDS-polyacrylamide gels and transferred to PVDF membranes (Millipore). The membranes were blocked with 5 % nonfat milk in Tris-buffered saline containing 0.1 % Tween-20 (TBST) and then incubated with primary antibodies specific for mouse anti-JEV E, goat anti-Dynamin-2 (sc-6400, Santa Cruz), or rabbit anti-ß-Actin (#4967,1:1000, Cell Signaling) overnight at 4 °C. Membranes were then washed three times and incubated with a horseradish peroxidase-conjugated secondary antibody (Proteintech Group, Wuhan, China) for 1 h at ambient temperature. Finally, protein bands were visualized by addition of the SuperSignal West Pico chemiluminescent substrate (Thermo, Rockford, IL), with β-actin as a control. The mean densities of the protein bands were measured by ImageJ software (National Institutes of Health, Bethesda, Maryland).
Plasmid construction and luciferase reporter assay
Luciferase reporter plasmids were constructed by cloning double-stranded synthetic oligonucleotides (Table
2) containing miRNA target sequences flanked by XhoI and NotI sites into the XhoI/NotI digested psiCHECK-2 Dual-Luciferase Expression Vector (Promega, Madison, WI). All constructs were verified by sequencing using the universal primers listed in Table
2. BHK-21 cells were co-transfected with 200 ng of the psiCHECK2 constructs and 30 nM of the miR-124 mimic or miR-negative control in each well of a 24-well plate. Twenty-four hours after transfection, firefly and Renilla luciferase activities were measured using the Dual-Glo Luciferase assay system (Promega) according to the manufacturer’s instructions. Normalized data were calculated as the quotient of Renilla/firefly luciferase activities. Three independent experiments were performed in triplicate.
Table 2
Primers used for luciferase reporter gene vector construct
DNM2-3UTR-S |
CA
CTCGAGAGAAACAGCACCAAGGGAG | 62 | 412 |
DNM2-3UTR-A |
TA
GCGGCCGCCAGGCACTTACAGTGGTCAA | | |
DNM2-3UTR-Mut1-A | GGACCTGGCATTCCGTGCTCTGGCAGCCGAGGCTG | | |
DNM2-3UTR-Mut1-S | GCTGCCAGAGCACGGAATGCCAGGTCCGGAAAACC | | |
DNM2-3UTR-Mut2-A | TAGCGGCCGCCGTGCCTTTACAGTGGTCAAACCGA | | |
JEV-E-S |
CA
CTCGAGAACTCAAAGGTGCTGGTC | 62 | 387 |
JEV-E-A |
TA
GCGGCCGCACCTCCTGTGGCTAAGAA | | |
JEV-NS4B-S |
CA
CTCGAGGTCTTGACCGTGGTTGGA | 60 | 378 |
JEV-NS4B-A |
TA
GCGGCCGCCGTTGTGAGGGTGACTTG | | |
Universal primer-S | CCACTTCAGCCAGGAGGA | 60 | 216 |
Universal primer-A | GGTCCGAAGACTCATTTAGA | | |
Flow cytometry
PK15 cells infected with JEV were washed one time with PBS, detached and transferred to 1.5 ml centrifuge tubes. The cells were centrifuged at 1000 rpm/min for 10 min and fixed with 4 % paraformaldehyde (Solarbio, Beijing, China) for 15 min at room temperature. After being permeabilized with Triton X-100(amresco, Solon, OH), the cells were incubated with mouse anti-JEV E antibody overnight at 4 °C. The cells were washed 3 times with PBS then incubated with Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) at 1:200 for 1 h at room temperature and protected from light. The cells were washed with PBS, resuspended in 500 μl PBS and analyzed using a FACScan flow cytometer with CellQuest pro software (BD Biosciences, San Jose, CA). The cells were counted as infected if their fluorescence density was greater than the intensity of the uninfected cells. The amount of infected cells relative to the untreated or siCtrl-transfected controls was given as percent infection. At least 10,000 cells were analyzed per sample. Three independent experiments were performed in triplicate.
Statistical analysis
The results were presented as the mean ± standard deviation (SD). Statistical significance was assessed by Student’s t-test, and statistical significance was ascribed when *p < 0.05, **p < 0.01.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (31501921, 31372302), the Zhejiang Provincial Natural Science Foundation of China (LQ15C170001) and the Research Development Foundation of Zhejiang A&F University (2014FR068). We acknowledge Professor Shengbo Cao (Huazhong Agriculture University, Wuhan, China) for generously providing the JEV E protein antibody and JEV attenuated strain.