miRNAs, which are extensively expressed in various organisms, comprise a class of endogenous non-coding RNAs and post-transcriptionally regulate gene expression via translational repression and/or degradation of mRNAs by binding to complementary sequences in the 3′ untranslated region (UTR) of mRNAs [1
]. MiRNAs are initially transcribed as long pri-miRNAs in the nucleus and then processed by the RNase III enzyme Drosha in the nucleus and Dicer in cytoplasm to be mature ~22 nt miRNAs [7
]. To date, >10,000 miRNAs have been annotated in 96 species [8
], including >1000 human miRNAs [9
]. It is believed that >50% of host mRNAs are regulated by one or more miRNAs, and individual miRNAs regulate >100 mRNAs [5
]. miRNAs are involved in almost all biological processes in eukaryotes, including cell differentiation, apoptosis and immunity [10
]. In addition, miRNAs are also involved in the modulation of gene expression and replication of viruses and play a pivotal role in host-virus interactions [1
]. Therefore, the study of miRNA-mediated host-virus interactions is important to understand the mechanisms of virus infection and host counteraction [14
Members of the family Rhabdoviridae, such as spring viremia of carp virus, infectious hematopoietic necrosis virus, viral hemorrhagic septicemia virus and Siniperca chuatsi rhabdovirus, have caused serious diseases in a wide variety of species in aquaculture [15
]. In 2014, a disease outbreak occurred in hybrid snakehead fish cultured at a farm in Guangdong province, China. From the diseased snakehead fishes, a virus was isolated and determined to be Vesiculovirus via genome sequencing. The genome of snakehead Vesiculovirus (SHVV) is about 11 kb in length and encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase protein (large protein, L) [18
]. In addition to the genome sequence, a snakehead fish cell line SSN-1, but not a zebrafish cell line (ZF4), was able to support efficient replication of SHVV [18
]. However, the pathogenic mechanism of SHVV infection is still poorly understood. Therefore, we aimed to identify the miRNA expression profile of SSN-1 cells upon SHVV infection in this study. In addition, several miRNAs of interest were chosen, and their roles in SHVV infection were further investigated.
Although miRNAs are widely expressed in a variety of organisms, their expression profiles vary greatly among tissues [21
]. For example, miR-122 is highly expressed in hepatocytes, but is absent in almost all other cells [22
]. Therefore, different miRNA expression profiles might be acquired when we use different tissues from the same organism. The miRNA expression profile of several teleost fishes has recently been identified in different tissues, including bighead carp and silver carp [23
], common carp [24
], blunt snout bream [25
] and epithelial cells of carp [14
]. Since SHVV could infect and replicate in several tissues of snakehead fish [18
], we choose SNN-1 cells, which are derived from a whole snakehead fry fish [26
], to study the differential miRNA expression profile of snakehead fish upon SHVV infection.
Upon infection, viruses encounter a large number of miRNAs expressed in host cells. Differentially-expressed miRNAs pre- and post-virus infection may be associated with viral infection [27
]. In this study, we identified 143 known and seven novel differentially-expressed miRNAs in SSN-1 cells upon SHVV infection at 24 h poi. Interestingly, all of the 143 known differentially-expressed miRNAs were downregulated (Table S3
). However, two of the seven novel miRNAs were upregulated and five were downregulated (Table 1
), and the reasons are not known. Functional confirmation of the 150 differentially-expressed miRNAs in SSN-1 cells to facilitate or repress virus multiplication is needed.
Multiple reports have revealed that miRNAs regulate virus replication by targeting key elements of innate immunity. miR-136 regulates host antiviral innate immunity by acting as an immune agonist of retinoic acid-inducible gene 1 (RIG-1) [28
]. miR-146 was identified as a regulator of enterovirus replication by targeting key elements of the MyD88 signal pathway [29
]. In addition, miRNAs also mediate antiviral defense by targeting viral transcripts. miR-122 stimulates translation of hepatitis C virus RNA through interaction with the 3′ UTRs of viral genome [30
]. miR-181 can directly impair porcine reproductive and respiratory syndrome virus replication via specifically binding to a conserved region in the downstream of open reading frame 4 of the viral genomic RNA [31
]. miR-let-7c inhibits H1N1 influenza virus replication by targeting the 3′ UTR of the M1 genome and represses M1 protein expression [32
]. In this study, three downregulated miRNAs that were predicted to target two or three genes of SHVV were selected to determine their effects on virus multiplication. All three miRNAs exhibited significant antiviral properties. In addition to binding to the SHVV genome, these three miRNAs were predicted to target tens of host genes (data not shown). Some of the predicted host genes are involved in antiviral signal pathways. Therefore, it is possible that these miRNAs utilize both direct and indirect ways to suppress SHVV multiplication. Further studies are needed to understand the exact mechanisms of the inhibition.
The miRNA was not exclusively host miRNA, many viruses can encode viral miRNAs, which not only facilitate viral replication, but also help the virus to counteract host immune response. Based on the ability of viral miRNA production, viruses can be divided into three categories: the herpesviruses, which encode multiple viral miRNAs; the nuclear DNA viruses, which may encode one or two miRNAs; the RNA viruses and cytoplasmic DNA viruses, which appear to lack any miRNAs [7
]. In this study, no miRNA encoded by SHVV was identified. The probable reason behind this seems to be that SHVV is an RNA virus.
4. Experimental Section
4.1. Virus, Cells and RNA Preparation
SHVV was isolated from diseased hybrid snakehead fish and stored at −80 °C. SSN-1 cells, kindly provided by Hong Liu from Shenzhen Animal & Plant Inspection and Quarantine Technology Center, were cultured in minimum essential medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Auckland, New Zealand) and penicillin (100 µg/mL), streptomycin (100 µg/mL) at 25 °C. To prepare RNA sample, SSN-1 cells were grown on four 6-well plastic dishes (Costar, Corning, NY, USA). Two 6-well plastic dishes was infected with 0.1 multiplicity of infection (MOI) of SHVV; the other two were used as uninfected controls with an equal volume of PBS. At 3 and 24 h poi, SHVV infected and non-infected SSN-1 cells were collected in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) for the total RNA extraction according to the manufacturer’s instructions.
4.2. Small RNA Library Construction
The RNA samples were subjected to 15% polyacrylamide gel electrophoresis (PAGE) for the enrichment of molecules with a length of 17–30 nt [14
]. After 5′ and 3′ adaptors were added, RT-PCR with adaptor-specific primers was used to generate a small RNA library from SHVV-infected and non-infected SSN-1 cells at 3 and 24 h poi. The small RNA library was then sequenced by illumine Hiseq2500.
4.3. Sequencing Data Analysis
The raw reads were counted and the clean reads of four samples, including SSN-1 cells (3 h), SSN-1 cells infected with SHVV (3 h), SSN-1 cells (24 h) and SSN-1 cells infected with SHVV (24 h), were acquired by excluding low-quality reads, 3′ adapter reads, 5′ adapter reads and poly(A) sequences. The length distribution of the clean reads was calculated, and the clean reads were aligned with the genome of zebra fish. Conserved miRNAs were identified by aligning the small RNAs of the four samples to the Zebrafish miRNAs data in miRBase Version 20.0 [33
4.4. Identification of Novel miRNAs
MiRDeep2 software was used to identify potential novel miRNAs from the four samples. Overlapping sequences were used to form longer sequences according to their alignments to known precursor sequences in the miRBase. The differentially-expressed novel miRNAs were summarized between SHVV-infected and non-infected SSN-1 cells.
4.5. Analysis of Differentially-Expressed miRNAs
The identified miRNAs in the four samples were analyzed to identify differentially-expressed miRNAs between SHVV-infected and non-infected SSN-1 cells. The fold change higher than 2 and a p-value <0.05 were the criteria to select differentially-expressed miRNAs.
4.6. Prediction of miRNA Targets on the SHVV Genome
The miRNA targets on the SHVV genome were predicted using Miranda software [34
4.7. RT-PCR and qRT-PCR Analysis of miRNAs
The predicted novel miRNAs were validated by RT-PCR as described previously [14
]. Five randomly-selected miRNAs were detected by qRT-PCR using the same RNA samples used for the construction of the miRNA library. Five forward primers were designed based on mature miRNA sequences (Table S5
). A 20-bp segment at the 3′ end of the 5S rRNA gene was amplified as an endogenous control to normalize template amounts. Quantitative PCR reactions were conducted in 20-µL volumes containing 1 µL diluted cDNA, 300 nM of each primer and 10 µL of the SYBR Master Mix with the following cycling conditions: 95 °C for 5 min, 45 cycles at 95 °C for 10 s, 58 °C for 10 s and 72 °C for 15 s, and ended with a 95 °C at 5 °C/s calefactive velocity to make the melt curve. All expression levels were normalized to the arithmetic mean of the selected 5S ribosomal RNA gene. Amplification results were analyzed using a comparative Ct
method, which uses a formula 2−ΔΔCt
to achieve results for relative quantification. Ct
represents the threshold cycle.
4.8. Effects of Three Randomly-Selected Differentially-Expressed miRNAs on Virus Multiplication
SSN-1 cells in 12-well plates were transfected with 100 nM mimics or 50 nM inhibitors of miR-130c-5p, miR-214, miR-216b, as well as mimic control or inhibitor control using Lipofectamine 2000 (Invitrogen, USA). The inhibitor is the single-strand RNA that is completely paired to miRNAs. At 24 h post-transfection, the cells were infected with 0.1 MOI of SHVV for 24 h. The cells were harvested to determine the effects of three miRNA mimics or inhibitors on virus mRNA expression by qRT-PCR and protein expression by Western blotting.