Cellular miR-130b inhibits replication of porcine reproductive and respiratory syndrome virus in vitro and in vivo

MicroRNAs (miRNAs) can impact viral infections by binding to sequences with partial complementarity on viral RNA transcripts, usually resulting in the repression of virus replication. In the present study, we identified a potential binding site for miR-130 in the 5′ untranslated region (bps 155-162) of the porcine reproductive and respiratory syndrome virus (PRRSV) genome. We found that the delivery of multiple miR-130 family mimics, especially miR-130b, resulted in inhibition of PRRSV replication in vitro. miR-130 was effective in inhibiting the replication of multiple type 2 PRRSV strains, but not against vSHE, a classical type 1 strain. miR-130 over-expression did not induce IFN-α or TNF-α expression in either uninfected or PRRSV-infected porcine alveolar macrophages. Results from luciferase reporter assays indicated that miR-130 directly targeted the PRRSV 5′ UTR. Intranasal inoculation of piglets with miR-130b exhibited antiviral activity in vivo and partially protected piglets from an otherwise lethal challenge with HP-PRRSV strain vJX143. Overall, these results demonstrate the importance of the miR-130 family in modulating PRRSV replication and also provide a scientific basis for using cellular miRNAs in anti-PRRSV therapies.

gene regulation in PRRSV pathogenesis [18][19][20][21][22][23][24] . Our previous study identified that mir-26a could suppress PRRSV replication by activating the type I interferon pathway 23 . Here we sought to identify additional antiviral miRNAs by computational analysis of the PRRSV genome. We found that miR-130 family members directly target the 5′ UTR of the PRRSV genome and inhibit viral replication both in vitro and in vivo. Over-expression of miR-130b inhibited multiple type 2 strains in a dose-dependent manner, but had no impact on the replication of type 1 strains. Our study reveals an example of an miRNA that modulates PRRSV replication and also highlights a host factor that could be used for RNAi-mediated antiviral therapeutic strategies.

Results
MiR-130 family members inhibit PRRSV replication. We used ViTa 25 to predict miRNA target sites in the genomes of the HP-PRRSV strain vJX143 and the classical PRRSV strain vAPRRS. The results indicated that miR-130 might target bps 155 to 162 in the viral genomic RNA through seed base pairing (Fig. 1A). We aligned the target sequences in 24 representative PRRSV strains, covering two genotype and different virulence. The target region was 100% conserved in 21 type 2 strains, which now circulate in most commercial swine industries throughout the world, but was absent in 3 type 1 strains (Fig. 1B). Since all members of the same miRNA family 4,26 (i.e., miRNAs with the same sequence at nucleotides 2 to 8) share the same predicted targets, we synthesized all the five members of miR-130 family, including miR-130a, miR-130b, miR-301a, miR-301b, and miR-454 (Table 1) based on the mature miRNA sequences annotated in miRBase (http://www.mirbase.org/) 27 . The first nucleotide at the 5′ end of miR-130 family members, except for miR-454, is C (Table 1) and the C is also complementary to the G in the viral genome (taking miR-130b as an example) (Fig. 1A), suggesting that the binding site length of miR-454 is one nucleotide shorter than that of other members.
We transfected chemically modified miR-130 mimics and mutants (80 nM miRNA) into MARC-145 cells, infected these cells with PRRSV vJX143 at an MOI of 0.01, and then examined virus production and viral gene expression by using virus titer assays and quantitative real-time PCR (qRT-PCR). MARC-145 cells transfected with miR-130 mimics yielded significantly lower PRRSV titers and ORF7 gene expression, as compared with cells transfected with the NC mimic ( Fig. 2A,B) at 36 h post-infection. Transfection of miR-130 mutants had no significant impact on ORF7 RNA levels and virus titers in MARC-145 cells (Fig. 2C,D), indicating that the miR-130 family has antiviral activity against PRRSV. miR-130a/b seemed to be more efficient suppressors than miR-301a/b ( Fig. 2A-D).
Since the miR-130 family is highly conserved between monkeys and pigs, we conducted the subsequent investigations in PAMs, which are the target cells of PRRSV infection in vivo. We analyzed the growth dynamics of HP-PRRSV isolate vJX143 in PAMs transfected with miR-130 family or NC mimics   IgG. Cellular nuclei were counterstained with DAPI (1 mg/ml). Data are the mean + standard deviation of three independent experiments. Statistical significance was analyzed using t-tests; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant. and found that miR-130b was the most efficient at suppressing viral growth, as compared with the other four members (Fig. 2E). Viral growth was suppressed about 1,000-fold in PAMs transfected with miR-130b during PRRSV infection after 72 h and about 100-fold in PAMs transfected with miR-454. These data were corroborated using an immunofluorescence assay in which we found that transfecting miR-130b reduced N protein expression in both PAMs and MARC-145 cells (Fig. 2F).
MiR-130b inhibits multiple type 2 strains in a dose-dependent manner, but does not inhibit a type 1 strain. To corroborate our findings above with miR-130b further, MARC-145 cells were transfected with increasing concentrations of miR-130b mimics (20, 40, 80 nM) and then infected with vAPRRS. Both PRRSV growth and ORF7 mRNA levels were inhibited as a function of the dose of miR-130b mimic (Fig. 3A,B). Consistent with this, transfecting the miR-130b mimic also reduced the accumulation of the PRRSV nucleocapsid (N) protein in a dose-dependent manner (Fig. 3C). To exclude the possibility that reduced PRRSV replication was due to potential toxicity of the miR-130b mimic, MARC-145 cells were transfected with the miR-130b mimic at different doses (40 nM, 80 nM, and 160 nM). No appreciable effect of the miR-130b mimic on cellular viability or morphology was observed (data not shown). The potential target region of miR-130b is 100% conserved in 21 type 2 strains, but is absent in 3 type 1 strains. We further investigated the antiviral activity of miR-130b against three other type 2 PRRSV (vAPRRS, vJX143, vJXM100) strains and a classical type 1 PRRSV strain (vSHE) in MARC-145 cells. Over-expression of the miR-130b mimic reduced virus titers and ORF7 gene expression in the 3 type 2 PRRSV strains, but not in vSHE (Fig. 4A,B). miR-130b had no impact on N protein expression from vSHE ( Fig. 4C,D).  MiR-130 family members directly target the PRRSV genome. We then determined whether the miR-130 family specifically targets the PRRSV genome to exert its antiviral effect (Fig. 1A). Among 20 different vectors containing various PRRSV cDNA fragments, only the relative luciferase activity driven by pGL3-5UTR was significantly reduced by the miR-130b mimic, as compared with cells transfected with the NC mimic (Fig. 5A). To verify further that the direct target sites (bp 155 to 162) in the viral genomic RNA were involved in the inhibition of PRRSV replication, we generated another reporter construct with mutations at positions corresponding to the miR-130 seed region (pGL3-5UTR-mut; Fig. 5B). All 5 miR-130 family members significantly inhibited the luciferase activity of pGL3-5UTR but did not suppress pGL3-5UTR-mut activity (Fig. 5C). Reciprocally, the 5 miR-130 mutants significantly inhibited the luciferase activity of pGL3-5UTR-mut but did not affect pGL3-5UTR activity (Fig. 5D). Thus, the miR-130 family directly targets the PRRSV genome.

MiR-130b does not affect IFN-α and TNF-α mRNA expression in
Intranasal delivery of miR-130b exhibits antiviral activity in vivo. Finally, to confirm whether the miR-130 family could be used in therapy, we tested the anti-PRRSV effects of miR-130b in piglets. The rectal temperature of each pig was recorded daily until 21 dpi. Pigs treated with miR-130b did not experience elevated rectal temperatures until 10 dpi, while the temperature of pigs in the NC treatment group rapidly rose to 40 °C at 3 dpi (Fig. 6A). Comparison of the mean rectal temperatures of the two groups revealed a temperature about 1 °C lower in the miR-130b treatment group, as compared with the NC treatment group (Fig. 6B). The lungs of pigs in the miR-130b treatment group showed fewer pathological changes than piglets in the NC treatment group (Fig. 6C). As a highly virulent strain, vJX143 typically causes 100% mortality by 10 dpi. However, 75% of piglets treated with miR-130b survived to 21 dpi (Fig. 6D). PRRSV RNA copy number was analyzed at 3, 7, 10, 14 and 21 dpi and was found to be about 1,000-fold lower in the miR-130b treatment group, as compared with the NC group (Fig. 6E).
Thus, it appears that PRRSV replication in vivo can be controlled by miR-130b.

Discussion
Given the breadth of miRNA-mediated regulation of host-virus interaction, the role of cellular miRNAs in PRRSV infection is of significant interest. In the current study, we found that miR-130 family members were strong inhibitors of PRRSV replication (Figs 1 and 2). Over-expression of miR-130b inhibited the replication of multiple type 2 PRRSV isolates in a dose-dependent manner (Figs 3 and 4). The antiviral activity of miR-130 was attributable to the direct targeting of the PRRSV 5′ UTR, rather than to a stimulation of IFN-α and TNF-α expression (Figs 4 and 5). Intranasal delivery of miR-130b exhibited antiviral activity in vivo and provided partial protection to piglets from an otherwise lethal challenge (Fig. 6).
Many computational and experimental approaches have shown that UTRs are common miRNA targets 4 . Previous studies reported that both miR-181 and miR-23 inhibited PRRSV replication by directly binding to the 3′ UTR of viral genomic and subgenomic RNAs 19,21 . We found that miR-130 instead targets the 5′ UTR of PRRSV (Fig. 1A). This region is highly conserved among type 2 PRRSV, which now circulates in most commercial swine industries through out the world 30 , but is absent in type 1 PRRSV (Fig. 1B).
The results from vJX143 growth assays in PAMs showed that miR-130a and miR-130b were more efficient at suppressing viral growth than were miR-301a, miR-301b, and miR-454 (Fig. 2E). The five miR-130 family members share the same seed region (2 ~ 8 nt at the 5′ end), but cause different inhibitory effects on PRRSV growth. We found that the first nucleotide at the 5′ end of miR-454 is different from other members (Table 1) and the binding site length of miR-454 is one nucleotide shorter, possibly accounting for the weak inhibition of PRRSV growth by miR-454.
Two previous studies implicated the miR-130 family in regulating the innate immune system by targeting TNF-α in cervical cancer cells 28 and by triggering the type I IFN pathway in the Huh-7.5.1 cell line 29 . By contrast, we found no significant change in either IFN-α and TNF-α expression in PAM cells transfected with miR-130b mimics (Fig. 4E), suggesting the miR-130b antiviral activity is more likely attributable to its direct targeting of viral RNA (Fig. 5). We attributed one reasonable cause of this finding to cell specificity. PAM cells as normal macrophage cells showed different behavior to cancer cells against the miR-130b over-expression. Also one miRNA could commonly modulate hundreds of target genes in mammals due to the short length of binding sites, always 8-10 nucleotides 26 . MiR-130 family still might affect other target genes in PAMs which need further experiments to identify. MiRNAs are considered to have potential efficacy as antiviral therapeutics 31 . The HP-PRRSV strain can cause high fever, high morbidity, and high mortality in pigs 30,32 . The intranasal inhalation delivery route has been used in many siRNA-mediated therapies against other respiratory viruses 33-35 . Guo et al. provided the only direct evidence that therapeutic miR-181c delivery could reduce the severity of infection in pigs infected with HP-PRRSV, but still cause 100% mortality 21 . In our study, intranasal delivery of miR-130b exhibited antiviral activity in vivo and provided partial protection to piglets from an otherwise lethal challenge of vJX143. Developing miR-130b as an antiviral therapeutic approach must be rather limited against those strains that have homology and emergence of viral resistance to the miRNA due to mutations in the target sequence is also possible. However, the animal experiments on miRNAs is opening exciting avenues for understanding host-pathogen interactions and for developing therapeutic approaches to treatment of infectious diseases, viral infections in particular.
Overall, our study demonstrates the importance of the miR-130 family in modulating PRRSV replication and also highlights the therapeutic possibility of using miRNAs to control PRRSV infections.

RNA isolation and qRT-PCR. Total intracellular RNA was isolated using TRIzol reagent (Invitrogen)
according to the manufacturer′ s instructions. PrimeScript TM 1st Strand cDNA Synthesis Kit (Takara) was used for reverse transcription. Quantitative RT-PCR (qPCR) analysis was performed using a Step-one Plus real-time PCR system (Applied Biosystems). The levels of ORF7 RNA, IFN-α and TNF-α mRNA were quantified using a SYBR Premix Ex Taq TM (Takara). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an endogenous control and all primers above are listed in Table 2. For detection of the miR-130b expression, a commercial miRcute miRNA First-Strand cDNA Synthesis and a miRcute miRNA qPCR Detection kit (TIANGEN) were used for reverse transcription and measuring miRNA abundance. The ubiquitously expressed U6 small nuclear RNA (TIANGEN) was used for normalization purpose. All PCR experiments were performed in triplicate and relative expression levels were analyzed using the Δ Δ Ct method 44 . Luciferase assays. Twenty pGL3 luciferase reporter plasmids containing different cDNA fragments encompassing the PRRSV genome were cloned downstream of the luciferase ORF 23 and used for luciferase assays to test and verify predicted miR-130 target regions in the PRRSV genome. For luciferase reporter assays, subconfluent BHK-21 cells cultured in 12-well plates were co-transfected with 500 ng/ well of the indicated reporter plasmid and 100 ng/well of pRL-CMV (as an internal control to normalize  transfection efficiency, Promega) along with the indicated amount of miR-130b or NC mimic. Cells were lysed 24 h later for determination of firefly luciferase activities using the Luciferase assay system (Promega). Data are presented as the relative luciferase activities in miR-130b mimic-transfected cells relative to NC mimic-transfected controls and are representative of three independent experiments. To generate a miR-130 target-mutated reporter construct (pGL3-5UTR-mut), mutations at positions corresponding to the miR-130 seed region were introduced using PstI restriction site and mutation PCR. Mutant plasmid (pGL3-5UTR-mut) was confirmed by DNA sequencing and the primer sequences are listed in Table 2. Luciferase activity in BHK-21 cell lysates were determined as described above except that miR-130 family mimics or mutants were cotransfected into cells with two reporter constructs (PGL3-5UTR or pGL3-5UTR-mut).
Animal experiments with intranasal delivery of miR-130b. The animal welfare committee of the Shanghai Veterinary Research Institute approved the animal experiments. The approve ID is SYXK-2011-0116. All animal studies were carried out in accordance with the approved guidelines and blinded to remove investigator bias. Twelve 4-week-old PRRSV-free piglets were obtained and divided randomly into three groups, i.e., four piglets in each group. Each treatment group was housed individually. We administered miR-130b or NC mimic (6 mg per dose) mixed with RNAi-Mate (GenePharma) in Opti-MEM ® I (Invitrogen) solution in a final volume of 2.5 ml intranasally to the piglets, and inoculated intranasally with 3 ml of diluted vJX143 (1 × 105 TCID 50 ) 6 h later, simulating the natural route of PRRSV infection. At 5 day post-infection (dpi), second deliveries of miR-130b or NC mimics were performed using the half dose and same route. The rectal temperature of each piglet was monitored daily until 21 dpi. Viral genomic RNA in serum samples from each piglet was isolated using a QIAprep viral RNA minikit (Qiagen) and viral load was detected at 3, 7, 10, 14, and 21 dpi using one-step RT-PCR. Specific primers for quantitative analysis of viral RNA copies and listed in Table 2.

Statistical analysis.
All experiments in figures were performed with at least three independent experiments. The appropriate statistical analyses were used and are presented in each figure legend. A P value of less than 0.05 was considered significant.