MicroRNA let-7f-5p Inhibits Porcine Reproductive and Respiratory Syndrome Virus by Targeting MYH9

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens in the swine industry. Current antiviral strategies do not effectively prevent and control PRRSV. Recent reports show that microRNAs (miRNAs) play vital roles in viral infections by post transcriptionally regulating the expression of viral or host genes. Our previous research showed that non-muscle myosin heavy chain 9 (MYH9) is an essential factor for PRRSV infection. Using bioinformatic prediction and experimental verification, we demonstrate that MYH9 expression is regulated by the miRNA let-7f-5p, which binds to the MYH9 mRNA 3′UTR and may play an important role during PRRSV infection. To understand how let-7f-5p regulates PRRSV infection, we analyzed the expression pattern of both let-7f-5p and MYH9 in porcine alveolar macrophages (PAMs) after infection with either highly pathogenic PRRSV (HP-PRRSV) or classical type PRRSV (N-PRRSV) using a deep sequencing approach with quantitative real-time PCR validation. Our results showed that both HP-PRRSV and N-PRRSV infection reduced let-7f-5p expression while also inducing MYH9 expression. Furthermore, let-7f-5p significantly inhibited PRRSV replication through suppression of MYH9 expression. These findings not only provide new insights into the pathogenesis of PRRSV, but also suggest potential new antiviral strategies against PRRSV infection.


Results
Prediction and identification of the miRNAs which regulate the MYH9 gene. Our previous studies indicate that MYH9 plays an important role in intercellular viral spread. In this study, we further investigated miRNAs which regulate MYH9 expression at the posttranscriptional level. MicroRNA prediction algorithms generally pair the "seed region" of the microRNA to a specific mRNA 3′ UTR sequence. To predict miRNAs which can bind to pig and monkey MYH9 3′ UTRs, computer-based sequence analysis using RNAhybrid and Segal Lab online microRNA prediction tools were used. We found that one pig miRNA and three monkey miRNAs exhibited potential 3′ UTR binding sites to pig and monkey MYH9 mRNAs, respectively. The miRNA identified as potentially targeting pig MYH9 mRNA was let-7f-5p, whereas miRNAs potentially targeting monkey MYH9 mRNA were let-7f-5p, mml-miR-638, and mml-miR-346.
To address the question of whether the candidate miRNAs could potentially regulate MYH9 expression, the 3′ UTRs of pig and monkey MYH9 were cloned downstream of the luciferase ORF in a reporter vector (Fig. 1a). The reporter plasmids containing either pig MYH9 3′ UTR (psiCheck2-pMYH9-WT) or monkey 3′ UTR (psi-Check2-mMYH9-WT) were transfected into HEK293FT cells along with synthetic various miRNA mimics or a negative control mimics, and luciferase activity was measured. As shown in Fig. 1b, luciferase activity was downregulated in the presence of either let-7f-5p or mml-miR346 mimics, resulting in respective decreases of 62% and 58% in luciferase expression relative to psiCheck2-pMYH9-WT expression. Downregulation of luciferase activity was not observed with overexpression of mml-miR-638 or the negative control (NC). Only let-7f-5p mimics significantly decreased luciferase expression (by 56%) relative to psiCheck2-mMYH9-WT (Fig. 1c). Notably, let-7f-5p dramatically inhibited luciferase expression for both psiCheck2-pMYH9-WT (Fig. 1b) and psiCheck2-mMYH9-WT (Fig. 1c). These observations, coupled with the fact that let-7f-5p is well conserved among different host species (Fig. 1d), led us to choose let-7f-5p for further validation studies.
To further demonstrate the direct interaction between let-7f-5p and MYH9 mRNA, immunoprecipitation (IP) analysis was carried out using anti-Ago2 antibody and cell lysates. PAMs were mock-infected or infected with the GD-HD strain at an MOI of 0.1. After 12 hours, Ago-2-bound entities within lysates from PRRSV-infected or non-infected PAMs were immunoprecipitated with anti-Ago2 monoclonal antibody. Next, let-7f-5p miRNA and MYH9 mRNA in the immunoprecipitates were detected by qRT-PCR. As shown in Fig. 4a, the let-7f-5p level associated with Ago2 protein was increased by 30.8-fold and 7.8-fold compared to that of the IgG isotype control in mock-infected and PRRSV-infected cells, respectively. However, the abundance of MYH9 mRNA increased by approximately 2.1-fold and 3.5-fold in mock-infected and PRRSV-infected cells, respectively, compared to that of the IgG isotype control (Fig. 4b). These results demonstrate that let-7f-5p directly interacts with MYH9 mRNA in the RISC.
Scientific RepoRts | 6:34332 | DOI: 10.1038/srep34332 Let-7f-5p regulates MYH9 expression through both mRNA degradation and translational repression. To further investigate whether let-7f-5p can regulate MYH9 expression, PAMs were transfected with let-7f-5p, NC mimics, siMYH9 (as a positive control), or NC siRNA; the expression of let-7f-5p and MYH9 mRNA were examined at the indicated times. As shown in Fig. 5, expression of let-7f-5p in the various groups was examined by qRT-PCR and normalized against U6 rRNA expression (Fig. 5a). The expression level of MYH9 mRNA in cells transfected with the siMYH9 and let-7f-5p mimics were lower than that of the NC group. Notably, when PAMs were transfected with 100 nM let-7f-5p mimics for 24 h, the expression level of MYH9 mRNA decreased by 55% compared with that of negative controls (Fig. 5b). At the same time, expression of MYH9 protein in PAMs, which were transfected with let-7f-5p mimics, siMYH9, or negative controls for 36 h, was also detected by western blot. Moreover, MYH9 expression in cells treated with let-7f-5p mimics or siMYH9 3′UTR and monkey MYH9 3′ UTR were cloned into 3′ UTR-luciferase reporter psiCheck2 vectors to create plasmids designated psiCheck2-pMYH9-WT and psiCheck2-mMYH9-WT, respectively. (b) HEK293FT cells were cotransfected with indicated mimics and psiCheck2-pMYH9-WT. (c) HEK293FT cells were cotransfected with indicated mimics and psiCheck2-mMYH9-WT. Thirty-six hrs later, cells were lysed and luciferase activity was measured. Results are expressed as mean ± SD of three independent experiments. p values were calculated using the Student's t test. **P < 0.01, ***P < 0.001. (d) Sequence homology of let-7f-5p to human, monkey, and pig.
was markedly reduced compared with negative control (Fig. 5c). Collectively, qRT-PCR and western blot assays revealed that let-7f-5p markedly inhibits the expression of MYH9 at both mRNA and protein levels.
To confirm the effect of let-7f-5p on MYH9 expression, PAMs were transfected with let-7f-5p inhibitor and the negative control, then the expression of let-7f-5p and MYH9 were examined at the indicated times. As shown in Fig. 6, let-7f-5p inhibitor transfection led to a significant reduction in let-7f-5p mRNA expression level (Fig. 6a). When PAMs were transfected with 200 nM let-7f-5p inhibitor for 24 h, the expression level of MYH9 mRNA was increased by 62% compared with that of negative control (Fig. 6b). Similarly, western blotting showed that the level of MYH9 expression was increased by transfection of 200 nM let-7f-5p inhibitor relative to that achieved by transfection of a negative control inhibitor (Fig. 6c). The 3′ UTR of pig MYH9 contains one seed-matched target site (highlighted) for let-7f-5p. (b) Schematic of the "seed region" match between let-7f-5p and pig MYH9 3′ UTR. The mutation of six nucleotides in the seed match is shown. The positions of six seed match sites for let-7f-5p in the pig MYH9 3′ UTR were replaced. (c) WT or MUT reporter plasmids were cotransfected with the let-7f-5p mimics, let-7f-5p-MUT mimics, or NC mimics into the HEK293FT cells. Reporter activities were determined thirty-six hrs post-transfection by dual-luciferase assays. Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student's t test. ***P < 0.001. (d) Sequence homology of the let-7f-5p binding sequence in the MYH9 3′ UTR of human, monkey, and pig.
Scientific RepoRts | 6:34332 | DOI: 10.1038/srep34332 A let-7f-5p decrease accompanied enhanced MYH9 expression in PRRSV-infected PAMs. In order to elucidate whether PRRSV infection affects the expression of let-7f-5p, microRNA let-7 family member expression in response to PRRSV infection was checked in PAMs. Small RNA deep sequencing was performed using libraries of RNA of mock-infected, HP-PRRSV (GD-HD)-, or N-PRRSV (CH-1a)-infected PAMs each at an MOI of 0.1 at 12 h post-infection (hpi). After deep sequencing data analysis, the numbers of let-7 family member reads of the three small RNA libraries were compared (Table 1). A striking observation was the downregulation of let-7f-5p miRNAs in both HP-and N-PRRSV-infected PAMs. Similar expression patterns were observed for several other let-7 miRNA members (Fig. 7a).
To further confirm the let-7f-5p expression profile associated with PRRSV infection, the levels of let-7f-5p in PAMs were detected using qRT-PCR. Total RNA was isolated from PAMs at the indicated time points and the PCR product of the ORF7 gene, a measure of the replication of the two PRRSV strains, was detected (Fig. 7b,c). Next, miRNA profiles were determined using a stem-loop-primer qRT-PCR. As shown in Fig. 7d,e, let-7f-5p was greatly reduced in PAMs infected with both PRRSV strains as early as 6 hpi and its expression remained lower in the PRRSV-infected PAMs than in mock and UV-inactivated virus controls at 12 hpi and 24 hpi. contains one seed matched target site (highlighted) for let-7f-5p. (b) Schematic of the "seed region" match between let-7f-5p and monkey MYH9 3′ UTR. The mutation of six nucleotides in the seed match is shown. The positions of six seed match sites for let-7f-5p in the monkey MYH9 3′ UTR were replaced. (c) WT or MUT reporter plasmids were cotransfected with the let-7f-5p mimics, let-7f-5p-MUT mimics, or NC mimics into HEK293FT cells. Reporter activities were determined thirty-six hrs post-transfection by dual-luciferase assays. Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student's t test. ***P < 0.001.
Scientific RepoRts | 6:34332 | DOI: 10.1038/srep34332 We next asked the question of whether the downregulation of let-7f-5p was linked to the concomitant upregulation of expression of its target, MYH9 mRNA, in PRRSV-infected PAMs. To investigate this, mRNA profiles were generated from the same set of samples as those used for qRT-PCR. MYH9 was transcriptionally upregulated as early as 6 hpi; it reached peak expression at 12 hpi (3.29-fold), and then its expression decreased gradually as the HP-PRRSV infection progressed (Fig. 7f). The relative expression of MYH9 mRNA was upregulated at 6 hpi and then reached a peak at 24 hpi (2.52-fold) following N-PRRSV infection (Fig. 7g). These results suggest that the downregulation of let-7f-5p likely upregulates MYH9 expression in PRRSV-infected PAMs.

PRRSV replication is suppressed by overexpression of let-7f-5p.
To investigate whether the activation of let-7f-5p affects PRRSV replication through targeting of the MYH9 gene, PAMs were transfected with increasing concentrations of let-7f-5p mimics (20, 50, 100 nM) or NC mimics (100 nM), followed by infection with the GD-HD strain at an MOI of 0.01. At 24 hpi, the cells were collected to determine relative ORF7 expression. PRRSV RNA copy number in cell culture supernatants were detected by qRT-PCR and cell lysates were also analyzed by western blotting. Both PRRSV growth and ORF7 mRNA levels were inhibited in a dose-dependent PAMs were subjected to Ago2-IP, the relative expression of let-7f-5p (a) and MYH9 mRNA (b) in the immunoprecipitates were determined by qRT-PCR. As a negative control, immunoprecipitation was performed using anti-Flag antibody beads (IgG). Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student's t test. *P < 0.05, ***P < 0.001. manner and cells transfected with a higher dose of let-7f-5p displayed a stronger antiviral effect. Notably, 100 nM let-7f-5p could markedly inhibit both MYH9 expression and viral replication, resulting in reduction in virus copy number by 83%, ORF7 mRNA by 80%, and reduction of protein expression levels (Fig. 8a-c).

Let-7f-5p inhibits both HP-PRRSV and N-PRRSV replication.
To further examine viral replication inhibition caused by let-7f-5p, PAMs were transfected with 100 nM let-7f-5p mimics or NC mimics. After 24 hpi with HP-PRRSV (GD-HD) or N-PRRSV (CH-1a) (each at an MOI of 0.01), the relative expression level of MYH9 Figure 5. Overexpression of let-7f-5p decreases MYH9 mRNA and protein levels in PAMs. PAMs were transfected with 100nM of MYH9-siRNA, NC-siRNA, let-7f-5p mimics, and NC-mimics. Relative expression level of let-7f-5p (a) and MYH9 mRNA (b) in PAMs determined by qRT-PCR. MYH9 protein expression (c) analyzed by western blot with α -tubulin as the control. Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student's t test. **P < 0.01, ***P < 0.001. mRNA and intracellular PRRSV ORF7 mRNA, extracellular PRRSV genome RNA, and culture supernatant viral titers were detected at the indicated time points. As shown in Fig. 9a,b, significant decreases were observed in MYH9 mRNA expression after let-7f-5p transfection. Moreover, transfected PAMs contained smaller amounts of PRRSV ORF7 mRNA at 12 hpi, 24 hpi, and 36 hpi, and lower virus genome copy numbers at 6 hpi, 12 hpi, 24 hpi, Figure 6. Inhibition of let-7f-5p enhances MYH9 mRNA and protein expression in PAMs. PAMs were transfected with indicated concentrations of MYH9-siRNA, NC-siRNA, let-7f-5p-inhibitor, and NC-inhibitor. Relative expression level of let-7f-5p (a) and MYH9 mRNA (b) in PAMs determined by qRT-PCR. MYH9 protein expression (c) analyzed by western blot, with α -tubulin as control. Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student's t test. *P < 0.05, **P < 0.01.

Discussion
MYH9, the gene for non-muscle myosin heavy chain 9, codes for a protein that exhibits multiple functions, including roles in cytokinesis, cell motility, and maintenance of cell shape 24 . A recent report demonstrates that MYH9 plays a key role in some virus infections. For example, herpes simplex virus 1 (HSV-1), Epstein-Barr virus (EBV), and PRRSV have been known to utilize MYH9 as a functional receptor that interacts with viral proteins 15,25,26 . Our previous work has shown that the MYH9 expression level apparently increased after PRRSV incubation with host cells 15 . Therefore, we hypothesized that regulatory factors control MYH9 expression.
MicroRNAs belong to a class of small non-coding RNAs which can modulate gene expression across a wide spectrum of biological systems 16 . MicroRNAs have emerged as important regulators in the complex interactions of host and virus at the posttranscriptional level 27,28 . Moreover, a number of cellular miRNAs have been found to regulate PRRSV infection through a variety of pathways [20][21][22] .
In the present study, we sought to determine which miRNAs could regulate MYH9 expression at the posttranscriptional level, while also regulating PRRSV replication. Using bioinformatics prediction and experimental verification, we demonstrated that let-7f-5p is a key regulator of MYH9. Results of luciferase activity assays showed that let-7f-5p regulated luciferase expression by targeting both pig and monkey MYH9 3′ UTR (Fig. 1). To identify let-7f-5p targets regulated through base pairing between the miRNA "seed region" and the 3′ UTR of the pig and monkey MYH9, the microRNA-mRNA relationship was validated using luciferase activity assays (Figs 2 and 3) and Ago2-IP analysis (Fig. 4). In addition, let-7f-5p and its target sites are both conserved among different host species (Figs 1c and 4a). The results show that MYH9 is a direct target for let-7f-5p and that let-7f-5p-mediated suppression of MYH9 is dependent on the MYH9 3′ -UTR sequence.
Previous studies indicated that regulation of gene expression by miRNAs can be explained by two mechanisms that result in either degradation of mRNA or suppression of translation 29,30 . Many miRNAs regulate expression of target genes using both pathways. For example, miR-24-3p downregulates HO-1 expression through both mRNA degradation and translational repression in MARC-145 cells 20 . Our results showed that overexpression of let-7f-5p in PAMs significantly downregulated both mRNA and protein levels of MYH9 (Fig. 5). MYH9 mRNA and protein levels were augmented in cells transfected with let-7f-5p inhibitor compared to cells transfected with control inhibitor (Fig. 6). These results suggest that let-7f-5p might regulate MYH9 expression through both mRNA degradation and translational repression.
For successful establishment of infection within the host, intracellular pathogens such as viruses need to evade the host-immune response, reshape the cellular environment, and utilize host resources to synthesize viral proteins and RNA 31,32 . In the hide-and-seek game between virus and host, viruses have evolved highly sophisticated mechanisms for their propagation and survival 28,33,34 . Increasing evidence suggests that viral infection can influence an expression profile of host miRNAs, either through inhibition of defensive host signaling against viral infection or by viral hijacking of host defenses to favor infection 28,35 . The let-7 family contains some members with highly conserved sequences in species ranging from worms to humans, which share several conserved mRNA targets 17,36 . To understand the expression patterns of let-7 family members during PRRSV infection, we profiled host miRNAs in PAMs during HP-PRRSV and N-PRRSV infections using a deep sequencing approach. PRRSV infection of PAMs by both HP-PRRSV and N-PRRSV strains downregulated the expression of let-7f-5p and several other let-7 family members, including let-7i-5p, let-7d-5p, and let-7g-5p, resulting in greater virus infectivity. Notably, although the members share the same "seed region", let-7 family members are each unique and encoded on different chromosomes. Furthermore, their expression levels and functions differ from one another during virus infection. The expression of let-7f-5p and MYH9 was further validated by quantitative real-time PCR. PRRSV infection markedly downregulated let-7f-5p expression and upregulated MYH9 in PAMs infected with both PRRSV strains (Fig. 7).  Based on the observation that let-7f-5p is downregulated during PRRSV infection, we propose that downregulation of let-7f-5p could, in a context-specific manner, offer an advantage to PRRSV. Collectively, these results demonstrate that there is an inverse correlation between the expression of let-7f-5p and expression of MYH9.  ORF7 (b,c), let-7f-5p (d,e), and MYH9 (f,g) mRNA relative expression levels were determined using qRT-PCR. Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001.
Moreover, we observed that let-7f-5p has a significant impact on PRRSV infection and this result is consistent with previous observations showing that microRNAs are small RNAs with great power; differential regulation by let-7 family miRNAs has previously been shown to participate in regulation of the immune response to many virus infections. For example, let-7b have been reported to inhibit hepatitis C virus (HCV) infectivity by interacting with the HCV genome 37 . Let-7a suppresses Kaposi's sarcoma-associated herpesvirus (KSHV) replication through targeting of MAP4K4 signaling pathways 38 . Let-7g inhibits hepatitis B virus (HBV) preS2 protein expression and generation of viral products 39 . Our results show that let-7f-5p can inhibit viral replication of both HP-PRRSV and N-PRRSV in PAMs (Figs 8 and 9). The potential for one miRNA to regulate numerous mRNAs and for one mRNA to be targeted by multiple miRNAs suggests that there may be other host genes regulated by let-7f-5p that limit PRRSV proliferation. Further research is needed to explore this possibility.
Here we report that MYH9 is the direct target of let-7f-5p, which regulates MYH9 expression via binding to its mRNA 3′ UTR, resulting in the downregulation of MYH9 expression at both the posttranscriptional and translational level. We demonstrate that overexpression of let-7f-5p can inhibit PRRSV replication through suppression of MYH9 expression. These findings reveal an example of a host microRNA that modulates PRRSV replication and also highlights how microRNAs could be used in microRNA-mediated antiviral therapeutic strategies.

Figure 9. Overexpression of let-7f-5p attenuates both HP-PRRSV and N-PRRSV replication in PAMs.
PAMs were transfected with 100 nM let-7f-5p or NC mimics, followed by infection with two PRRSV strains: GD-HD and CH-1a (MOI = 0.01). (a,b) Relative expression level of MYH9 mRNA was evaluated by qRT-PCR. PRRSV replication was evaluated by qRT-PCR for intracellular PRRSV ORF7 RNA (c,d) and extracellular PRRSV genome RNA in the supernatants (e,f), and viral titers in the supernatants were measured by TCID 50 at the indicated time points (g,h). Results are expressed as mean ± SD of three independent experiments. p values were calculated using Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001. Cellular delivery of microRNA mimics, inhibitor, or siRNAs. Cells were cultured in 24-well plates for 12 h and transfected with mimics, inhibitor, siRNAs, or negative control siRNA synthesized by GenePharma (Shanghai, China). X-tremeGENE siRNA Transfection Reagent (Roche, Basle, Switzerland) was used, following manufacturers' instructions. Each treatment was performed in at least triplicate. The sequences of mimics, inhibitor, and siRNAs are shown in Table 2.

Plasmids construction.
To construct the psiCheck-2 (Promega, Madison, WI, USA) target luciferase reporter plasmid, the 802 bp 3′ UTR of pig MYH9 and 1,083 bp 3′ UTR of monkey MYH9 were amplified using PCR of cDNA of PAMs and MARC-145 cells, then cloned into the 3′ UTR of the Renilla luciferase gene using the XhoI and NotI restriction sites of the psiCheck-2 vector. Target mutations were made to the "seed region" of the let-7f-5p binding site in porcine MYH9 (pMYH9) and murine MYH9 (mMYH9), then the oligonucleotides were synthesized, annealed into double strands, and cloned into the psiCheck-2 vector. The primer sequences used for psiCheck-2 luciferase reporter plasmid construction are shown in Table 3.

RISC immunoprecipitation assays.
PAMs were mock-infected or infected with the GD-HD strain at an MOI of 0.1, harvested and lysed using cell lysis buffer (1% NP-40, 150 mM NaCl, 10 mM Tris-HCl, pH 7.8 with 1 mM EDTA) containing a proteinase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), and used for RISC immunoprecipitation assays at 12 hpi. Briefly, protein G Sepharose beads (Invitrogen, Carlsbad, CA, USA) were incubated with rat anti-Ago2 monoclonal antibody (clone 2E12-1C9; Abnova, Taiwan) or with anti-Flag antibody (as an isotype control) and mixed by rotation overnight at 4 °C followed by three washes with cell lysis buffer to remove non-specifically bound material. Cell lysates from PRRSV-infected or non-infected PAMs were added to the beads and incubated for 4 h at 4 °C. RISC-associated RNAs were isolated from the beads using RNAiso   Table 2 and the U6 endogenous control served as an internal standard for quantification. Use the following conditions for the qRT-PCR reactions: 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s, and annealing and elongation at 60 °C for 30 s. Fluorescence emission was measured at the end of each extension step. Relative expression was analyzed using the comparative threshold cycle (Ct) method. To detect relative PRRSV N and MYH9 mRNA levels, total RNA was reverse transcribed using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China) with a mixture of oligo (dT) and random primers, following manufacturer's instructions. Quantitative real-time PCR was performed as described above. The fold changes in target gene expression levels were determined using the comparative threshold cycle (Ct), with HPRT1 as reference (housekeeping control gene). The primers used for qRT-PCR amplification are listed in Table 2.
To determine supernatant PRRSV genome RNA copy numbers, a 372 bp fragment of the PRRSV ORF7 sequence was cloned into the T-tagged site of the pMD18-T vector (TaKaRa, Dalian, China), following manufacturer's instructions. Standard curves were generated from serial ten-fold dilutions of the plasmid constructs. PRRSV RNA genome absolute quantities were calculated by normalization to the standard curve.
Western blot analysis. PRRSV structural protein N or cellular MYH9 protein were detected by western blotting. Briefly, cells were lysed in NP-40 lysis buffer supplemented with protease and phosphatase inhibitor. Proteins were separated on SDS-PAGE gels and then electro-transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% milk in phosphate buffered saline (PBS) with Tween-20 (PBST containing 0.025% Tween-20) at 4 °C for 2 h, and then blotted with the following primary antibodies: mouse anti-PRRSV N protein monoclonal antibody (6D10), mouse antibodies against MYH9 (Sigma-Aldrich, psiCheck2-pMYH9-3UTR-Mut-R 5′ -GGCCGCGGCTGGGGGGGGCAGAGGGTGACTCCACACCGCGTGTGATTGCCAACAGCAC-3′ Table 3. Primers used for qRT-PCR and psiCheck2 vector construction. St. Louis, MO, USA), or anti-α -tubulin antibody (Abcam, Cambridge, MA, USA) for 2 h at room temperature. After washing with PBST three times, the blots were subsequently incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Jackson Laboratories, West Grove, PA, USA) as the secondary antibody. Finally, the proteins were visualized using enhanced chemiluminescence (ECL) reagent (Pierce, Rockford, IL, USA), following manufacturer's instructions.

Virus titration.
To determine cell culture supernatant virus titers, MARC-145 cells were seeded into 96-well plates before virus infection. Cells grown to 80% confluence were infected with serial dilutions of each supernatant containing virus progeny in DMEM and added to each well in replicates of eight. After 2-3 days of incubation at 37 °C, cytopathic effect (CPE) was observed microscopically. The 50% tissue culture infectious dose (TCID 50 ) assay end point was calculated using the Reed-Muench method.
Statistical analysis. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA) and significant differences between the two groups were determined using two-tailed Student's t-tests. P < 0.05 was considered statistically significant.