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• W3155357773 abstract "Porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating pathogen in the swine industry worldwide. miRNAs are reported to be involved in virus–host interaction. Here, we used high-throughput sequencing and miRNA inhibitors to screen possible miRNAs that can inhibit PRRSV infection on its target cell, porcine alveolar macrophages. We observed that miR-218 was downregulated upon virus infection, and knockdown of miR-218 significantly enhanced PRRSV replication. Overexpression of miR-218 resulted in a decrease in PRRSV replication, and this overexpression did not alter viral genomic RNA levels, but rather increased antiviral interferon signaling. Further analysis revealed that miR-218 regulated PRRSV replication by directly targeting porcine suppressor of cytokine signaling 3 (SOCS3), a JAK2 kinase inhibitor. Knockdown of the endogenous SOCS3 expression led to augmentation of type I interferon genes and resulted in decreased PRRSV replication, and vice versa. During PRRSV infection in vivo and in vitro, cellular miR-218 expression was downregulated and SOCS3 expression was upregulated, further supporting the inverse correlation between miR-218 and SOCS3 expression. The data on SOCS3 depletion in combination with miR-218 inhibition suggested that the antiviral activity of miR-218 required the SOCS3-mediated signaling pathway. Similarly, miR-218 negatively regulated PRRSV replication in Marc-145 cells, as well as the replication of porcine epidemic diarrhea virus and transmissible gastroenteritis virus in Vero and ST cells respectively. Taken together, these results demonstrate that PRRSV-induced miR-218 downregulation serves to inhibit the type I interferon response and may provide a novel therapeutic target for treatment of PRRSV and other viral infections. Porcine reproductive and respiratory syndrome virus (PRRSV) is a devastating pathogen in the swine industry worldwide. miRNAs are reported to be involved in virus–host interaction. Here, we used high-throughput sequencing and miRNA inhibitors to screen possible miRNAs that can inhibit PRRSV infection on its target cell, porcine alveolar macrophages. We observed that miR-218 was downregulated upon virus infection, and knockdown of miR-218 significantly enhanced PRRSV replication. Overexpression of miR-218 resulted in a decrease in PRRSV replication, and this overexpression did not alter viral genomic RNA levels, but rather increased antiviral interferon signaling. Further analysis revealed that miR-218 regulated PRRSV replication by directly targeting porcine suppressor of cytokine signaling 3 (SOCS3), a JAK2 kinase inhibitor. Knockdown of the endogenous SOCS3 expression led to augmentation of type I interferon genes and resulted in decreased PRRSV replication, and vice versa. During PRRSV infection in vivo and in vitro, cellular miR-218 expression was downregulated and SOCS3 expression was upregulated, further supporting the inverse correlation between miR-218 and SOCS3 expression. The data on SOCS3 depletion in combination with miR-218 inhibition suggested that the antiviral activity of miR-218 required the SOCS3-mediated signaling pathway. Similarly, miR-218 negatively regulated PRRSV replication in Marc-145 cells, as well as the replication of porcine epidemic diarrhea virus and transmissible gastroenteritis virus in Vero and ST cells respectively. Taken together, these results demonstrate that PRRSV-induced miR-218 downregulation serves to inhibit the type I interferon response and may provide a novel therapeutic target for treatment of PRRSV and other viral infections. Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, single-stranded positive RNA virus, belonging to the family Arteriviridae of the order Nidovirales (1Cavanagh D. Nidovirales: A new order comprising Coronaviridae and arteriviridae.Arch. Virol. 1997; 142: 629-633PubMed Google Scholar, 2Meulenberg J.J. PRRSV, the virus.Vet. Res. 2000; 31: 11-21PubMed Google Scholar). PRRSV is the etiological agent of porcine reproductive and respiratory syndrome (PRRS), which is characterized by reproductive failure in sows and severe respiratory symptoms in piglets and growing pigs. PRRS was first described in the United States in 1987 and in Europe in 1990 (3Collins J.E. Benfield D.A. Christianson W.T. Harris L. Hennings J.C. Shaw D.P. Goyal S.M. McCullough S. Morrison R.B. Joo H.S. Gorcyca D.C. Isolation of swine infertility and respiratory syndrome virus (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs.J. Vet. Diagn. Invest. 1992; 4: 117-126Crossref PubMed Scopus (601) Google Scholar, 4Meulenberg J.J. Hulst M.M. de Meijer E.J. Moonen P.L. den Besten A. de Kluyver E.P. Wensvoort G. Moormann R.J. Lelystad virus, the causative agent of porcine epidemic abortion and respiratory syndrome (PEARS), is related to LDV and EAV.Virology. 1993; 192: 62-72Crossref PubMed Scopus (623) Google Scholar), and since then this disease has spread around most pig-producing countries and has become an economically devastating disease in the swine industry worldwide. To control this disease, researchers have developed different vaccines. However, due to the high antigenic heterogeneity of PRRSV, the use of current vaccines has some limitations (5Rappe J.C. García-Nicolás O. Flückiger F. Thür B. Hofmann M.A. Summerfield A. Ruggli N. Heterogeneous antigenic properties of the porcine reproductive and respiratory syndrome virus nucleocapsid.Vet. Res. 2016; 47: 117Crossref PubMed Scopus (10) Google Scholar, 6Vu H.L.X. Pattnaik A.K. Osorio F.A. Strategies to broaden the cross-protective efficacy of vaccines against porcine reproductive and respiratory syndrome virus.Vet. Microbiol. 2017; 206: 29-34Crossref PubMed Scopus (25) Google Scholar). Therefore, it is worthwhile to explore the immune regulatory molecules against PRRSV infection from the host’s perspective. miRNAs, a class of endogenous noncoding RNAs of ∼22 nucleotides, play important roles in the regulation of gene expression at the posttranscriptional level. miRNAs are initially transcribed from the genome as primary miRNAs and processed into the final single-stranded mature miRNAs through a series of intermediates by biogenesis machinery. Mature miRNAs are then incorporated into RNA-induced silencing complex where miRNAs bind to their target mRNAs and result in mRNA destabilization and/or translational repression (7Dong H. Lei J. Ding L. Wen Y. Ju H. Zhang X. MicroRNA: Function, detection, and bioanalysis.Chem. Rev. 2013; 113: 6207-6233Crossref PubMed Scopus (686) Google Scholar, 8Mohr A.M. Mott J.L. Overview of microRNA biology.Semin. Liver Dis. 2015; 35: 3-11Crossref PubMed Scopus (467) Google Scholar). In animals, the 5'-proximal seed region (at nucleotides 2–8) of miRNAs binds to complementary sequences within the 3'-untranslated region (3'UTR) of the target mRNA (9Eulalio A. Huntzinger E. Izaurralde E. Getting to the root of miRNA-mediated gene silencing.Cell. 2008; 132: 9-14Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar). It is estimated that more than half of the protein coding genes in mammals can be regulated by miRNAs (10Chekulaeva M. Filipowicz W. Mechanisms of miRNA-mediated post-transcriptional regulation in animal cells.Curr. Opin. Cell Biol. 2009; 21: 452-460Crossref PubMed Scopus (524) Google Scholar), thus miRNAs can participate in a series of cellular processes including DNA replication and reparation, cell proliferation and differentiation, and ontogenesis (11Krol J. Loedige I. Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay.Nat. Rev. Genet. 2010; 11: 597-610Crossref PubMed Scopus (3059) Google Scholar, 12Bartel D.P. MicroRNAs: Target recognition and regulatory functions.Cell. 2009; 136: 215-233Abstract Full Text Full Text PDF PubMed Scopus (14172) Google Scholar, 13Vidigal J.A. Ventura A. The biological functions of miRNAs: Lessons from in vivo studies.Trends Cell Biol. 2015; 25: 137-147Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). In addition, miRNAs are also involved in the repertoire of virus–host interactions and affect viral replication (14Barbu M.G. Condrat C.E. Thompson D.C. Bugnar O.L. Cretoiu D. Toader O.D. Suciu N. Voinea S.C. MicroRNA Involvement in signaling pathways during viral infection.Front Cell Dev Biol. 2020; 8: 143Crossref PubMed Scopus (23) Google Scholar, 15Duan X. Wang L. Sun G. Yan W. Yang Y. Understanding the cross-talk between host and virus in poultry from the perspectives of microRNA.Poult. Sci. 2020; 99: 1838-1846Crossref PubMed Scopus (4) Google Scholar, 16Trobaugh D.W. Klimstra W.B. MicroRNA regulation of RNA virus replication and pathogenesis.Trends Mol. Med. 2017; 23: 80-93Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). During virus infection, type Ⅰ interferons (IFNs) with antiviral activity, such as IFN-α and IFN-β, are produced by fibroblasts and monocytes (17Muller U. Steinhoff U. Reis L.F. Hemmi S. Pavlovic J. Zinkernagel R.M. Aguet M. Functional role of type I and type II interferons in antiviral defense.Science. 1994; 264: 1918-1921Crossref PubMed Scopus (1895) Google Scholar). Once released, type I IFNs bind to their cognate receptors on target cells, which activate the Jak-STAT signaling pathway to induce transcription of IFN-stimulated genes (ISGs) (18Schoggins J.W. Rice C.M. Interferon-stimulated genes and their antiviral effector functions.Curr. Opin. Virol. 2011; 1: 519-525Crossref PubMed Scopus (685) Google Scholar, 19Sadler A.J. Williams B.R. Interferon-inducible antiviral effectors.Nat. Rev. Immunol. 2008; 8: 559-568Crossref PubMed Scopus (1398) Google Scholar, 20Schneider W.M. Chevillotte M.D. Rice C.M. Interferon-stimulated genes: A complex web of host defenses.Annu. Rev. Immunol. 2014; 32: 513-545Crossref PubMed Scopus (1291) Google Scholar). Recent evidence reveals that miRNAs can regulate the replication of several viruses through managing the production of IFNs and ISGs (21Forster S.C. Tate M.D. Hertzog P.J. MicroRNA as type I interferon-regulated transcripts and modulators of the innate immune response.Front Immunol. 2015; 6: 334Crossref PubMed Scopus (81) Google Scholar, 22Wong R.R. Abd-Aziz N. Affendi S. Poh C.L. Role of microRNAs in antiviral responses to dengue infection.J. Biomed. Sci. 2020; 27: 4Crossref PubMed Scopus (19) Google Scholar). Likewise, a few miRNAs, such as miRNA-23, miR-26a, miR-30c, and miRNA-373, attribute to modulate PRRSV replication by targeting IFN or its signaling pathways (23Zhang Q. Huang C. Yang Q. Gao L. Liu H.C. Tang J. Feng W.H. MicroRNA-30c modulates type I IFN responses to facilitate porcine reproductive and respiratory syndrome virus infection by targeting JAK1.J. Immunol. 2016; 196: 2272-2282Crossref PubMed Scopus (52) Google Scholar, 24Li L. Wei Z. Zhou Y. Gao F. Jiang Y. Yu L. Zheng H. Tong W. Yang S. Zheng H. Shan T. Liu F. Xia T. Tong G. Host miR-26a suppresses replication of porcine reproductive and respiratory syndrome virus by upregulating type I interferons.Virus Res. 2015; 195: 86-94Crossref PubMed Scopus (42) Google Scholar, 25Chen J. Shi X. Zhang X. Wang A. Wang L. Yang Y. Deng R. Zhang G.P. MicroRNA 373 facilitates the replication of porcine reproductive and respiratory syndrome virus by its negative regulation of type I interferon induction.J. Virol. 2017; 91e01311-16Crossref PubMed Scopus (28) Google Scholar). Since the details of miRNA-mediated regulation of viral replication have just begun to emerge, a comprehensive investigation of their roles in PRRSV pathogenesis will contribute to a better understanding of host–pathogen interactions. In the present study, we obtained the differently expressed miRNA profiles by deep sequencing of HP-PRRSV-infected alveolar macrophages. Based on the screening data, we have investigated miR-218 induction during PRRSV infection in vitro and in vivo. We found that miR-218 negatively regulates PRRSV replication by targeting SOCS3, thus clarifying one of the molecular mechanisms underlying PRRSV pathogenesis. To investigate the miRNAs involved in the host response against PRRSV infection, we utilized a high-throughput deep sequencing to profile miRNA in porcine alveolar macrophages (PAMs) at 24 h postinfection (hpi) of a HP-PRRSV strain HuN4. Mock-treated PAMs were used as control. The top 100 expressed miRNAs obtained from mock-treated PAMs are shown in Figure 1A. Comparing with previously reported miRNAs related to PRRSV infection, most of them are present in this figure, such as miR-23, miR-378, miR-26a, miR-22, let-7f, miR-29a, miR-181, miR-30c, miR-505, and miR-125b (26Du T. Nan Y. Xiao S. Zhao Q. Zhou E.M. Antiviral strategies against PRRSV infection.Trends Microbiol. 2017; 25: 968-979Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), suggesting that our deep sequencing data are reliable. After virus treatment, most miRNAs were downregulated, as shown in the volcano plot (Fig. 1B). Among them, seven miRNAs including miR-339-5p, miR-99b, miR-365-5p, miR-378, miR-345, miR-27b-3p, and miR-218 all ranked inside the top 100 and were significantly downregulated (Fig. 1C). Like our findings, Zhen et al. reported that miR-378 was downregulated upon PRRSV infection as well (27Zhen Y. Wang F. Liang W. Liu J. Gao G. Wang Y. Xu X. Su Q. Zhang Q. Liu B. Identification of differentially expressed non-coding RNA in porcine alveolar macrophages from Tongcheng and large white pigs Responded to PRRSV.Sci. Rep. 2018; 8: 15621Crossref PubMed Scopus (6) Google Scholar). The PRRSV replication in PAMs was confirmed by western blot analysis (Fig. 1D). Next, the effect of these seven miRNAs on PRRSV infection was explored by blocking their endogenous expression using specific inhibitors. PAMs were transfected with each of seven miRNA inhibitors or negative control for 24 h, and cells were then inoculated with PRRSV at an MOI of 0.1 for 24 h. Relative quantitative PCR (qPCR) results showed that inhibition of miR-378, miR-345, miR-27b-3p, or miR-218 had significantly increased the expression levels of viral RNA (Fig. 1E), indicating that these four miRNAs negatively regulate PRRSV replication. Consistent with our data, miR-378 has been reported to be an effective suppressor against PRRSV replication (27Zhen Y. Wang F. Liang W. Liu J. Gao G. Wang Y. Xu X. Su Q. Zhang Q. Liu B. Identification of differentially expressed non-coding RNA in porcine alveolar macrophages from Tongcheng and large white pigs Responded to PRRSV.Sci. Rep. 2018; 8: 15621Crossref PubMed Scopus (6) Google Scholar). Since the miR-218 was significantly downregulated upon virus infection and the miR-218 inhibitor resulted in an increase in viral RNA, we further investigated the regulatory function of miR-218 inhibitor on PRRSV replication. The virus titers of collected cell supernatants were measured by TCID50 assay, and the result showed that treatment with miR-218 inhibitor resulted in a marked increase in PRRSV titers in contrast to treatment with miRNA negative control (Fig. 2A). Meanwhile, the commercial miRNA mimic was used to overexpress miR-218 in PAMs. At 24 h post miR-218 mimic transfection, PAMs were infected with PRRSV for an additional 24 h. The qPCR results showed that overexpression of miR-218 significantly reduced viral RNA load compared with the negative control (Fig. 2B). The results of TCID50 assay also displayed that miR-218 overexpression led to a significant decrease in viral titers (Fig. 2C). No toxicity of miR-218 mimic at concentrations of <150 nM was evident for PAMs (Fig. S1). These findings indicate that miR-218 has the potential to inhibit PRRSV replication in PAMs. To determine whether low-virulent PRRSV has a similar effect on miR-218 expression, PAMs were treated with attenuated live PRRSV vaccine strain HuN4-F112, which is a Marc-145-adaptive strain and cannot efficiently replicate in PAMs (28Tian Z.J. An T.Q. Zhou Y.J. Peng J.M. Hu S.P. Wei T.C. Jiang Y.F. Xiao Y. Tong G.Z. An attenuated live vaccine based on highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) protects piglets against HP-PRRS.Vet. Microbiol. 2009; 138: 34-40Crossref PubMed Scopus (96) Google Scholar). The result showed that the expression level of miR-218 was not reduced in HuN4-F112-treated PAMs compared with the control group (Fig. 2D). Similarly, UV-inactivated HuN4 did not result in the decrease of miR-218 expression (Fig. 2E), indicating that virus replication is required for the downregulation of miR-218. Furthermore, we identified the dynamic changes of miR-218 expression at different stages of virus infection. As shown in Figure 2F, the virus attachment did not affect miR-218 expression; as shown in Figure 2G, the downregulation of miR-218 occurred after the virus entered the cell and during its biosynthesis (after two hpi). Altogether, our findings indicate that virus replication-mediated miR-218 downregulation facilitates PRRSV replication. Previous studies have demonstrated that miRNA-mediated viral regulation can happen by directly targeting the viral genome or by targeting host genes involved in viral replication (29Bernier A. Sagan S.M. The Diverse roles of microRNAs at the Host⁻Virus Interface.Viruses. 2018; 10: 440-465Crossref Scopus (38) Google Scholar, 30Skalsky R.L. Cullen B.R. Viruses, microRNAs, and host interactions.Annu. Rev. Microbiol. 2010; 64: 123-141Crossref PubMed Scopus (470) Google Scholar). During the replication cycle of PRRSV, the positive-sense RNA genome is transcribed into an intermediate, negative-sense RNA molecule, which functions as templates for the synthesis of positive-sense genomic RNA (31Fang Y. Snijder E.J. The PRRSV replicase: Exploring the multifunctionality of an intriguing set of nonstructural proteins.Virus Res. 2010; 154: 61-76Crossref PubMed Scopus (248) Google Scholar). Thus, to uncover the potential function of miR-218 during PRRSV replication, we compared the miR-218 seed sequence with PRRSV genomic RNA and found that there were at least two paired regions between miR-218 and negative/positive-sense viral RNA (Fig. 3A). To test whether miR-218 directly targets viral RNA and acts as a direct antiviral factor, we infected PAMs with PRRSV for 2 h and then transfected miR-218 mimic for 8 h and followed by detecting its effect on viral RNAs. As shown in Figure 3, B and C, overexpression of miR-218 did not affect the expression levels of negative-sense genomic RNA and positive-sense genomic RNA, suggesting that miR-218 regulates PRRSV replication not through directly targeting viral genomic RNA. Given that miR-218 can regulate PRRSV replication in PAMs, it is worthwhile to further investigate the molecular mechanisms. Type I IFN is the key innate immune cytokine produced in large quantities by cells to trigger antiviral function (32Mesev E.V. LeDesma R.A. Ploss A. Decoding type I and III interferon signalling during viral infection.Nat. Microbiol. 2019; 4: 914-924Crossref PubMed Scopus (95) Google Scholar), we next determined whether miR-218 could modify innate immune response pathways of type I IFN. PAMs were treated with miR-218 mimic for 24 h, and cells were then collected for detecting the transcriptional levels of IFN-β and several ISGs. The results showed that the mRNA levels of IFN-β and several ISGs including ISG15, 2′-5′-oligoadenylate synthetase-like protein (OASL), GBP1, IFIT1, and IFITM3 were increased in the presence of miR-218 mimic compared with the negative control (Fig. 3D). These findings suggest that miR-218 regulates PRRSV replication through modulating type I IFN response. To explore the molecular mechanism by which miR-218 regulates type I IFN response, TargetScan and miRDB were used to determine the potential target gene of miR-218. One putative binding site for miR-218 was identified in the 3’UTR of porcine suppressor of cytokine signaling 3 (SOCS3), a critical regulator of infection and inflammation (33Carow B. Rottenberg M.E. SOCS3, a Major regulator of infection and inflammation.Front Immunol. 2014; 5: 58Crossref PubMed Scopus (218) Google Scholar). The pairing region between the miR-218 seed sequence and SOCS3 3'UTR seed binding site was shown in Figure 4A. To validate the direct association between SOCS3 and miR-218, a region of SOCS3 3'UTR containing the predicted target site of miR-218 was cloned into a dual-luciferase reporter vector pmirGLO, and the same 3'UTR region with mutations in the seed binding site was also constructed into the same vector to obtain the mutant plasmid (Supplementary material S1). HEK-293 T cells were cotransfected with each of an empty vector, wild-type SOCS3 3'UTR plasmid or mutant plasmid in the presence of miR-218 mimic or miRNA negative control for 48 h. The results showed that miR-218 mimic significantly inhibited the luciferase activity of wild-type SOCS3 3'UTR plasmid-transfected cells relative to miRNA control. In contrast, transfection with mutant SOCS3 3'UTR abolished the inhibition effect of miR-218 mimic on the luciferase activity (Fig. 4B), suggesting that miR-218 can directly interact with SOCS3 3’UTR. To further verify the regulatory effect of miR-218 on SOCS3, PAMs were transfected with miR-218 mimic for 24 h and 48 h, and the mRNA and protein expression levels of SOCS3 were determined. The qPCR results showed that SOCS3 mRNA level was significantly reduced following the transfection with miR-218 mimic (Fig. 4C). Western blot analysis also revealed that treatment with miR-218 mimic markedly reduced SOCS3 protein expression (Fig. 4D). These data demonstrate that miR-218 negatively regulates SOCS3 expression at the transcriptional level by targeting SOCS3 3’UTR. Next, we determined whether there is a correlation between miR-218 and SOCS3 during PRRSV infection in vitro and in vivo. After incubating PAMs with mock or PRRSV for different time points, the expression levels of miR-218 and SOCS3 were assessed by qPCR. As shown in Figure 4E, the expression levels of miR-218 decreased within 24 hpi. Whereas the expression levels of SOCS3 significantly increased at 6 hpi and reached a peak at 24 hpi. These results suggest that the expression of SOCS3 is negatively correlated with miR-218 during PRRSV infection in vitro. To confirm the in vitro data, in vivo samples were also examined. Total RNA was extracted from the lung of pigs that were inoculated with mock, vaccine strain HuN4-F112, and HP-PRRSV strain HuN4 for 21 days as previously described (28Tian Z.J. An T.Q. Zhou Y.J. Peng J.M. Hu S.P. Wei T.C. Jiang Y.F. Xiao Y. Tong G.Z. An attenuated live vaccine based on highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) protects piglets against HP-PRRS.Vet. Microbiol. 2009; 138: 34-40Crossref PubMed Scopus (96) Google Scholar). The expression levels of miR-218 and SOCS3 were determined by qPCR. The levels of miR-218 were significantly lower in HuN4-inoculated pigs compared with mock controls and HuN4-F112 pigs. On the contrary, infection by HuN4 resulted in an increase in SOCS3 expression levels in comparison with the control group or HuN4-F112 group (Fig. 4F). Taken together, these results demonstrate that virus-induced miR-218 downregulation leads to an increase of SOCS3 expression, which might impair the antiviral response to facilitate PRRSV replication. Moreover, the effect of SOCS3 on PRRSV replication was investigated using genetic modification methods. The SOCS3 mRNA level in PAMs transfected with SOCS3-specific siRNA was significantly decreased relative to control siRNA. Western blot analysis of detergent lysates collected from cells transfected with SOCS3 siRNA also revealed a clear reduction in the level of SOCS3 protein (Fig. 5A). At 24 h post siRNA transfection, PAMs were inoculated with PRRSV for an additional 24 h. We observed that the viral RNA levels were greatly decreased in the SOCS3-specific transfection group compared with the control siRNA group (Fig. 5B). Knockdown of endogenous SOCS3 with siRNA also reduced virus titer as measured by TCID50 assay (Fig. 5C). To confirm siRNA data, another SOCS3-specific siRNA was introduced to repeat the above experiments. The results showed that the SOCS3 siRNA resulted in the decrease of virus RNA level and titers (Fig. S2). To further confirm the effect of SOCS3 on PRRSV replication, we tried to overexpress SOCS3 in primary PAMs, but the expression level of SOCS3 was not detectable (data not shown), which might be due to low transfection efficiency. Therefore, the immortalized PAMs were used to overexpress SOCS3, and the cells were then infected with PRRSV. Western blot analysis confirmed that SOCS3 was overexpressed successfully in the immortalized PAMs (Fig. 5D). As shown in Figure 5E, SOCS3 overexpression increased the levels of viral RNA in contrast to the vector control. TCID50 assay results confirmed the positive effect of SOCS3 overexpression on virus titer in comparison with the vector control (Fig. 5F). These data indicate that SOCS3 negatively regulates PRRSV replication. Since miR-218 negatively regulates type I IFN response, we assessed the effect of SOCS3 on the signaling pathway of type I IFN by modifying SOCS3 expression in PAMs. Here, the mRNA levels of IFN-β and several ISGs including ISG15, OASL, and GBP1 were analyzed by qPCR. The results showed that depletion of endogenous SOCS3 expression significantly enhanced the mRNA levels of IFN-β and three ISGs, ISG15, OASL, and GBP1, in contrast to the control siRNA treatment (Figs. 5G and S3). To confirm the effect of SOCS3 on type I IFN signaling pathway, the immortalized PAMs were transfected with a eukaryotic expression plasmid containing SOCS3 and followed by IFN-β stimulation. We observed that the mRNA levels of IFN-β, ISG15, OASL, and GBP1 were significantly decreased in SOCS3 transfection group compared with the control group (Fig. 5H). Overall, these findings suggest that PRRSV induced-miR-218 downregulation upregulates SOCS3 expression, which may serve as a negative regulator of type I IFN response to promote PRRSV replication. To elucidate the role of SOCS3 in the antiviral function of miR-218, we carried out miR-218 and SOCS3 RNA interference knockdown experiments. We confirmed that inhibition of miR-218 effectively promoted PRRSV replication in PAMs, as shown by viral RNA and virus titer. Whereas depletion of SOCS3 by specific siRNA remarkably suppressed PRRSV replication (Fig. 6, A and B). When the miR-218 was inhibited by its inhibitor in SOCS3-depleted cells, the increase of both viral RNA and virus titer by miR-218 inhibitor was blocked by the depletion of SOCS3 expression (Fig. 6, A and B). These results indicate that SOCS3 is beneficial for PRRSV replication, and the virus-induced miR-218 downregulation impairs the host antiviral response through the SOCS3-mediated signaling pathway. To verify the negative regulation of PRRSV replication by miR-218, we examined the antiviral effect of miR-218 in Marc-145 cells. Marc-145 cells were transfected with miR-218 inhibitor for 24 h, and cells were then infected with PRRSV at an MOI of 0.1 for 24 h. Cell monolayers were fixed for IFA analysis, and we observed notably more PRRSV-positive cells with miR-218 inhibitor than the control group (Fig. 7A). The levels of viral RNA were increased in miR-218 inhibitor-treated group compared with the control group (Fig. 7B). Transfection with miR-218 inhibitor consequently augmented viral titers as measured by TCID50 (Fig. 7C). Similarly, when Marc-145 cells were transfected with miR-218 mimic, the number of PRRSV-positive cells was lower in miR-218 mimic-treated cells than in control-treated cells (Fig. 7D). The viral RNA levels were significantly decreased with miR-218 mimic treatment (Fig. 7E), and the reduced titers of virus in infected cell cultures containing miR-218 mimic were confirmed by measuring TCID50 (Fig. 7F). These data indicate that suppression of PRRSV replication by miR-218 in Marc-145 cells is consistent with that in PAMs, suggesting that the evolutionarily conserved miR-218 might have a similar function in antiviral effect in different cell lines. Next, we evaluated the antiviral effect of the evolutionarily conserved miR-218 in two other members of the order Nidovirales, PEDV (porcine epidemic diarrhea virus) and TGEV (transmissible gastroenteritis virus). Vero E6 cells were transfected with miR-218 mimic for 24 h, and cells were then inoculated with PEDV at an MOI of 0.1 for another 24 h. Cells and supernatant were harvested for detecting viral RNA levels by qPCR and viral titers by TCID50 respectively. The results showed that overexpression of miR-218 significantly inhibited PEDV replication as indicated by RNA levels and viral titers (Fig. 7G). ST cells were also treated with miR-218 mimic as above, and cells were infected with TGEV at an MOI of 0.1 for 24 h. As shown in Figure 7H, miR-218 overexpression also reduced TGEV loads as measured by qPCR and TCID50. These results indicate that the evolutionarily conserved miR-218 may have a broad-spectrum antiviral property. The first line of host defense against viruses is the innate immune system, in which IFNs are glycoproteins with strong antiviral and immunomodulatory activities. The published data suggest that PRRSV is susceptible to IFNs in vivo and in vitro (34Brockmeier S.L. Loving C.L. Eberle K.C. Hau S.J. Buckley A. Van Geelen A. Montiel N.A. Nicholson T. Lager K.M. Interferon alpha inhibits replication of a live-attenuated porcine reproductive and respiratory syndrome virus vaccine preventing development of an adaptive immune response in swine.Vet. Microbiol. 2017; 212: 48-51Crossref PubMed Scopus (9) Google Scholar, 35Luo R. Fang L. Jin H. Jiang Y. Wang D. Chen H. Xiao S. Antiviral activity of type I and type III interferons against porcine reproductive and respiratory syndrome virus (PRRSV).An" @default.
• W3155357773 created "2021-04-26" @default.
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• W3155357773 title "Downregulation of miR-218 by porcine reproductive and respiratory syndrome virus facilitates viral replication via inhibition of type I interferon responses" @default.
• W3155357773 cites W1600022398 @default.
• W3155357773 cites W1975931383 @default.
• W3155357773 cites W1998142094 @default.
• W3155357773 cites W2001476170 @default.
• W3155357773 cites W2004829503 @default.
• W3155357773 cites W2005217652 @default.
• W3155357773 cites W2019009111 @default.
• W3155357773 cites W2031913105 @default.
• W3155357773 cites W2035509260 @default.
• W3155357773 cites W2036627785 @default.
• W3155357773 cites W2041381307 @default.
• W3155357773 cites W2042004955 @default.
• W3155357773 cites W2047355421 @default.
• W3155357773 cites W2057633846 @default.
• W3155357773 cites W2059117644 @default.
• W3155357773 cites W2059357290 @default.
• W3155357773 cites W2071700214 @default.
• W3155357773 cites W2081912130 @default.
• W3155357773 cites W2082742317 @default.
• W3155357773 cites W2098128349 @default.
• W3155357773 cites W2107277218 @default.
• W3155357773 cites W2108771986 @default.
• W3155357773 cites W2124371255 @default.
• W3155357773 cites W2133402546 @default.
• W3155357773 cites W2140013858 @default.
• W3155357773 cites W2150013528 @default.
• W3155357773 cites W2150361201 @default.
• W3155357773 cites W2162674813 @default.
• W3155357773 cites W2164708446 @default.
• W3155357773 cites W2173574671 @default.
• W3155357773 cites W2257616042 @default.
• W3155357773 cites W2322565284 @default.
• W3155357773 cites W2468978895 @default.
• W3155357773 cites W2508325336 @default.
• W3155357773 cites W2519423713 @default.
• W3155357773 cites W2522303900 @default.
• W3155357773 cites W2552238048 @default.
• W3155357773 cites W2557180350 @default.
• W3155357773 cites W2563350820 @default.
• W3155357773 cites W2592473983 @default.
• W3155357773 cites W2596527460 @default.
• W3155357773 cites W2604833888 @default.
• W3155357773 cites W2686300806 @default.
• W3155357773 cites W2746352180 @default.
• W3155357773 cites W2767323151 @default.
• W3155357773 cites W2783739334 @default.
• W3155357773 cites W2793246968 @default.
• W3155357773 cites W2888657086 @default.
• W3155357773 cites W2896263258 @default.
• W3155357773 cites W2899995656 @default.
• W3155357773 cites W2913203394 @default.
• W3155357773 cites W2931678155 @default.
• W3155357773 cites W2975692296 @default.
• W3155357773 cites W2981162155 @default.
• W3155357773 cites W2999538459 @default.
• W3155357773 cites W3006797558 @default.
• W3155357773 cites W3009424226 @default.
• W3155357773 cites W3014083212 @default.
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