MicroRNA-373 Facilitated The Replication Of Porcine Reproductive And Respiratory Syndrome Virus(PRRSV) By Its Negative Regulation Of Type ? Interferon Induction

Porcine reproductive and respiratory syndrome(PRRS) is caused by PRRS virus(PRRSV) which can cause reproductive failure in pregnant sows and piglets respiratory diseases. PRRSV has been causing huge economic losses and seriously endangering to swine industry worldwide. PRRSV could suppress or escape the host innate and acquired immune system, in particular, including the suppression of Type ? interferon(Type ? IFNs, IFN-I) to form a persistent infection. Therefore, there is an urgent need for further investigating on persistent infection and immunosuppression of PRRSV. It will provide a theoretical basis for the prevention and control of PRRS.MicroRNAs(miRNAs) are small non-coding RNA which regulate gene expression by post-transcriptional. Emerging evidence have indicated that miRNAs influenced viral replication through targeting the viral genome or regulating of host antiviral response. Previous studies have indicated that dysregulation of miRNAs may be involved in the immunosuppression of PRRSV. However, the mechanisms that PRRSV regulated the miRNAs expression and the roles of miRNAs in regulating PRRSV replication have not been clear. Thus, using high-throughput RNA sequencing(RNA-Seq), we found PRRSV infection significantly changed the miRNA expression profiling in MARC-145 cell, in which miR-373 expression was the most obvious difference. Then, we verified the miR-373 expression level by q RT-PCR in PRRSV infected MARC-145 cells, and found PRRSV could significantly upregulate the expression of miR-373. So how PRRSV upregulate miR-373 expression, how miR-373 influence PRRSV replication and what is the mechanism? In this paper, the above scientific issues were conducted exhaustive research, and the results were as follows:PRRSV promoted miR-373 transcription through elevating Sp1 expression. miR-373 promoter truncation, bioinformatic analysis and mutation assay showed that the Sp1(specificity protein 1, Sp1) was an important transcription factor of miR-373 gene. And the EMSA and Ch IP assay confirmed that Sp1 was capable to binding to miR-373 promoter. Next, we found that Sp1 was significantly increased in PRRSV infected MARC-145 cells, and Sp1 si RNA could inhibit PRRSV to upregulate miR-373 expression, in contrast, overexpression of Sp1 could promot PRRSV to upregulate miR-373 expression. These results indicates that PRRSV could promot miR-373 transcription through elevating Sp1 expression. Through the overexpression of viral proteins and promoter reporter experiments, we found that PRRSV nonstructural proteins nsp9 and structural protein N could enhance miR-373 promoter activity and expression, and nsp9 and N protein could promot Sp1 expression. These results suggested that the nsp9 and N proteins of PRRSV could promot miR-373 transcription through elevating Sp1 expression.miR-373 facilitated the replication of PRRSV through negative regulation of IFN-I induction. The transfection experiments of miR-373 mimic and inhibitor showed that miR-373 mimic could significantly improve PRRSV load in MARC-145 cells and PAMs, while miR-373 inhibitor could obviously inhibit PRRSV replication. It suggested that miR-373 could facilitate PRRSV replication. Further research demonstrated that Sp1 could significantly promot PRRSV replication, and that was dependent on miR-373. The selection and identification results showed that NFIA, NFIB, IRAK1, IRAK4, IRF1 and IRF9 were target genes of miR-373. IRAK1, IRAK4, IRF1 are necessary signaling proteins which involve in IFN-I production, and the further results indicated that miR-373 could inhibit IFN-? production. Thus, miR-373 could inhibit IFN-I signaling pathway by targeting the key molecules of NFIA, NFIB, IRAK1, IRAK4, IRF1 and IRF9 in IFN-I signaling pathway, thereby facilitate PRRSV replication.NFIA and NFIB promoted IFN-I production and inhibited PRRSV replication. Overexpression of NFIA and NFIB could enhance IFN-? promoter activity and expression, while downregulation of NFIA and NFIB could inhibit IFN-? promoter activity and expression. This indicated that NFIA and NFIB could regulate IFN-? promoter activity and expression. Followed EMSA and Ch IP showed that NFIA/NFIB could combine with IFN-? promoter. Therefore, the above results indicated that NFIA and NFIB may be IFN-? transcription factor and could regulate transcript expression of IFN-?. In addition, overexpressed NFIA and NFIB could inhibit PRRSV replication, while downregulation of NFIA and NFIB could promot PRRSV replication. This results showed that NFIA and NFIB may be an antiviral protein which could suppress PRRSV replication. In the present paper, we first discovered NFIA and NFIB could inhibit RNA virus(PRRSV) replication.In summary, in this paper, we described a new mechanism for PRRSV how to regulate and use miR-373 to escape IFN-I mediated antiviral immune response, and we first discovered and proved NFIA and NFIB were the novel proteins for inducing IFN-? production and both of them could inhibit PRRSV replication. The results of this study has important reference significance to elucidate PRRSV immunosuppression and persistent infection mechanism, and also provide a new target for the prevention and treatment of PRRS.