The Effect Of Celluar MicroRNA And Viral Small RNA On Porcine Reproductive And Respiratory Syndrome Virus Infection In Porcine Alveolar Macrophages

Porcine reproductive and respiratory syndrome(PRRS) is one of the most highly infectious swine diseases, resulting in great economic losses every year affecting the swine industry worldwide. PRRS is characterized by respiratory disease, weight loss and poor growth performance, as well as late-term abortions. Porcine reproductive and respiratory syndrome virus(PRRSV), the causative agent of PRRS, is a single-stranded, positive-sense RNA virus of the genus Arterivirus, in the family Arteriviridae within the order Nidovirales. A highly pathogenic strain of PRRSV(HP-PRRSV) was associated with devastating atypical PRRS outbreaks in China in 2006. Characterized by high fever, high morbidity, and high mortality in pigs of all ages, HP-PRRSV coexists in China with the long-established low pathogenic classical North American type PRRSV strains(N-PRRSV). Because existing antiviral strategies have been ineffective in providing sustainable protection, especially against highly pathogenic strains, it is imperative that new measures are developed to control widespread virus infection.PRRSV strains have a restricted repertoire of susceptible hosts, limited by a viral requirement for specific receptors on host cells. Despite the restricted cell tropism of PRRSV, the virus is able to replicate in several non-permissive cell lines, indicating that additional proteins may facilitate virus entry. Recently, non-muscle myosin heavy chain 9(MYH9) was implicated in PRRSV infection. Through formation of intercellular nanotube connections that can transport infectious viral RNA from cell to cell, MYH9 facilitates intercellular viral spread, while allowing PRRSV to escape the host’s neutralizing antibody response. Our previous work demonstrated that MYH9 also guides the infection process after virus particles attach to cell surface receptors, culminating in completion of subsequent un-coating events required for PRRSV genomic release within the host cell. Although MYH9 plays important role in PRRSV infection, the regulation mechanism of MYH9 during PRRSV replication in PRRSV-permissive cells is still unknown which need to be further studied.MicroRNAs(miRNAs) are small non-coding single-stranded RNAs which regulate gene expression at the post-transcriptional level by either inducing mRNA degradation or inhibiting mRNA translation. Recent evidence indicates that miRNAs encoded by the host or virus can directly modulate virus replication, as well as alter the host cell response to infection in either a proviral or antiviral manner. The majority of the known virus-encoded mi RNAs are derived from DNA viruses and only a few of them fromRNA viruses. Therefore, naturally occurring miRNAs derived fromRNA viruses have not been widely acknowledged. Recent studies have confirmed that cytoplasmic RNA viruses are capable of expressing miRNA-like viral small RNAs. However, it is unknown whether PRRSV, a small enveloped linear, positive-sense single-stranded RNA virus, encodes mi RNAs/vsRNAs. In this thesis, cellular microRNA which targeting MYH9 and vsRNAs derived from PRRSV genome have been identificated and their effects on PRRSV infection in porcine alveolar macrophages(PAMs) are validated. The results of this study were as follows: 1. MicroRNA let-7f-5p Inhibits Porcine Reproductive and Respiratory Syndrome Virus by Targeting MYH9Using bioinformatic prediction and experimental verification, we demonstrate that MYH9 expression is regulated by the microRNA let-7f-5p, which binds to the MYH9 mRNA 3’UTR and regulates MYH9 expression through both mRNA degradation and translational repression. To understand how let-7f-5p regulates PRRSV infection, we analyzed the expression pattern of both let-7f-5p and MYH9 in PAMs after infection with either HP-PRRSV or N-PRRSV using Stem-Loop qRT-PCR validation. Our results showed that both HP-PRRSV and N-PRRSV infection reduced let-7f-5p expression while also inducing MYH9 expression. Furthermore, overexpression of let-7f-5p significantly inhibited PRRSV replication through suppression of MYH9 expression. 2. MicroRNA-like viral small RNA from porcine reproductive and respiratory syndrome virus negatively regulates viral replication by targeting the viral nonstructural protein 2Four viral small RNAs(vsRNAs) were mapped to the stem-loop structures in the ORF1 a, ORF1 b and GP2 a regions of the PRRSV genome by bioinformatics prediction and experimental verification. Of these, the structures with the lowest minimum free energy(MFE) values predicted for PRRSV-vsRNA1 corresponded to typical stem-loop, hairpin structures. Northern blot hybridization with a PRRSV-vsRNA1 probe produced distinct bands which matched the size of the sequenced small RNA obtained from PRRSV-infected cells. The time-dependent increase in the abundance of PRRSV-vsRNA1 mirrored the gradual upregulation of PRRSV RNA expression. Inhibition of PRRSV-vsRNA1 function led to significant increases in viral replication. Transfection with PRRSV-vsRNA1 mimics significantly inhibited PRRSV replication in PAMs. Knockdown of proteins associated with cellular miRNA biogenesis demonstrated that Drosha and Argonaute(Ago2) are involved in PRRSV-vsRNA1 biogenesis. Moreover, PRRSV-vsRNA1 bound specifically to the nonstructural protein 2(NSP2)-coding sequence of PRRSV genome RNA. Collectively, the results reveal that PRRSV encodes a functional PRRSV-vsRNA1 which auto-regulates PRRSV replication by directly targeting and suppressing viral NSP2 gene expression.In summary, 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 and overexpression of let-7f-5p can inhibit PRRSV replication through suppression of MYH9 expression. We also demonstrate for the first time that PRRSV utilizes cellular miRNA processing machinery to encode functional microRNA-like vsRNAs. PRRSV-vsRNA1 negatively regulates PRRSV replication by directly targeting the viral NSP2 sequence. These findings not only provide new insights into the mechanism of the pathogenesis of PRRSV, but also explore a potential avenue for controlling PRRSV infection using microRNAs.