Study On MicroRNAs Function Of Pseudorabies

As an important member of alphaherpesvirus, suid herpesvirus1(PRV), known as pseudorabies virus (PRV) or Aujeszky’s disease virus, causes heavy economic burden for pig industry and wild animals worldwide. PRV is able to establish life-long latency in peripheral nervous system such as trigeminal ganglion (TG).In acute/lytic infection, the tegument protein VP16triggers transcription of the immediate-early (IE) genes and then initiates replication of virus via a DNA-binding complex formed together with the cellular proteins Oct-1and HCF-1. The major histocompatibility complex (MHC) class I genes recognizes and presents virus-derived peptides in infected cells.The viral miRNAs not only interfere with protective host innate or adaptive immune and apoptosis but also target viral genes to regulate the life cycle of virus. Recently, we have reported that the long latency-associated transcript (LLT) PRV encodes11mature miRNAs in infected porcine kidney (PK-15) cells. However, the functions of these miRNAs are not determined. The present study tries to discover the function of the novel miRNAs in regulating the expression of several viral key genes and one immunity gene. With this effect, we try to deepen the understanding of the role of miRNA in the pathogenesis. This study will provide some novel therapeutic targets for prophylaxis and treatment of alphaherpesvirus-associated diseases. The detailed research includes the following aspects:1. we found MHC I and VP16were the potential targets of prv-mir-LLT7, there were two binding sites of prv-mir-LLT7in the3’UTR of VP16; the prv-mir-LLT1, prv-mir-LLT7, prv-mir-LLT8, prv-mir-LLT9and prv-mir-LLT11can target the IE180predicted by miRanda.2. The down regulation of these two genes were validated by luciferase reporter assay, quantitative RT-PCR, western blot and flow cytometry. Prv-mir-LLT7was transcribed in an infection-phase dependent manner. Overexpression of prv-mir-LLT7attenuated the production of IE180, resulting in a lower replication level of virus. Blocking prv-mir-LLT7rescue the expression of both the MHC class I and the VP16genes.3. The expression of PRV prv-mir-LLT7was determined by qRT-PCR. The prv-mir-LLT7attenuated the replication of PRV by western blot and plaque assay.4. The prv-mir-LLT1, prv-mir-LLT9and prv-mir-LLT11down regulated the expression of IE180gene validated by luciferase reporter assay and western blot.Together, this result showed that the MHC I and VP16can be down regulated by the prv-mir-LLT7. The IE180can be down regulated by prv-mir-LLT1, prv-mir-LLT9and prv-mir-LLT11. We infer that alphaherpesvirus develops delicate and economical strategy to maintain appropriate infection state.