Mechanism Of ICP4-driven MiR-101 Expression And Regulation Of MiR-101on HSV-1 Replication

【Objective】micro RNAs(mi RNAs) are endogenous 18~26 nt RNAs that play important posttranscriptional regulation on target gene expression in animals and plants by binding 3’UTR of m RNA. It has been reported that mi RNAs are involoved in antiviral response. On the other hand, viruses also can downregulate or upregulate the expression of cellular mi RNAs. The mi RNAs of the host cells is closely related to viral infection. Our previous results showed that HSV-1 infection significantly induced the expression of mi R-101 in He La. Cyclooxygenase 2(COX2) and G-rich RNA sequence binding factor 1(GRSF1), which are the direct target gene of mi R-101 may impact HSV-1 replication. We have noted that there are two loci of the mi R-101 gene in the human genome: pre-mi R-101-1 and pre-mi R-101-2 which located in chromosome 1 and chromosome 9. Our previous results showed that upregulation of mi R-101 by HSV-1 infection mainly attribute to activation of pre-mi R-101-2. The aim of this study was to explore the mechanism of hsa-mi R-101-2 induced by HSV-1 infection and the mechanism of hsa-mi R-101-2 on regulation of HSV-1 replication, to further clarify the interactions between mi RNAs of host cell and HSV-1.【Method】 This project mainly focus on two aspects: the mechanism of mi R-101 induced by HSV-1 infection and the mechanism of mi R-101-mediated regulation of HSV-1 replication.1. First, luciferase report assay was applied to detect the activation of the cloned promoter of hsa-mi R-101-2 under HSV-1 infection and to identify regulative factors encoded by HSV-1 on that promoters. Then, we used q RT-PCR and western blot assay to detect the expression level of hsa-mi R-101-2 and its host gene under HSV-1 infection and alteration of HSV-1-encoded infected-cell polypeptide 4(ICP4) by overexpression and RNAi. Finally, chromatin immunoprecipitation assay and electrophoretic mobility shift assay were used to determine whether HSV-1-encoded protein ICP4 directly interacts with hsa-mi R-101-2 promoter.2. To further determine the specific role of the direct target gene of mi R-101, COX2 and GRSF1 on HSV-1 replication, virus titration, immunofluorescence and western blot assay were used to study the specific pathways of the COX2 and GRSF1 affecton HSV-1 replication. Then we detect COX2 effect on the expression of interferon through the q RT-PCR. By western blot, RNA immune coprecipitation and gel electrophoresis migration assay to determine the way of GRSF1 promoting HSV-1 capsid protein expression.【Results】1. Luciferase report assay showed that the activity of hsa-mi R-101-2 promoter is obviously induced under HSV-1 infection.2. Luciferase report assay showed that the activity of hsa-mi R-101-2 promoter is enhanced under overexpression of ICP4. q RT-PCR results showed that overexpression of ICP4 promote the expression of hsa-mi R-101-2 and its host gene.3. The chromatin immunoprecipitation assay and electrophoretic mobility shift assay results showed that ICP4 binds to the region of hsa-mi R-101-2 promoter.4. The results of virus titration, immunofluorescence andwestern blot assay showed that GRSF1 promotes the HSV-1 replication, but COX2 inhibits.5. q RT-PCR assay showed that COX2 promotes the expression level of type I/III interferon.6. Western blot assay showed that GRSF1 enhances the expression level of HSV-1 capsid protein p40.7. RNA immunoprecipitation assay and RNA electrophoretic mobility shift assay showed that GRSF1 binds to m RNA of p40.【Conclusion】 Collectly, according to thses results, we concluded as following:1. Induction of mi R-101 expression by HSV-1 infection mainly depends on ICP4 of HSV-1. ICP4 can directly bind to the sequence of hsa-mi R-101-2 promoter and activate its transcription as a transcriptional factor.2. COX2, a direct target gene of mi R-101 inhibits HSV-1 replication by promoting the expression level of type I/III interferon.3. GRSF1, a direct target gene of mi R-101 promotes the translation expression level of p40 by binding to its m RNA, which promotes HSV-1 replication...