عملکرد ضد ویروسی مسیر microRNA در سلول‌های Spodoptera frugiperda (Sf9) آلوده با Autographa californica multiple nucleopolyhedrovirus

Baculoviruses are important insect pathogen with narrow host rang and great potential in pest control. As a result, they can be considered as promising agents for use as biopesticides. Therefore understanding the molecular mechanisms underlying Baculovirus-host insect interaction would help to produce and apply these viruses more efficiently in biological control programs. MicroRNA pathway is an important epigenetic mechanism in eukaryote cells. Organisms utilize miRNA pathway to regulate gene expression and altered the gene expression pattern in different situation. Given high transcriptional reprogramming has occurred after viral infection in insects, we hypothesized that miRNA pathway may have functioned in this interaction. In the present study we investigated the importance of microRNA pathway in the interaction of Spodoptera frugiperda cells (Sf9) and Autographa californica multiple nucleopolyhedrovirus (AcMNPV). To do this, we obtained sequence of important genes in the miRNA pathway (i.e.Ago1, Dcr1, Exp5 and Ran) from National Center for Biotechnology Information (NCBI). Specific primers targeting these genes were designed using primer3plus and Oligo7 softwares. To assess the gene expression in the mock and AcMNPV-infected cells, total RNA was extracted from the mock and the infected cells at 4, 8, 16, 24, 48 hours post infection (hpi). The RNA samples were subjected to cDNA synthesis and RT-qPCR was carried out utilizing the specific primers and Rpl27 primer as the reference gene. Our results showed that the expression levels of Ago1, Dcr1 and Exp5 were increased at 16 hpi whereas the expression level of Ran gene was reduced at 24 hpi. Reduction in expression of Ran protein prevents miRNA transfer from nuclei to cytoplasm thereby decreases cellular miRNA levels in the cytoplasm. However, it seems that increasing in expression levels of other genes are to compensate Ran downregulation. For functional analysis of Ago1 and Dcr1, RNAi was used to suppress Ago1 and Dcr1 expression. DsRNA was synthesized using MEGA Script kit according the manufacturer’s instruction. DsRNA of Green Fluorescent Protein (dsGFP) was also generated and used as the control. After confirmation of Ago1 and Dcr1 suppression by RNAi, cells were infected with AcMNPV and the viral DNA replication quantified with qPCR method using ie-1primer. RNAi of Ago1 and Dcr1 genes enhanced viral replication highlighting the antiviral effect of microRNA pathway. Overall our results indicated that microRNA pathway has important role in cellular antiviral response.