Small interfering RNA pathway contributes to antiviral immunity in Spodoptera frugiperda (Sf9) cells following Autographa californica multiple nucleopolyhedrovirus infection

摘要

Autographs californica multiple nucleopolyhedrovirus (AcMNPV) is a well-known virus in the Baculoviridae family. Presence of the p35 gene in the AcMNPV genome as a suppressor of the short interfering RNA (siRNA) pathway is a strong reason for the importance of the siRNA pathway in the host cellular defense. Given that, here we explored the roles of Dicer-2 (Dcr2) and Atgonaute 2 (Ago2) genes, key factors in the siRNA pathway in response to AcMNPV infection in Spodoptera frugiperda Sf9 cells. The results showed that the transcript levels of Dcr2 and Ago2 increased in response to AcMNPV infection particularly over 16 h post infection suggesting induction of the siRNA pathway. Reductions in the expression levels of Dcr2 and Ago2 by using specific dsRNAs in Sf9 cells modestly enhanced production of viral genomic DNA which indicated their role in the host antiviral defense. Using deep sequencing, our previous study showed a large number of small reads (siRNAs of 20 nucleotides) from AcMNPV-infected Sf9 cells that were mapped to some of the viral genes (hot spots). Down regulation of Dcr2 in Sf9 cells resulted in enhanced expression levels of the selected virus hotspot genes (i.e. ORF9 and ORF-148), while the transcript levels of virus cold spots (i.e. ORF-18 and ORF-25) with no or few siRNAs mapped to them did not change. Overexpression of AcMNPV p35 as a suppressor of RNAi and anti-apoptosis gene in Sf9 cells increased virus replication. Also, replication of mutant AcMNPV lacking the p35 gene was significantly increased in Sf9 cells with reduced transcript levels of Dcr2 and Ago2, highlighting the antiviral role of the siRNA pathway in Sf9 cells. Together, our results demonstrate that Dcr2 and Ago2 genes contribute in efficient antiviral response of Sf9 cells towards AcMNPV, and in turn, the AcMNPV p35 suppresses the siRNA pathway, besides being an antiapoptotic protein.