Department of Entomology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran.
Australian Infectious Disease Research Centre, School of Biological Sciences, The University of Queensland, Brisbane, QLD, Australia.
Insect Biochem Mol Biol. 2018 Oct;101:24-31. doi: 10.1016/j.ibmb.2018.07.004. Epub 2018 Jul 31.
Autographa 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 Argonaute 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. ORF-9 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.
美洲棉铃虫多粒包埋型核型多角体病毒（AcMNPV）是杆状病毒科中一种众所周知的病毒。 AcMNPV 基因组中 p35 基因的存在作为短干扰 RNA（siRNA）途径的抑制剂，是 siRNA 途径在宿主细胞防御中重要性的一个有力原因。有鉴于此，我们在这里探讨了 Dicer-2（Dcr2）和 Argonaute 2（Ago2）基因在杆状病毒科中的作用，这些基因是 siRNA 途径的关键因素，以响应 Spodoptera frugiperda Sf9 细胞中的 AcMNPV 感染。结果表明，Dcr2 和 Ago2 的转录水平在 AcMNPV 感染后特别是在感染后 16 小时增加，表明 siRNA 途径的诱导。在 Sf9 细胞中使用特异性 dsRNA 降低 Dcr2 和 Ago2 的表达水平，适度增加了病毒基因组 DNA 的产生，这表明它们在宿主抗病毒防御中的作用。通过深度测序，我们之前的研究表明，从 AcMNPV 感染的 Sf9 细胞中获得了大量小读长（约 20 个核苷酸的 siRNA），这些小读长可映射到一些病毒基因（热点）。 Sf9 细胞中 Dcr2 的下调导致所选病毒热点基因（即 ORF-9 和 ORF-148）的表达水平升高，而没有或很少有 siRNA 映射到它们的病毒冷点（即 ORF-18 和 ORF-25）的转录水平没有变化。 Sf9 细胞中 AcMNPV p35 的过表达作为 RNAi 和抗细胞凋亡基因的抑制剂，可增加病毒复制。此外，在 Dcr2 和 Ago2 转录水平降低的 Sf9 细胞中，缺乏 p35 基因的突变型 AcMNPV 的复制显著增加，突出了 siRNA 途径在 Sf9 细胞中的抗病毒作用。总之，我们的结果表明，Dcr2 和 Ago2 基因有助于 Sf9 细胞对 AcMNPV 的有效抗病毒反应，而 AcMNPV p35 除了作为抗细胞凋亡蛋白外，还抑制 siRNA 途径。
