School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China.
Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China.
Insect Sci. 2020 Jun;27(3):449-462. doi: 10.1111/1744-7917.12671. Epub 2019 Apr 16.
Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus-encoded microRNAs (miRNAs) have been proven to play important roles in host-pathogen interactions. In this study we identified a BmCPV-derived miRNA-like 21 nt small RNA, BmCPV-miR-1, from the small RNA deep sequencing of BmCPV-infected silkworm larvae by stem-loop quantitative real-time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV-miR-1 at the 5' untranslated region. It was found that the expression of BmCPV-miR-1 and its target gene BmIAP were both up-regulated in BmCPV-infected larvae. At the same time, it was confirmed that BmCPV-miR-1 could up-regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV-miR-1 mimics could up-regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV-infected larvae, BmCPV-miR-1 mimics could be further up-regulated and inhibitors could lower the virus-mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV-miR-1 mimics could up-regulate and inhibitors down-regulate their replication in the infected silkworm. These results implied that BmCPV-miR-1 could inhibit cell apoptosis in the infected silkworm through up-regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.
家蚕细胞质多角体病毒（BmCPV）是经济昆虫家蚕的主要病原体。病毒编码的 microRNAs（miRNAs）已被证明在宿主-病原体相互作用中发挥重要作用。在本研究中，我们通过茎环定量实时 PCR（qPCR）从小 RNA 深度测序中鉴定出一种源自 BmCPV 感染的家蚕幼虫的 BmCPV 衍生的 miRNA-like 21 nt 小 RNA，BmCPV-miR-1，并通过 qPCR 和慢病毒表达系统研究了其功能。BmCPV-miR-1 的靶基因预测软件 miRanda 和 Targetscan 均预测凋亡抑制蛋白（BmIAP）基因是其靶基因之一，BmCPV-miR-1 在其 5'非翻译区具有结合位点。研究发现，BmCPV-miR-1 和其靶基因 BmIAP 的表达均在 BmCPV 感染的幼虫中上调。同时，通过慢病毒表达系统和转染 mimics 在 HEK293T 细胞和 BmN 细胞中证实，BmCPV-miR-1 可以上调 BmIAP 基因的表达。此外，BmCPV-miR-1 mimics 可以上调家蚕中肠和脂肪体中 BmIAP 基因的表达水平。在 BmCPV 感染幼虫的中肠中，BmCPV-miR-1 mimics 可以进一步上调，而抑制剂可以降低病毒介导的 BmIAP 基因的表达。以病毒基因组 RNA 片段 S1 和 S10 为指标，BmCPV-miR-1 mimics 可以上调，而抑制剂可以下调其在感染家蚕中的复制。这些结果表明，BmCPV-miR-1 可以通过上调 BmIAP 表达来抑制感染家蚕中的细胞凋亡，为病毒的复制提供了更好的细胞环境。
