School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China.
Sericulture Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China.
Insect Mol Biol. 2021 Dec;30(6):552-565. doi: 10.1111/imb.12725. Epub 2021 Jul 30.
Bombyx mori cypovirus (BmCPV) is an important pathogen of silkworm (B. mori), the economically beneficial insect. The mechanism of its interaction with host immune defence system in the process of infection is still not yet completely clear. Researches have demonstrated that virus-encoded microRNAs (miRNA) play a crucial role in regulating host-pathogen interaction, but few reports are available so far on miRNAs encoded by insect viruses, especially the RNA viruses. In this study, a putative miRNA encoded by the 10th segment of BmCPV genomic RNA, BmCPV-miR-10, was identified and functionally analysed. The expression of the putative BmCPV-miR-10 could be detected via stem-loop RT-PCR (reverse transcription-Polymerase Chain Reaction) in the midgut of silkworm larvae infected with BmCPV. BmCSDE1 (B. mori cold shock domain E1 protein) gene was predicted to be a candidate target gene for BmCPV-miR-10 with the miRNA binding site located in 3' untranslated region of its mRNA. The regulation effect of the putative BmCPV-miR-10 on BmCSDE1 was verified in HEK293 cells by lentiviral expression system, in BmN cells by transfecting BmCPV-miR-10 mimics. The qRT-PCR (quantitative real-time PCR) results showed that the putative BmCPV-miR-10 could suppress the expression of BmCSDE1. By injection of BmCPV-miR-10 mimics into the silkworm larvae infected with BmCPV, it was further proved that the putative BmCPV-miR-10 could suppress the expression of BmCSDE1 in vivo, then inhibit the expression of BmApaf-1 (B. mori apoptotic protease activating factor 1), while enhance the replication of BmCPV genomic RNAs to a certain extent. These results implied that the putative BmCPV-miR-10 could down-regulate the expression of BmCSDE1, then suppress the expression of BmApaf-1, thereby created a favourable intracellular environment for virus replication and proliferation.
家蚕质型多角体病毒（BmCPV）是一种重要的家蚕病原体，也是一种经济有益的昆虫。其在感染过程中与宿主免疫防御系统相互作用的机制尚不完全清楚。研究表明，病毒编码的 microRNAs（miRNA）在调控宿主-病原体相互作用中起着至关重要的作用，但目前关于昆虫病毒，特别是 RNA 病毒编码的 miRNA 的报道还很少。本研究鉴定并分析了 BmCPV 基因组 RNA 第 10 节编码的一种假定 miRNA，即 BmCPV-miR-10。通过茎环 RT-PCR（逆转录-聚合酶链反应）检测，在感染 BmCPV 的家蚕幼虫中可以检测到假定的 BmCPV-miR-10 的表达。BmCSDE1（家蚕冷休克结构域 E1 蛋白）基因被预测为 BmCPV-miR-10 的候选靶基因，其 mRNA 的 3'非翻译区存在 miRNA 结合位点。通过慢病毒表达系统在 HEK293 细胞中，通过转染 BmCPV-miR-10 模拟物在 BmN 细胞中，验证了假定的 BmCPV-miR-10 对 BmCSDE1 的调控作用。qRT-PCR（实时定量 PCR）结果表明，假定的 BmCPV-miR-10 可以抑制 BmCSDE1 的表达。通过向感染 BmCPV 的家蚕幼虫注射 BmCPV-miR-10 模拟物，进一步证明了假定的 BmCPV-miR-10 可以在体内抑制 BmCSDE1 的表达，从而抑制 BmApaf-1（家蚕凋亡蛋白酶激活因子 1）的表达，同时在一定程度上增强 BmCPV 基因组 RNA 的复制。这些结果表明，假定的 BmCPV-miR-10 可以下调 BmCSDE1 的表达，从而抑制 BmApaf-1 的表达，从而为病毒复制和增殖创造了有利的细胞内环境。
