家蚕病毒 miRNA BmCPV-miR-10 的功能分析及其对病毒复制的影响,Insect Molecular Biology

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家蚕病毒 miRNA BmCPV-miR-10 的功能分析及其对病毒复制的影响
Insect Molecular Biology ( IF 2.3 ) Pub Date : 2021-07-22 , DOI: 10.1111/imb.12725
Y Wang ₁ , S Lin ₁ , Z Zhao ₁ , P Xu ₁ , K Gao ₁ , H Qian _{1,

2} , Z Zhang ₁ , X Guo _{1,

2}

Affiliation

Bombyx mori cypovirus (BmCPV) 是家蚕( B. mori )的重要病原体，家蚕是经济益虫。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，病毒编码的微小RNA（miRNA）在调节宿主-病原体相互作用中起着至关重要的作用，但迄今为止关于昆虫病毒尤其是RNA病毒编码的miRNA的报道很少。在本研究中，鉴定了 BmCPV 基因组 RNA 第 10 段 BmCPV-miR-10 编码的推定 miRNA，并对其进行了功能分析。可以通过茎环 RT-PCR（逆转录聚合酶链反应）在感染 BmCPV 的家蚕幼虫的中肠中检测推定的 BmCPV-miR-10 的表达。BmCSDE1（家蚕冷休克域E1蛋白）基因被预测为BmCPV-miR-10的候选靶基因，其miRNA结合位点位于其mRNA的3'非翻译区。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 细胞中验证了推定的 BmCPV-miR-10 对BmCSDE1的调节作用。qRT-PCR（定量实时PCR）结果表明，假定的BmCPV-miR-10 可以抑制BmCSDE1的表达。通过将BmCPV-miR-10模拟物注射到感染BmCPV的家蚕幼虫体内，进一步证明推定的BmCPV-miR-10在体内可以抑制BmCSDE1的表达，进而抑制BmAPAF-1的表达。( B. mori凋亡蛋白酶激活因子 1)，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，进而抑制BmPaf-1的表达，从而为病毒复制和增殖创造有利的细胞内环境。

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Functional analysis of a putative Bombyx mori cypovirus miRNA BmCPV-miR-10 and its effect on virus replication

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.

更新日期：2021-07-22

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