Pan Zhong-Hua, Wu Ping, Gao Kun, Hou Cheng-Xiang, Qin Guang-Xing, Geng Tao, Guo Xi-Jie
School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212018, China; Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, China; School of Biology & Basic Medical Science, Soochow University, Suzhou 215123, China.
School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212018, China; Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, China.
Virus Res. 2017 Apr 2;233:86-94. doi: 10.1016/j.virusres.2017.03.009. Epub 2017 Mar 8.
Viral microRNAs (miRNAs) have been demonstrated to play important roles in virus-host interactions. Some RNA virus-encoded miRNAs have been reported to promote viral replication and may be used as potential drug targets. Bombyx mori cypovirus (BmCPV), an important pathogen of silkworm, is a double-stranded RNA virus frequently causing serious damages in sericulture. Research on miRNA encoded by BmCPV may be useful to elucidate the BmCPV-host interaction and to develop new anti-viral methods. In our previous study, small RNA libraries of the midgut of BmCPV-infected silkworm have been generated by deep sequencing and several BmCPV-encoded putative miRNAs were predicted. In this study, two putative miRNAs encoded by BmCPV were identified and then validated by stem-loop qRT-PCR and northern blot. They are BmCPV-miR-3 encoded by the third genomic RNA segment of BmCPV (478-497bp) and BmCPV-miR-5 encoded by the fifth genomic RNA segment (2481-2500bp), both are 20bp and encoded by ORF regions. miRNA expression could be detected as early as 5h after BmCPV infection, and the expression level of BmCPV-miR-3 is much higher than that of BmCPV-miR-5 in the course of infection. Three potential target genes were predicted in the host genome, two for BmCPV-miR-3 and one for BmCPV-miR-5, but just one in the virus genome for BmCPV-miR-3 only, with the binding sites all in coding regions. Dual luciferase assay and qRT-PCR indicated that BmCPV-miR-3 could down-regulate the expression of both its two target genes, but no regulatory effect by BmCPV-miR-5 on its target gene was detected. In contrast, BmCPV-miR-3 could up-regulate the viral target. This is the first report that an insect double stranded RNA virus may generate miRNAs and the results obtained will benefit the future study of the functions of BmCPV-encoded miRNAs on viral replication and virus-host interaction.
病毒微小RNA（miRNA）已被证明在病毒与宿主的相互作用中发挥重要作用。据报道，一些RNA病毒编码的miRNA可促进病毒复制，可能用作潜在的药物靶点。家蚕质型多角体病毒（BmCPV）是家蚕的一种重要病原体，是一种双链RNA病毒，经常在养蚕业中造成严重损害。对BmCPV编码的miRNA进行研究可能有助于阐明BmCPV与宿主的相互作用，并开发新的抗病毒方法。在我们之前的研究中，通过深度测序生成了感染BmCPV的家蚕中肠的小RNA文库，并预测了几种BmCPV编码的假定miRNA。在本研究中，鉴定了两种由BmCPV编码的假定miRNA，然后通过茎环qRT-PCR和Northern印迹进行验证。它们是由BmCPV的第三个基因组RNA片段（478 - 497bp）编码的BmCPV-miR-3和由第五个基因组RNA片段（2481 - 2500bp）编码的BmCPV-miR-5，两者均为20bp且由开放阅读框区域编码。在BmCPV感染后5小时即可检测到miRNA表达，并且在感染过程中BmCPV-miR-3的表达水平远高于BmCPV-miR-5。在宿主基因组中预测了三个潜在靶基因，BmCPV-miR-3有两个，BmCPV-miR-5有一个，但在病毒基因组中仅BmCPV-miR-3有一个，其结合位点均在编码区。双荧光素酶测定和qRT-PCR表明，BmCPV-miR-3可以下调其两个靶基因的表达，但未检测到BmCPV-miR-5对其靶基因的调节作用。相反，BmCPV-miR-3可以上调病毒靶标。这是关于昆虫双链RNA病毒可能产生miRNA的首次报道，所获得的结果将有利于未来对BmCPV编码的miRNA在病毒复制和病毒 - 宿主相互作用方面功能的研究。
