MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China.
MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People's Republic of China
J Virol. 2018 Aug 29;92(18). doi: 10.1128/JVI.00503-18. Print 2018 Sep 15.
RNA interference (RNAi) is widely used in gene knockdown analysis and as a tool to screen host genes involved in viral infection. Owing to the limitations of transducing cells with synthetic small interfering RNAs (siRNAs), lentiviral short hairpin RNA (shRNA) vectors are more widely used. However, we found that stable transduction with lentiviral shRNA vectors inhibited hepatitis C virus (HCV) propagation in human hepatoma cells. We found by microRNA (miRNA) microarray analysis that this inhibition was induced by the alteration of host miRNA expression. In addition to one miRNA (miR-196b-5p) previously reported to be involved in HCV infection, other miRNAs (miR-216a-5p, -216b-5p, 217, and -30b-5p) were found to influence HCV infection in this study. Further studies suggested that this effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration site of the lentiviral vector might determine the change in miRNA expression. Moreover, the upregulation of JUN contributed to the dysregulation of miR-216a-5p, -216b-5p, and -217 in stably transduced cells. Although the changes in miRNA expression were beneficial for inhibiting HCV infection in our study, this off-target effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy. We found that stable transduction with lentiviral shRNA was able to nonspecifically inhibit HCV infection by the dysregulation of host miRNAs. Previous studies showed that the overexpression of shRNAs oversaturated the host miRNA pathways to inhibit HCV infection. In contrast, the miRNA machinery was not affected in our study. Knockout studies suggested that the nonspecific effect was independent of the transcription of shRNAs. The lentiviral vector itself and the integration sites in the host genome determined the changes in miRNAs. Stable transduction with lentiviral vectors was able to increase the expression of JUN, which in turn upregulated miR-216a-5p, miR-216b-5p, and miR-217. miR-216a-5p and miR-216b-5p might inhibit HCV by suppressing the host autophagic machinery. Our study suggested a novel nonspecific effect of lentiviral vectors, and this side effect should be considered when transduction with lentiviral vectors is performed for other purposes, especially in therapy.
RNA 干扰 (RNAi) 广泛应用于基因敲低分析和作为筛选病毒感染宿主基因的工具。由于合成的小干扰 RNA (siRNA) 转导细胞的局限性，慢病毒短发夹 RNA (shRNA) 载体应用更广泛。然而，我们发现慢病毒 shRNA 载体的稳定转导抑制了人肝癌细胞中的丙型肝炎病毒 (HCV) 复制。通过 microRNA (miRNA) 微阵列分析发现，这种抑制是由宿主 miRNA 表达的改变引起的。除了先前报道与 HCV 感染有关的一个 miRNA (miR-196b-5p) 外，本研究还发现其他 miRNAs (miR-216a-5p、-216b-5p、217 和 -30b-5p) 影响 HCV 感染。进一步的研究表明，这种效应不依赖于 shRNAs 的转录。慢病毒载体本身及其整合位点可能决定 miRNA 表达的变化。此外，JUN 的上调导致稳定转导细胞中 miR-216a-5p、-216b-5p 和 -217 的失调。虽然在本研究中，miRNA 表达的变化有利于抑制 HCV 感染，但在使用慢病毒载体进行其他目的转导时，应考虑这种脱靶效应，尤其是在治疗中。我们发现，慢病毒 shRNA 的稳定转导通过宿主 miRNAs 的失调能够非特异性地抑制 HCV 感染。先前的研究表明，shRNAs 的过表达使宿主 miRNA 途径饱和从而抑制 HCV 感染。相反，在本研究中 miRNA 机制不受影响。敲除研究表明，这种非特异性效应不依赖于 shRNAs 的转录。慢病毒载体本身及其在宿主基因组中的整合位点决定了 miRNAs 的变化。慢病毒载体的稳定转导能够增加 JUN 的表达，进而上调 miR-216a-5p、miR-216b-5p 和 miR-217。miR-216a-5p 和 miR-216b-5p 可能通过抑制宿主自噬机制抑制 HCV。本研究提示了慢病毒载体的一种新的非特异性效应，在使用慢病毒载体进行其他目的转导时，尤其是在治疗中，应考虑这种副作用。
