带有尿苷凸起的免疫刺激性siRNA可有效抑制HBV和激活先天免疫

StephenW

Immunostimulatory siRNA with a uridine bulge leads to potent inhibition of HBV and activation of innate immunity

    Tingyu Lan, Zhiqiang Wei, Yulin He, Song Wan, Li Liu, Bin Cheng, Ruimin Li, Hongxia Chen, Guohua Liu & Zhongji Meng

Virology Journal volume 18, Article number: 37 (2021) Cite this article

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Abstract
Background

Hepatitis B virus (HBV) infection is difficult to cure. HBV-specific immune tolerance plays a key role in HBV persistence, and enhancing cellular and humoral immunity will improve the control of HBV infection. The purpose of the study was to explore the anti-HBV and immunostimulatory effects of msiRNAs that introduce unpaired uridine bulges in the passenger strand.
Methods

msiRNAs targeting the HBV S and X genes were designed and named msiHBs and msiHBx, respectively. HepG2 cells were cotransfected with siRNA or msiRNA and the HBV replication-competent plasmid pHY106-wta or pHY106-X15. HepG2.215 cells were transfected with siRNA or msiRNA. The levels of HBsAg, HBeAg, and the cytokines TNF-α, IFN-α, IFN-β, IL-1α, and IL-6 in the culture supernatant was detected by ELISA. The levels of intracellular HBV RNA, nuclear HBV replication intermediates, and HBV DNA in the supernatant were measured by quantitative RT-PCR and PCR. The levels of HBV replication intermediates were detected by Southern blotting. Peripheral blood mononuclear cells were transfected with siRNA or msiRNA, and the levels of secreted cytokines IFN-α and IFN-β were detected by ELISA. The bioactivity of type I interferons in the supernatants was detected by the virus protection assay.
Results

msiHBx treatment led to a significant decrease in HBsAg (to a negative level) and HBV DNA (95.5%) in the supernatant and intrahepatocellular HBV replication intermediates (89.8%) in HepG2 cells with transient HBV replication and in HepG2.2.15 cells. There was no significant difference between msiHBx and siHBx in terms of the reduction in HBV proteins and HBV replication (P > 0.05). Compared with siHBx, msiHBx treatment of HepG2 cells transfected with the HBV replication-competent plasmid led to a significant increase in the levels of the antiviral cytokines TNF-α (3.3-fold), IFN-α (1.4-fold), and IFN-β (2.5-fold) (P < 0.01), without upregulation of the proinflammatory cytokines IL-1α and IL-6. The virus protection assay results showed msiHBx-mediated type I interferons effectively protected L929 cells against ECMV infection.
Conclusions

msiHBx could effectively inhibit HBV expression and replication and induce an antiviral innate immune response without proinflammatory activation. The dual RNAi and immunostimulatory activity of msiRNAs may play an important role in the control of HBV infection.

StephenW

带有尿苷凸起的免疫刺激性siRNA可有效抑制HBV和激活先天免疫

    兰婷玉，魏志强，何玉林，宋万，刘力，程斌，李瑞敏，陈红霞，刘国华＆孟仲吉

Virology Journal卷18，文章编号：37（2021）引用本文

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抽象的
背景

乙型肝炎病毒（HBV）感染难以治愈。 HBV特异性免疫耐受在HBV持久性中起关键作用，增强细胞和体液免疫将改善对HBV感染的控制。这项研究的目的是探讨msiRNA的抗HBV和免疫刺激作用，该作用在过客链中引入了未配对的尿苷凸起。
方法

设计了靶向HBV S和X基因的msiRNA，分别命名为msiHBs和msiHBx。用siRNA或msiRNA和具有HBV复制能力的质粒pHY106-wta或pHY106-X15共转染HepG2细胞。用siRNA或msiRNA转染HepG2.215细胞。通过ELISA检测培养上清液中的HBsAg，HBeAg和细胞因子TNF-α，IFN-α，IFN-β，IL-1α和IL-6的水平。通过定量RT-PCR和PCR测量上清液中的细胞内HBV RNA，核HBV复制中间体和HBV DNA的水平。通过Southern印迹检测HBV复制中间体的水平。用siRNA或msiRNA转染外周血单个核细胞，并通过ELISA检测分泌的细胞因子IFN-α和IFN-β的水平。通过病毒保护测定法检测上清液中I型干扰素的生物活性。
结果

msiHBx治疗导致具有短暂HBV复制的HepG2细胞和HepG2.2.15细胞的上清液和肝细胞内HBV复制中间体的HBsAg（降至阴性水平）和HBV DNA显着降低（95.5％）。在减少HBV蛋白和减少HBV复制方面，msiHBx和siHBx之间没有显着差异（P> 0.05）。与siHBx相比，msiHBx处理可转染HBV复制质粒的HepG2细胞导致抗病毒细胞因子TNF-α（3.3倍），IFN-α（1.4倍）和IFN-α的水平显着增加。 β（2.5倍）（P <0.01），而没有促炎性细胞因子IL-1α和IL-6的上调。病毒保护检测结果表明，msiHBx介导的I型干扰素有效保护L929细胞免受ECMV感染。
结论

msiHBx可以有效抑制HBV的表达和复制，并诱导抗病毒的先天性免疫反应而无促炎性激活。 msiRNA的双重RNAi和免疫刺激活性可能在控制HBV感染中起重要作用。

StephenW

https://virologyj.biomedcentral. ... 985-021-01509-z.pdf