Background & aims

Hepatitis B virus (HBV) and D virus (HDV) co-infections cause the most severe form of viral hepatitis. HDV induces an innate immune response, but it is unknown how the host cell senses HDV and if this defense affects HDV replication. We aim to characterize interferon (IFN) activation by HDV, identify the responsible sensor and evaluate the effect of IFN on HDV replication.

Methods

HDV and HBV susceptible hepatoma cell lines and primary human hepatocytes (PHH) were used for infection studies. Viral markers and cellular gene expression were analyzed at different time points after infection. Pattern recognition receptors (PRRs) required for HDV-mediated IFN activation and the impact on HDV replication were studied using stable knock-down or overexpression of the PRRs.

Results

Microarray analysis revealed that HDV but not HBV infection activated a broad range of interferon stimulated genes (ISGs) in HepG2^NTCP cells. HDV strongly activated IFN-β and IFN-λ in cell lines and PHH. HDV induced IFN levels remained unaltered upon RIG-I or TLR3 knock-down, but were almost completely abolished upon MDA5 depletion. Conversely, overexpression of MDA5 but not RIG-I and TLR3 in Huh7.5^NTCP cells partially restored ISG induction. During long-term infection, IFN levels gradually diminished in both HepG2^NTCP and HepaRG^NTCP cell lines. MDA5 depletion had little effect on HDV replication despite dampening HDV-induced IFN response. Moreover, treatment with type I or type III IFNs did not abolish HDV replication.

Conclusions

Active replication of HDV induces an IFN-β/λ response, which is predominantly mediated by MDA5. This IFN response and exogenous IFN treatment have only a moderate effect on HDV replication in vitro indicating the adaption of HDV replication to an IFN activated state.

Lay summary

In contrast to HBV, infection with HDV induces a strong IFN-β/λ response in innate immune competent cell lines. MDA5 is the key sensor for the recognition of HDV replicative intermediates. An IFN-activated state did not abrogate HDV replication in vitro indicating resistance to self-induced innate immune responses and therapeutic IFN treatment.

中文翻译：

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MDA5感测D型肝炎病毒复制并在肝细胞中诱导IFN-β/λ反应

背景与目标

乙型肝炎病毒（HBV）和D型病毒（HDV）合并感染是最严重的病毒性肝炎形式。HDV会诱发先天性免疫反应，但尚不清楚宿主细胞如何感测HDV，以及这种防御是否影响HDV复制。我们旨在表征HDV干扰素（IFN）的激活，确定负责任的传感器，并评估IFN对HDV复制的影响。

方法

HDV和HBV易感性肝癌细胞系和原代人肝细胞（PHH）用于感染研究。在感染后的不同时间点分析病毒标志物和细胞基因表达。使用稳定的敲除或过表达的PRR研究了HDV介导的IFN激活所需的模式识别受体（PRR）及其对HDV复制的影响。

结果

基因芯片分析显示，HDV而非HBV感染激活了HepG2 ^NTCP细胞中广泛的干扰素刺激基因（ISG）。HDV在细胞系和PHH中强烈激活IFN-β和IFN-λ。HDV诱导的IFN水平在RIG-1或TLR3敲低后保持不变，但在MDA5耗尽时几乎完全被消除。相反，Huh7.5 ^NTCP细胞中MDA5的过表达而不是RIG-1和TLR3的过表达部分恢复了ISG的诱导。在长期感染期间，HepG2 ^NTCP和HepaRG ^NTCP细胞系中的IFN水平逐渐降低。尽管减弱了HDV诱导的IFN反应，但MDA5消耗对HDV复制几乎没有影响。而且，用I型或III型IFN治疗不能消除HDV复制。

结论

HDV的主动复制诱导IFN-β/λ反应，该反应主要由MDA5介导。这种IFN反应和外源性IFN治疗在体外对HDV复制仅具有中等作用，表明HDV复制适应IFN激活状态。

放置摘要

与HBV相反，HDV感染在先天免疫感受态细胞系中诱导强烈的IFN-β/λ反应。MDA5是识别HDV复制中间体的关键传感器。IFN激活状态不能在体外消除HDV复制，表明对自身诱导的先天免疫应答和IFN治疗具有抵抗力。