Background & Aims

Hepatitis C virus (HCV) infections most often result in chronic outcomes, although the virus constantly produces replication intermediates, in particular double-stranded RNA (dsRNA), representing potent inducers of innate immunity. We aimed to characterize the fate of HCV dsRNA in hepatocyte cultures to identify mechanisms contributing to viral persistence in presence of an active innate immune response.

Methods

We analyzed hepatocyte-based culture models for HCV for induction of innate immunity, secretion of virus positive- or negative-strand RNA, and viral replication using different quantification methods and microscopy techniques. Expression of pattern recognition receptors was reconstituted in hepatoma cells by lentiviral transduction.

Results

HCV-infected cells secrete substantial amounts of virus positive- and negative-strand RNAs in extracellular vesicles (EVs), toward the apical and basolateral domain of hepatocytes. Secretion of negative-strand RNA was independent from virus production, and viral RNA secreted in EVs contained higher relative amounts of negative-strands, indicating that mostly virus dsRNA is released. A substantial part of viral replication complexes and dsRNA was found in the endosomal compartment and multivesicular bodies, indicating that secretion of HCV replication intermediates is mediated by the exosomal pathway. Block of vesicle release in HCV-positive cells increased intracellular dsRNA levels and increased activation of toll-like receptor 3, inhibiting HCV replication.

Conclusions

Using hepatocyte-based culture models for HCV, we found a portion of HCV dsRNA intermediates to be released from infected cells in EVs, which reduces activation of toll-like receptor 3. This represents a novel mechanism how HCV evades host immune responses, potentially contributing to viral persistence.

中文翻译：

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丙型肝炎病毒复制中间体的分泌减少了肝细胞中Toll样受体3的激活

背景与目标

丙型肝炎病毒（HCV）感染通常会导致慢性后果，尽管该病毒会不断产生复制中间体，尤其是双链RNA（dsRNA），代表先天免疫的有效诱导剂。我们旨在表征HCV dsRNA在肝细胞培养物中的命运，以鉴定在存在活跃的先天免疫应答的情况下有助于病毒持久性的机制。

方法

我们使用不同的定量方法和显微镜技术分析了基于肝细胞的HCV培养模型，用于诱导先天免疫，病毒正链或负链RNA的分泌以及病毒复制。通过慢病毒转导在肝癌细胞中重构模式识别受体的表达。

结果

HCV感染的细胞在细胞外囊泡（EVs）中向肝细胞的顶端和基底外侧区域分泌大量病毒正链和负链RNA。负链RNA的分泌与病毒的产生无关，并且电动汽车中分泌的病毒RNA包含较高的负链相对量，这表明大部分病毒dsRNA被释放。在内体区室和多囊泡体中发现了很大一部分病毒复制复合物和dsRNA，表明HCV复制中间体的分泌是由外体途径介导的。HCV阳性细胞中囊泡释放的阻滞增加了细胞内dsRNA的水平，并增加了Toll样受体3的活化，从而抑制了HCV复制。

结论

使用基于肝细胞的HCV培养模型，我们发现一部分HCV dsRNA中间体会从EV的受感染细胞中释放出来，从而减少了Toll样受体3的活化。这代表了HCV规避宿主免疫反应的新机制，可能有助于对病毒的持久性。