IRG, EA 4517, Immunopathology and Infection Research Grouping, CHR North Felix Guyon and University of La Reunion, St Denis, Ile de la Reunion, France.
Virol J. 2011 Sep 8;8:432. doi: 10.1186/1743-422X-8-432.
Chikungunya Virus (ChikV) surprised by a massive re-emerging outbreak in Indian Ocean in 2006, reaching Europe in 2007 and exhibited exceptional severe physiopathology in infants and elderly patients. In this context, it is important to analyze the innate immune host responses triggered against ChikV. Autophagy has been shown to be an important component of the innate immune response and is involved in host defense elimination of different pathogens. However, the autophagic process was recently observed to be hijacked by virus for their own replication. Here we provide the first evidence that hallmarks of autophagy are specifically found in HEK.293 infected cells and are involved in ChikV replication.
To test the capacity of ChikV to mobilize the autophagic machinery, we performed fluorescence microscopy experiments on HEK.GFP.LC3 stable cells, and followed the LC3 distribution during the time course of ChikV infection. To confirm this, we performed electron microscopy on HEK.293 infected cells. To test the effect of ChikV-induced-autophagy on viral replication, we blocked the autophagic process, either by pharmacological (3-MA) or genetic inhibition (siRNA against the transcript of Beclin 1, an autophagic protein), and analyzed the percentage of infected cells and the viral RNA load released in the supernatant. Moreover, the effect of induction of autophagy by Rapamycin on viral replication was tested.
The increasing number of GFP-LC3 positive cells with a punctate staining together with the enhanced number of GFP-LC3 dots per cell showed that ChikV triggered an autophagic process in HEK.293 infected cells. Those results were confirmed by electron microscopy analysis since numerous membrane-bound vacuoles characteristic of autophagosomes were observed in infected cells. Moreover, we found that inhibition of autophagy, either by biochemical reagent and RNA interference, dramatically decreases ChikV replication.
Taken together, our results suggest that autophagy may play a promoting role in ChikV replication. Investigating in details the relationship between autophagy and viral replication will greatly improve our knowledge of the pathogenesis of ChikV and provide insight for the design of candidate antiviral therapeutics.
基孔肯雅病毒（ChikV）于 2006 年在印度洋大规模爆发，令人惊讶，2007 年到达欧洲，并在婴儿和老年患者中表现出异常严重的病理生理学。在这种情况下，分析宿主对 ChikV 产生的先天免疫反应非常重要。自噬已被证明是先天免疫反应的一个重要组成部分，并且参与宿主防御消除不同的病原体。然而，最近发现病毒会劫持自噬过程来进行自身复制。在这里，我们首次提供证据表明，自噬的特征标志专门存在于 HEK.293 感染的细胞中，并参与 ChikV 的复制。
为了测试 ChikV 调动自噬机制的能力，我们对 HEK.GFP.LC3 稳定细胞进行荧光显微镜实验，并在 ChikV 感染过程中跟踪 LC3 的分布。为了证实这一点，我们对感染细胞进行电子显微镜检查。为了测试 ChikV 诱导的自噬对病毒复制的影响，我们通过药理学（3-MA）或基因抑制（针对自噬蛋白 Beclin 1 转录本的 siRNA）阻断自噬过程，并分析感染细胞的百分比和释放到上清液中的病毒 RNA 负荷。此外，还测试了 Rapamycin 诱导自噬对病毒复制的影响。
具有点状染色的 GFP-LC3 阳性细胞数量增加，每个细胞中的 GFP-LC3 点数量增加，表明 ChikV 在 HEK.293 感染细胞中触发了自噬过程。这些结果通过电子显微镜分析得到证实，因为在感染细胞中观察到大量膜结合的空泡，这些空泡是自噬体的特征。此外，我们发现自噬的抑制，无论是通过生化试剂还是 RNA 干扰，都显著降低了 ChikV 的复制。
总之，我们的结果表明自噬可能在 ChikV 复制中起促进作用。详细研究自噬与病毒复制之间的关系将极大地提高我们对 ChikV 发病机制的认识，并为设计候选抗病毒治疗药物提供启示。
