Regulation of Hepatitis B Virus Infection by Rab5, Rab7, and the Endolysosomal Compartment
Regulation of Hepatitis B Virus Infection by Rab5, Rab7, and the Endolysosomal Compartment
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DOI：
10.1128/jvi.00393-13
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发表时间：
2013-06-01
影响因子：
5.4
通讯作者：
Branza-Nichita, Norica
中科院分区：
文献类型：
--
作者：
Macovei, Alina;Petrareanu, Catalina;Branza-Nichita, Norica
尽管在破译人类B型肝炎病毒（HBV）进入宿主细胞方面取得了重要进展，但生命周期早期步骤的许多方面仍然完全不清楚。内吞作用后，HBV必须通过复杂的内吞途径网络到达细胞核并启动复制。除了将病毒颗粒引导到复制位点之外，内体囊泡可能在感染中起关键作用，为病毒脱壳和核衣壳释放提供适当的环境。在这项工作中，我们调查了HBV颗粒在允许细胞内的运输。在HepaRG细胞中，使用稳定的和可诱导的短发夹RNA（shRNA）表达系统调节关键Rab蛋白（参与导致不同细胞内位置的特定途径）的表达。通过共聚焦显微镜和流式细胞术使用特异性标记物证明了新开发细胞的运输特性。结果表明，HBV感染强烈依赖于Rab 5和Rab 7的表达，表明HBV从早期到成熟内体的转运是病毒生命周期中的一个步骤所必需的。这可能涉及二硫键连接的包膜蛋白的还原，因为内吞途径的氧化还原电位的改变导致感染的抑制。HBV感染细胞的亚细胞分级显示，病毒颗粒被进一步转运到溶酶体。有趣的是，感染并不依赖于溶酶体活性，这表明运输到这个隔间是一个“死胡同”的路线。总之，这些数据增加了我们对HBV-宿主细胞相互作用控制感染早期阶段的理解。
Despite important progress toward deciphering human hepatitis B virus (HBV) entry into host cells, many aspects of the early steps of the life cycle remained completely obscure. Following endocytosis, HBV must travel through the complex network of the endocytic pathway to reach the cell nucleus and initiate replication. In addition to guiding the viral particles to the replication site, the endosomal vesicles may play a crucial role in infection, providing the appropriate environment for virus uncoating and nucleocapsid release. In this work, we investigated the trafficking of HBV particles internalized in permissive cells. Expression of key Rab proteins, involved in specific pathways leading to different intracellular locations, was modulated in HepaRG cells, using a stable and inducible short hairpin RNA (shRNA) expression system. The trafficking properties of the newly developed cells were demonstrated by confocal microscopy and flow cytometry using specific markers. The results showed that HBV infection strongly depends on Rab5 and Rab7 expression, indicating that HBV transport from early to mature endosomes is required for a step in the viral life cycle. This may involve reduction of disulfide bond-linked envelope proteins, as alteration of the redox potential of the endocytic pathway resulted in inhibition of infection. Subcellular fractionation of HBV-infected cells showed that viral particles are further transported to lysosomes. Intriguingly, infection was not dependent on the lysosomal activity, suggesting that trafficking to this compartment is a "dead-end" route. Together, these data add to our understanding of the HBV-host cell interactions controlling the early stages of infection.