Chen Tzu-Chun, Hsieh Chung-Han, Sarnow Peter
Department of Microbiology & Immunology, School of Medicine, Stanford University, Stanford, California, United States of America.
Department of Neurosurgery, School of Medicine, Stanford University, Stanford, California, United States of America.
PLoS Pathog. 2015 Aug 25;11(8):e1005116. doi: 10.1371/journal.ppat.1005116. eCollection 2015 Aug.
The small GTPase Rab27a has been shown to control membrane trafficking and microvesicle transport pathways, in particular the secretion of exosomes. In the liver, high expression of Rab27a correlates with the development of hepatocellular carcinoma. We discovered that low abundance of Rab27a resulted in decreased hepatitis C virus (HCV) RNA and protein abundances in virus-infected cells. Curiously, both cell-associated and extracellular virus yield decreased in Rab27a depleted cells, suggesting that reduced exosome secretion did not cause the observed effect. Instead, Rab27a enhanced viral RNA replication by a mechanism that involves the liver-specific microRNA miR-122. Rab27a surrounded lipid droplets and was enriched in membrane fractions that harbor viral replication proteins, suggesting a supporting role for Rab27a in viral gene expression. Curiously, Rab27a depletion decreased the abundance of miR-122, whereas overexpression of miR-122 in Rab27a-depleted cells rescued HCV RNA abundance. Because intracellular HCV RNA abundance is enhanced by the binding of two miR-122 molecules to the extreme 5' end of the HCV RNA genome, the diminished amounts of miR-122 in Rab27a-depleted cells could have caused destabilization of HCV RNA. However, the abundance of HCV RNA carrying mutations on both miR-122-binding sites and whose stability was supported by ectopically expressed miR-122 mimetics with compensatory mutations also decreased in Rab27a-depleted cells. This result indicates that the effect of Rab27a depletion on HCV RNA abundance does not depend on the formation of 5' terminal HCV/miR-122 RNA complexes, but that miR-122 has a Rab27a-dependent function in the HCV lifecycle, likely the downregulation of a cellular inhibitor of HCV gene expression. These findings suggest that the absence of miR-122 results in a vulnerability not only to exoribonucleases that attack the viral genome, but also to upregulation of one more cellular factor that inhibit viral gene expression.
小GTP酶Rab27a已被证明可控制膜运输和微囊泡运输途径，尤其是外泌体的分泌。在肝脏中，Rab27a的高表达与肝细胞癌的发展相关。我们发现Rab27a丰度降低导致病毒感染细胞中丙型肝炎病毒（HCV）RNA和蛋白质丰度下降。奇怪的是，Rab27a缺失的细胞中细胞相关病毒产量和细胞外病毒产量均下降，这表明外泌体分泌减少并非导致上述现象的原因。相反，Rab27a通过一种涉及肝脏特异性微小RNA miR-122的机制增强病毒RNA复制。Rab27a围绕脂滴并富集于含有病毒复制蛋白的膜组分中，表明Rab27a在病毒基因表达中起支持作用。奇怪的是，Rab27a缺失会降低miR-122的丰度，而在Rab27a缺失的细胞中过表达miR-122可挽救HCV RNA丰度。由于两个miR-122分子与HCV RNA基因组的极端5'端结合可增强细胞内HCV RNA丰度，Rab27a缺失细胞中miR-122量的减少可能导致HCV RNA不稳定。然而，在两个miR-122结合位点均携带突变且其稳定性由异位表达的具有补偿性突变的miR-122模拟物支持的HCV RNA丰度，在Rab27a缺失的细胞中也降低了。这一结果表明，Rab27a缺失对HCV RNA丰度的影响不依赖于5'末端HCV/miR-122 RNA复合物的形成，而是miR-122在HCV生命周期中具有Rab27a依赖性功能，可能是下调一种细胞内HCV基因表达抑制剂。这些发现表明，miR-122的缺失不仅导致易受攻击病毒基因组的外切核糖核酸酶影响，还导致一种以上抑制病毒基因表达的细胞因子上调。
