Hepatitis C Virus NS5A Protein Promotes the Lysosomal Degradation of Hepatocyte Nuclear Factor 1α via Chaperone-Mediated Autophagy
Hepatitis C Virus NS5A Protein Promotes the Lysosomal Degradation of Hepatocyte Nuclear Factor 1α via Chaperone-Mediated Autophagy
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DOI：
10.1128/jvi.00639-18
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发表时间：
2018-07-01
影响因子：
5.4
通讯作者：
Shoji, Ikuo
中科院分区：
文献类型：
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作者：
Matsui, Chieko;Deng, Lin;Shoji, Ikuo
丙型肝炎病毒（HCV）感染与2型糖尿病密切相关。我们报道，HCV 感染通过与 HCV 非结构蛋白 5A (NS5A) 蛋白相互作用，诱导肝细胞核因子 1 α (HNF-1 α) 溶酶体降解，从而抑制 GLUT2 基因表达。 NS5A 引起的 HNF-1 α 选择性降解的分子机制尚不清楚。分子伴侣介导的自噬（CMA）是一种选择性溶酶体降解途径。在这里，我们研究了 CMA 是否参与 HCV 感染细胞中 HNF-1 α 的选择性降解，并观察到 HNF-1 α 从氨基酸 (aa) 130 到 aa 134 的五肽与 CMA 靶向基序（也称为 KFERQ 基序）的规则相匹配。胞质伴侣蛋白、70 kDa 的热休克同源蛋白 (HSC70) 和溶酶体膜蛋白、溶酶体相关膜蛋白 2A 型 (LAMP-2A) 是 CMA 的关键成分。免疫沉淀分析显示，HNF-1 α 与 HSC70 发生免疫共沉淀，而 HNF-1 α 的 Q130A 突变（第 130 位 Q 突变为 A）破坏了与 HSC70 的相互作用，表明 HNF-1 α 的 CMA 靶向基序对于与 HSC70 的关联非常重要。免疫沉淀分析显示，NS5A 量的增加增强了 HNF-1 α 与 HSC70 的关联。为了确定 LAMP-2A 是否在 HNF-1 α 蛋白的降解中发挥作用，我们通过 RNA 干扰敲低了 LAMP-2A mRNA；这种通过小干扰 RNA (siRNA) 的敲除恢复了 HCV J6/JFH1 感染细胞中 HNF-1 α 蛋白的水平。该结果表明 HNF-1 α 的降解需要 LAMP-2A。免疫荧光研究揭示了 NS5A 和 HNF-1 α 在溶酶体中的共定位。根据我们的研究结果，我们提出 HCV NS5A 与 HSC70 相互作用，并将 HSC70 招募到 HNF-1 α，从而通过 CMA 促进 HNF-1 α 的溶酶体降解。 重要性 许多病毒使用蛋白质降解系统，例如泛素蛋白酶体途径或自噬途径，以促进病毒传播和病毒发病机制。我们研究了丙型肝炎病毒 (HCV) NS5A 蛋白诱导的肝细胞核因子 1 α (HNF-1 α) 选择性溶酶体降解的机制细节。通过定点诱变，我们证明 HNF-1 α 在 HNF-1 α 的 POU 特异性结构域内含有五肽分子伴侣介导的自噬 (CMA) 靶向基序。 CMA 靶向基序对于与 HSC70 的关联非常重要。 NS5A 引起的 HNF-1 α 降解需要 LAMP-2A。我们提出，HCV NS5A 与 CMA 机制的关键组成部分 HSC70 相互作用，并将 HSC70 招募到 HNF-1 α 上，以靶向 HNF-1 α 进行 CMA 介导的溶酶体降解，从而促进 HCV 发病机制。我们发现 HCV NS5A 在 HNF-1 α 的 CMA 依赖性降解中发挥作用。我们的结果可能有助于更好地了解 CMA 在 HCV 发病机制中的作用。
Hepatitis C virus (HCV) infection is closely associated with type 2 diabetes. We reported that HCV infection induces the lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1 alpha) via interaction with HCV nonstructural protein 5A (NS5A) protein, thereby suppressing GLUT2 gene expression. The molecular mechanisms of selective degradation of HNF-1 alpha caused by NS5A are largely unknown. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway. Here, we investigated whether CMA is involved in the selective degradation of HNF-1 alpha in HCV-infected cells and observed that the pentapeptide spanning from amino acid (aa) 130 to aa 134 of HNF-1 alpha matches the rule for the CMA-targeting motif, also known as KFERQ motif. A cytosolic chaperone protein, heat shock cognate protein of 70 kDa (HSC70), and a lysosomal membrane protein, lysosome-associated membrane protein type 2A (LAMP-2A), are key components of CMA. Immunoprecipitation analysis revealed that HNF-1 alpha was coimmunoprecipitated with HSC70, whereas the Q130A mutation (mutation of Q to A at position 130) of HNF-1 alpha disrupted the interaction with HSC70, indicating that the CMA-targeting motif of HNF-1 alpha is important for the association with HSC70. Immunoprecipitation analysis revealed that increasing amounts of NS5A enhanced the association of HNF-1 alpha with HSC70. To determine whether LAMP-2A plays a role in the degradation of HNF-1 alpha protein, we knocked down LAMP-2A mRNA by RNA interference; this knockdown by small interfering RNA (siRNA) recovered the level of HNF-1 alpha protein in HCV J6/JFH1-infected cells. This result suggests that LAMP-2A is required for the degradation of HNF-1 alpha. Immunofluorescence study revealed colocalization of NS5A and HNF-1 alpha in the lysosome. Based on our findings, we propose that HCV NS5A interacts with HSC70 and recruits HSC70 to HNF-1 alpha, thereby promoting the lysosomal degradation of HNF-1 alpha via CMA.IMPORTANCE Many viruses use a protein degradation system, such as the ubiquitin-proteasome pathway or the autophagy pathway, for facilitating viral propagation and viral pathogenesis. We investigated the mechanistic details of the selective lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1 alpha) induced by hepatitis C virus (HCV) NS5A protein. Using site-directed mutagenesis, we demonstrated that HNF-1 alpha contains a pentapeptide chaperone-mediated autophagy (CMA)-targeting motif within the POU-specific domain of HNF-1 alpha. The CMA-targeting motif is important for the association with HSC70. LAMP-2A is required for degradation of HNF-1 alpha caused by NS5A. We propose that HCV NS5A interacts with HSC70, a key component of the CMA machinery, and recruits HSC70 to HNF-1 alpha to target HNF-1 alpha for CMA-mediated lysosomal degradation, thereby facilitating HCV pathogenesis. We discovered a role of HCV NS5A in CMA-dependent degradation of HNF-1 alpha. Our results may lead to a better understanding of the role of CMA in the pathogenesis of HCV.