LMBD1蛋白質參與C型肝炎病毒的增殖

Abstract

C 型肝炎病毒感染是慢性肝炎，肝硬化及肝癌的主要病因，影響全球約兩億人口。C 型肝炎病毒核心蛋白質形成C 型肝炎病毒顆粒的核衣殼。核心蛋白質在內質網生成並形成二聚體，成熟的核心蛋白質才座落至脂滴(lipid droplets) 表面。雖然已知核心蛋白質具有許多功能，包括RNA 結合能力，而且與脂滴形成有關，C 型肝炎病毒顆粒如何組裝之機制還不是很清楚。脂滴為一細胞內的胞器，不僅參與脂肪儲存，也涉及細胞內訊號傳遞和調節細胞內之囊泡運輸。最近的研究顯示，C 型肝炎病毒的生命週期需要C 型肝炎病毒核心蛋白質和脂滴之間的交互作用。在病毒感染的細胞中，核心蛋白質會座落在脂滴表面。在脂滴表面以及緊密圍繞脂滴的內質網膜上，核心蛋白質會自行組裝以驅動病毒顆粒之出芽過程。lmbrd1 基因位於染色體6q13 上，可轉譯出位於細胞質之LMBD1 蛋白質，其可能具有在溶酶體上負責將維生素B12 輸出至細胞質之功能。在本篇論文中，藉由免疫螢光檢測發現C 型肝炎病毒核心蛋白質與LMBD1 共同位在脂滴表面或接近脂滴的內質網膜上，形成環狀結構。當lmbrd1 基因被knockdown 時，會改變核心蛋白質的細胞分佈。利用免疫沉澱和Ni2+ pull-down 實驗確認LMBD1 與核心蛋白質之間的交互作用。由於C 型肝炎病毒核心蛋白質和脂滴之間的交互作用被認為與病毒組裝相關，因此，LMBD1 可能在C 型肝炎病毒的生活史上扮演重要角色。藉由subgenomic replicon 及具感染性之genomic replicon 系統的研究顯示宿主細胞蛋白質LMBD1 參與C 型肝炎病毒生活史中之複製(replication)、組裝(assembly) 及病毒釋出(release) 的過程。當lmbrd1 knockdown 時，subgenomic replicon 之複製會下降，但genomic replicon 的複製卻上升。進一步研究結果顯示當lmbrd1 knockdown時，病毒顆粒的組裝及釋出會受到抑制。這些結果建議LMBD1 不只影響病毒之複製，且於核心蛋白質的參與下亦會影響病毒之組裝及釋出。

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, affecting approximately 200 million people worldwide. HCV core protein forms the nucleocapsid of the HCV particle. The core protein dimer is formed at the endoplasmic reticulum where the core protein is initially produced and processed, and then located to the surface of lipid droplets (LDs). Although many functions of the core protein, including RNA binding ability and formation of LDs have been reported, how the HCV particle is assembled is not well understood. LDs are intracellular organelles involved not only in lipid storage but also in cell signaling and the regulation of intracellular vesicular trafficking. Recent studies suggest that the interaction between HCV core protein and LDs are required for the life cycle of HCV. In infected cells, HCV core protein is associated with the surface of LDs and the endoplasmic reticulum membranes closely surrounding these droplets. The self-assembly of the core protein drives virion budding. lmbrd1 gene located on chromosome 6q13 encodes cytoplasmic LMBD1 protein that functions as a putative lysosomal membrane exporter for vitamin B12. In this study, immunofluorescence assay demonstrated that HCV core protein co-localized with LMBD1, forming a ring-like structure on the surface of LDs. The subcellular distribution of the core protein was disturbed when lmbrd1 gene was knocked down. Immunoprecipitation and Ni2+ pull-down assay demonstrated a specific interaction of the core protein with LMBD1. Due to interactions between the core protein and LDs are considered to be associated with HCV assembly, LMBD1 was proposed to play an essential role in HCV morphogenesis. Studies performed with HCV subgenomic replicon and infectious genomic replicon systems indicate that LMBD1 is involved in the regulation of HCV life cycle including replication, assembly and release. When lmbrd1 was knocked down, replication was diminished in the subgenomic replicon system, but enhanced in the infectious replicon system. Further studies demonstrated a suppression of HCV morphogenesis when lmbrd1was knocked down. These data suggest that LMBD1 not only regulates virus replication, but also affects virus assembly and release in the presence of HCV core protein.