- 學位論文
LMBD1蛋白質對C型肝炎病毒RNA複製的調節作用
Cellular Protein LMBD1 Regulates RNA Replication of Hepatitis C Virus
摘要
C 型肝炎病毒(Hepatitis C virus，HCV)主要經由血液和體液傳染，此病毒感 染會造成C 型肝炎，屬於全球性的感染疾病。目前全球大約有一億五千萬人是慢 性C 型肝炎患者。HCV 在感染宿主細胞後，會在內質網的周邊形成適合病毒複 製的特殊網狀結構，並且在細胞中的油滴(lipid droplet)上進行病毒顆粒的組裝。 本實驗室先前的研究發現，在細胞中過量表現LMBD1 重組蛋白質時，此蛋白質 與細胞中的油滴有明顯共位(co-localization)的現象，此外，在細胞中同時送入 LMBD1 與HCV 之core 蛋白質的表現質體後也可以觀察到LMBD1 與core 蛋白 質的共位現象。更進一步以shRNA 將lmbrd1 基因踢弱(knockdown)後，再過量 表現HCV 的core 蛋白質時，發現在踢弱lmbrd1 基因的細胞中，core 蛋白質的 分布會散開在細胞質中，無法保持在細胞核附近聚集的狀態。為了瞭解LMBD1 蛋白質與HCV 的相關性，本研究首先以免疫共沉澱的方式證實LMBD1 蛋白質 與core 蛋白質具有交互作用，接著以Huh7.5 細胞建立的感染性HCV 細胞培養 系統(infectious HCV cell culture system，infectious HCVcc system)探討LMBD1 蛋 白質在HCV 的生活史中所扮演的角色。以HCVcc conditional medium 感染lmbrd1 基因受到踢弱的Huh7.5 細胞，RT-qPCR 的結果發現在細胞內病毒的負向基因體 有明顯升高的情況。進一步研究顯示，LMBD1 蛋白質並不參與HCV 貼附與進 入宿主細胞的過程，而是參與在HCV 之病毒基因體的複製過程。然而在帶有病 毒次基因體(subgenome replicon)的細胞株中踢弱lmbrd1 基因時卻發現subgenome replicon 之負向基因體的複製受到抑制。另一方面，當過量表現LMBD1 重組蛋 白質在lmbrd1 基因受到踢弱的細胞中時，不論是在HCV 細胞培養系統或在HCV subgenome replicon 系統中，都具有增強HCV RNA 複製的現象。LMBD1 蛋白質 對HCV 基因體的調控是否和core 蛋白質與LMBD1 蛋白質的交互作用有關，有 待更進一步的探討。
並列摘要
Hepatitis C virus infection is mainly transmitted through blood and body fluids. The viral infection can cause the occurrence of hepatitis C which belongs to the global infection disease. There are 150 million people suffered from chronic hepatitis C infection in the world. HCV infection induces formation of membranous web around the endoplasmic reticulum where the replication of genome takes place. In our previous studies, ectopically expressed LMBD1 recombinant protein was found to localize on the surface of lipid droplets. In addition, colocalization of LMBD1 and HCV core protein was observed. Furthermore, distribution of the core protein became scattering when lmbrd1 was knocked down. Towards understanding the association of LMBD1 with HCV in this study, the interaction between LMBD1 and HCV core protein was confirmed by co-immunoprecipitation assay. The infectious HCV cell culture system (HCVcc system) was established to elucidate the roles of LMBD1 in HCV life cycle. Results from RT-qPCR demonstrated an enhancement of HCV anti-genomic RNAlevel in lmbrd1 knockdown cells. This is mainly due to an effect on the viral genome replication rather than the viral attachment and entry. However, the replication of viral RNA was reduced in lmbrd1 knockdown HCVR subgenome replicon cells. On the other hand, HCV RNA replication was enhanced in both lmbrd1 knockdown HCVcc and HCVR system when overexpressing LMBD1 protein. Whether the involvement of LMBD1 in HCV RNA replication is regulated through the interaction between LMBD1 and core protein needs to be further investigated.