LMBRD1基因產物在C型肝炎病毒RNA複製及病毒組裝中扮演的角色

Abstract

C型肝炎病毒 (hepatitis C virus, HCV) 主要經由血液和體液傳染，感染後會造成C型肝炎，屬於全球性的感染疾病。HCV具有正向單股RNA基因體，在感染宿主細胞後,會以其正向單股RNA基因體進行轉譯，並在內質網的周邊形成適合病毒複製的特殊網狀結構，藉由細胞中油滴的幫助，進行病毒顆粒的組裝。本實驗室先前的研究發現，在細胞中表現LMBD1重組蛋白質時，此蛋白質與細胞中的油滴有明顯共位的現象。此外，在細胞中同時送入LMBD1以及HCV core蛋白質的表現質體後，也可觀察到LMBD1和core蛋白質的共位現象。利用共同免疫沉澱分析發現LMBD1以及core兩蛋白質在細胞中有交互作用。另外，在踢弱 (knockdown) LMBRD1基因表現的細胞感染HCV後，其細胞中的HCV負向基因體RNA量有上升的趨勢。為了更深入的瞭解LMBRD1基因與HCV基因體複製與病毒增殖的相關性，本研究利用HCVR次基因體細胞以及在HCV細胞培養系統中所產生的病毒 (HCVcc) 進行研究。結果顯示，在HCVR細胞中LMBRD1基因表現缺失會造成HCV的RNA負向基因體顯著降低，而其基因產物LMBD1以及NESI對於HCV基因體RNA複製皆有正向的調控作用。此外，HCV core蛋白質表現在踢弱LMBRD1基因的細胞中會使得HCV負向基因體量增加，推測core蛋白質可能會透過LMBD1蛋白質的幫助，穩定基因體RNA複製推進至病毒包裹的動態平衡。另一方面，在踢弱LMBRD1基因表現的情況下，被HCVcc感染的細胞所產生出來的病毒顆粒數量並無明顯差異，但其感染能力明顯降低。LMBRD1基因的表現影響了HCV基因體的複製以及病毒顆粒的組裝，然而其中的機轉仍然有待進一步的研究。

Hepatitis C virus (HCV) is mainly transmitted by blood and body fluids. It causes hepatitis C after infection, which is a worldwide infectious disease. HCV possesses positive single-strand RNA genome. Upon infection with a host cell, the positive single-strand RNA acts as a template and translates to viral proteins. In the HCV-infected cells, HCV induces formation of a special membrane structure around the endoplasmic reticulum suitable for the replication of viral genome. In addition, assembly of HCV particles is helped by lipid droplets. Previous studies from our laboratory demonstrated that the LMBD1 recombinant proteins colocalized with the lipid droplet marker ADRP in the cells. Co-localization of LMBD1 with the viral core protein was also detected when co-expression of LMBD1 and HCV core protein. Co-immunoprecipitation analysis revealed that LMBD1 protein interacts with core protein. In addition, the level of HCV antigenomic RNA tends to be higher in HCVcc-infected Huh7.5 cells to which LMBRD1 gene has been knocked-down (Huh-shLMBRD1) compared to the control Huh7.5 cells to which LUC gene has been knocked-down (Huh-shLUC). In this study, HCVR subgenomic replicon cells and HCVcc infection system were applied to further examine the roles of LMBRD1 gene on HCV replication and assembly. The results show a significant lower level of HCV antigenomic RNA in LMBRD1-knockdown HCVR cells compared to the control LUC-knockdown cells, both LMBRD1 gene products LMBD1 and NESI had positive effects on HCV replication. In addition, HCV core protein expression in LMBRD1-knockdown HCVR cells increased the levels of HCV antigenomic RNA, suggesting that core protein may interact with LMBD1 to stablize the turn over between viral replication and assembly. On the other hand, HCVcc produced from HCVcc-infected Huh-shLMBRD1 cells (HCVcc-shLMBRD1) had no significant difference in the number of virus particles compared to the control HCVcc produced from HCVcc-infected Huh-shLUC cells (HCVcc-shLUC) but significantly reduced the infectivity. Expression of LMBRD1 gene affects HCV replication and assembly, the detailed mechanism remains to be further elucidated.