- 學位論文
探討具雙重功能之ESCRT-0因子HGS影響B型肝炎病毒轉錄與核心蛋白顆粒釋放之能力
The dual role of an ESCRT-0 component HGS in HBV transcription and naked capsid secretion
摘要
Endosomal sorting complexes required for transport (ESCRT) 路徑負責細胞內受泛素化修飾 (ubiquitinated) 之蛋白的辨識及運送。已知多種病毒皆利用ESCRT路徑以有效釋放病毒顆粒。為了進一步探討ESCRT路徑對於B型肝炎病毒生活史的影響，我們利用小片段核醣核酸siRNA抑制ESCRT 因子表現，並偵測B型肝炎病毒DNA複製與病毒顆粒生成能力的變化。此篩選找出許多可影響B型肝炎病毒複製的ESCRT分子，其中我們也特別針對ESCRT-0中的蛋白HGS (HRS, hepatocyte growth factor-regulated tyrosine kinase substrate) 進行深入探討。表現量異常的HGS可抑制B型肝炎病毒的轉錄與DNA複製能力，進而影響病毒體的生成；此外，利用高壓流體力學 (hydrodynamic) 注射方式將HGS表現質體送入老鼠體內，亦能顯著抑制肝細胞與血清中的B型肝炎病毒表現量。有趣的是，大量表現的HGS可同時促進B型肝炎核心蛋白顆粒 (naked capsids) 的釋放，無論是空心蛋白顆粒或是包裹未成熟HBV基因體 ss-DNA、RNA之核心蛋白顆粒。利用HBV突變株進行實驗，亦發現核心蛋白上富含精氨酸之C端區域，對於空心病毒體 (empty virion)的生成十分重要。不管HGS是否大量表現，C端缺失之B肝病毒核心蛋白 (HBc 1-147) 皆僅可形成空心蛋白顆粒，而無法被表面抗原(HBsAg)包裹成可釋放的病毒體。由實驗結果推測，HGS大量促進細胞中尚未成熟的核心蛋白顆粒釋放，阻斷它們在細胞中進行DNA複製、進而形成成熟基因體的機會，因此阻礙完整病毒體的生成。同時，HGS和核心蛋白之結合與其泛素化修飾無關，兩者亦傾向共處於細胞膜周圍而非零星聚集於核內體。以上研究顯示適當表現量的HGS對於B型肝炎病毒而言十分重要；除了影響病毒轉錄能力外， HGS亦可藉由促進核心蛋白顆粒釋放以干擾完整病毒體的生成。根據HGS之多功能角色，B型肝炎病毒體與核心蛋白顆粒顯然使用不同的釋放途徑。
並列摘要
The endosomal sorting complexes required for transport (ESCRT) is an important cellular machinery for the sorting and trafficking of ubiquitinated cargos. It is also known that a number of viruses may utilize the ESCRT modules for egress. To investigate the relationship between ESCRT and hepatitis B virus (HBV), we conducted an siRNA screening of ESCRT components for their potential effect on HBV replication and virion release. We identified a number of ESCRT factors required for HBV replication, and focused our study here on HGS (HRS, hepatocyte growth factor-regulated tyrosine kinase substrate) in the ESCRT-0 complex. Aberrant levels of HGS suppressed HBV transcription, replication and virion secretion. Hydrodynamic delivery of HGS in a mouse model significantly suppressed viral replication in the liver and virion secretion in the serum. Surprisingly, overexpression of HGS stimulated the release of HBV naked capsids, irrespective of their viral RNA, DNA, or empty contents. Mutant core protein (HBc 1-147) containing no arginine-rich domain (ARD) failed to secrete empty virions with or without HGS. In contrast, empty naked capsids of HBc 1-147 could still be promoted for secretion by HGS. HGS exerted a strong positive effect on the secretion of naked capsids, at the expense of a reduced level of virions. The association between HGS and HBc appears to be ubiquitin-independent. Furthermore, HBc is preferentially co-localized with HGS near the cell periphery, instead of near the punctate endosomes in the cytoplasm. In summary, our work demonstrated the importance of an optimum level of HGS in HBV propagation. In addition to an effect on HBV transcription, HGS can diminish the pool size of intracellular nucleocapsids with ongoing genome maturation, probably in part by promoting the secretion of naked capsids. The secretion routes of HBV virions and naked capsids can be clearly distinguished based on the pleiotropic effect of HGS involved in the ESCRT-0 complex.
參考文獻
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