(1)探討SHP-1磷酸酶對B型肝炎病毒基因表現之影響 (2)以細胞模型探討野生型及剪接突變型B型肝炎病毒之感染力

Abstract

(1)慢性B型肝炎為導致肝癌發生之重要因子，而B型肝炎病毒(HBV)之X蛋白質(HBx)所引起的細胞內訊息傳遞路徑失調被認為是B型肝炎引發肝癌的機制之一。目前唯一許可之肝癌治療藥物蕾莎瓦膜衣錠 (Sorafenib) 可能透過SHP-1磷酸酶在肝臟內抑制由HBx所促進之致癌性路徑。許多文獻指出SHP-1為可能之抑癌因子以及癌症治療藥之作用靶點。截至目前已有許多磷酸酶被發現可能參與調控HBV複製以及基因表現，然而目前仍未有研究致力於探討SHP-1與HBV之間的關係。本篇研究利用慢病毒所攜帶的小髮夾RNA降低細胞內SHP-1的表現量，接著再進一步觀察HBV之基因產物使否受到影響。結果顯示在SHP-1表現量降低或其磷酸酶活性受抑制時皆會降低HBV之基因產物。相反地，在細胞內大量表現具磷酸酶活性之SHP-1時病毒之基因產物增加。因此，推斷SHP-1正向調控HBx所刺激之HBV基因表現可能透過其磷酸酶活性。然而目前為止SHP-1調控HBV基因表現之詳細機制仍未明。意外地，相較於其他基因產物，在SHP-1表現量降低時HBx的表現量維持可能源自於其半衰期的延長。這些因半衰期延長而累積之HBx是否為造成HBV之基因產物降低的原因還有待更多的實驗來證明。在本研究第一次闡述了細胞中SHP-1在HBV基因表現中扮演支持性角色，凸顯了開發特異性SHP-1抑制劑作為未來抗HBV藥物之可能性。 (2)慢性B 型肝炎病毒(HBV)感染至今仍為全球健康之一大負擔。在HBV 生活史中，除了一般所熟知的轉錄產物外，還有一群剪接型RNAs (SpRNAs)。儘管這些SpRNAs在先前文獻中被認為與第一型干擾素之療效與肝癌發生之風險有關，它們的生物意義仍不清楚。有研究指出在質體轉染之系統中有無SpRNAs的存在皆不影響HBV之複製。有鑑於剪接相關之蛋白對於人類免疫缺陷型病毒第一型之感染力有重要影響，我們團隊先前利用HBV感染人肝嵌合鼠的模型發現剪接功能缺失之HBV，相較野生型HBV在感染力方面有大幅下降。本研究致力於在細胞膜型中模擬近似HBV 在自然界中感染環境以及探討剪接相關之病毒蛋白是否參與病毒顆粒之組裝。持續以含二甲基亞碸(DMSO)之培養液培養細胞可增加HBV 在體外細胞模型之感染力。除此之外，額外補充HBV之表面抗原可提高有套膜之病毒顆粒，幫助體外細胞模型所產之HBV病毒更趨近其在自然界中之型態。在所有SpRNAs中，SP1所佔之比例最高，SP1所轉譯出的C 端半胱氨酸(cysteine)缺少之core蛋白質(HBc^(-Cys))在先前實驗結果中被發現可彌補剪接功能缺失之HBV感染力。本研究發現HBc^(-Cys)與全長HBc蛋白質位於同個沉降層，推測HBc^(-Cys)可能參與組裝病毒之蛋白質外殼。除此之外，野生型HBV病毒顆粒中單分子形式之HBc 明顯較剪接位點487突變之病毒顆粒中多，可能與HBc^(-Cys)參與病毒顆粒組裝所導致的蛋白質外殼完整性不同有關。這些結果顯示蛋白質外殼完整性上的差異可能影響病毒感染進程中進入細胞或是脫去蛋白質外殼的步驟。這些結果支持先前在人肝嵌合鼠上所發現之現象，也提供可操控之體外細胞感染模式，以利後續相關機制之探討。

(1)Chronic hepatitis B virus (HBV) infection is the major risk of hepatocellular carcinoma (HCC) in the endemic area. Dysregulation of cellular signaling pathways by HBV X (HBx) protein is one of the HBV-induced carcinogenic mechanisms. Sorafenib, the only approved drug for HCC treatment, may inhibit the HBx-enhanced oncogenic androgen pathway in livers through SH2 domain-containing phosphatase-1 (SHP-1). Accumulated evidences have indicated SHP‐1 is a potent tumor suppressor and druggable target in preclinical models. Several phosphatases were reported to participate in HBV gene expression and replication, while the interaction between HBV and SHP‐1 before HCC has not yet been explored. In this study, we knocked down endogenous SHP-1 by short hairpin RNA-bearing lentiviruses in the replicon-transfected cells and infected cells, and then determined the loss of function effect of SHP-1 on HBV transcripts and protein amounts. Our results showed that knockdown of SHP-1 or inhibition of its phosphatase activity resulted in the reduction of both HBV mRNA and protein levels in the presence of HBx. In contrast, overexpression of constitutively active form of SHP-1 but not the phosphatase-dead mutant led to the elevation of viral transcripts and proteins. Therefore, these results suggested the positive regulatory role of SHP-1 toward the HBx-enhanced HBV gene expression may through its phosphatase activity. In molecular details, this SHP-1-evoked enhancement was still uncertain though. Interestingly, in contrast with the reduced viral mRNAs, HBx protein amount was somehow maintained due to its prolonged stability in the cells when SHP-1 was knocked down. Further studies about whether SHP-1 assists HBV gene expression through temporally restraining the half-life of HBx protein are ongoing to be addressed. Hence, our current study illustrated for the first time about the role of cellular SHP-1 in supporting HBV gene expression, thus highlighting specific SHP-1 inhibitors for the development of potential anti-HBV compounds in the future. (2) Chronic infection of hepatitis B virus (HBV) remains a severe burden of global public health. In the viral life cycle, HBV produced a panel of spliced RNAs (SpRNAs) in addition to common contiguous transcripts. However, the biological functions of these SpRNAs are still unclear, although they have been reported to be correlated with interferon treatment response and risk of hepatocellular carcinoma. Previous transfection study showed that these SpRNAs were dispensable for viral replication, as the splicing-defect mutant replicated at a comparable level as wild-type (WT). Given that the splicing-related proteins are essential for augmented infectivity of human immunodeficiency virus type 1, our preliminary results indicated that the splicing-defect HBV showed impaired infectivity over 100 to 1000-fold compared with WT in humanized liver chimera mice. In this study, we tried to refine an in vitro HBV infection culture system which mimics natural condition and evaluated whether critical splicing-related viral proteins appeared in infectious viral particles. Continuous dimethyl sulfoxide treatment enhanced HBV infectivity in cultured cells. In addition, exogenous supplementation of HBV surface protein increased the amount of enveloped virions collected from the media of replicon-transfected cells, and thus helped produce naturally-mimicked infectious particles for viral entry. Among HBV SpRNAs, SP1 is the most abundant species. In infectious HBV particles, the SP1-encoded HBV core minus cysteine (HBc^(-Cys)) protein, which was preliminarily approved to recover the infectivity of splicing-defect mutant, was found to be co-localized with the full-length core in similar sedimentation layers, implying that HBc^(-Cys) may assemble into secreted virions. In addition, the integrity of WT capsid attributed to the incorporation of HBc^(-Cys) might be different from that of the splicing-deficient mutant, as the remaining amount of core protein monomers were higher in WT viral particles under oxidized environment compared with those in the mutant. This result implied that the cysteine-mediated disulfide bridges for regulating capsid integrity may influence the steps of viral entry and uncoating processes at the early phase of HBV infection. Therefore, our present study refined the culture system for HBV infection and provided a manageable model which resembled to natural condition for mechanism study.