探討B型肝炎病毒細野生型和剪接突變型病毒顆粒在細胞培養中的完整性及感染力

Abstract

慢性HBV感染仍是全球性的公共衛生問題。迄今為止，全長3.2 kb的HBV病毒可經由轉錄出四個主要的未剪接型unspliced RNA以及十六種剪接型spliced RNA (SpRNA) 。而spliced RNA裡發現最多的spliced 產物為SP1，其功能對於HBV 病毒複製仍然不清楚。研究發現SP1 所編碼出的新蛋白中，C端半胱胺酸(cysteine) 缺少之core 蛋白質 (HBc-cys) 會干擾HBV蛋白質外殼 (nucleocapsid) 的形成，這與感染過程中HBV核殼分解和病毒複製有關。從而發現增加HBc-cys的表現在HBV複製過程中扮演了一定的角色。先前的研究已報導這些spliced RNA對於轉染系統中的病毒複製並不重要。在動物實驗的初步結果指出，在人肝嵌合鼠中(chimera mice) ，缺乏splicing的HBV與野生型 HBV相比顯示出感染力受損。此外，在處理 β-巰基乙醇 (mercaptoethanol) 之下，相較野生型HBV病毒顆粒中單分子型式的HBc明顯多於剪接位點487突變的病毒顆粒。這個結果可能顯示蛋白質外殼之形成會受到胱胺酸之間的雙硫建差異而影響整個外殼的完整性。 在本篇研究中，我們試圖去探討HBV spliced RNA產物SP1所編碼的HBc-cys的功能作用。利用細胞實驗去了解缺乏HBc-cys的HBV是否會影響病毒初期感染進程中進入細胞或脫去蛋白質外殼的步驟。因此我們的結果顯示與spliced-deficient HBV相比，野生型HBV PF-rcDNA透過DNA脫蛋白以及蛋白質外殼結構變化在細胞質中測到的量比較多。

Chronic HBV infection still remains a global public health problem. Up to date, the 3.2 kb HBV transcribes four major unspliced RNA as well as 16 types of spliced RNAs (SpRNAs) which have been identified. The most abundant spliced variant Sp1, which its functions still remain obscure. Among the novel protein it encodes, HBV core minus one cysteine (HBc-Cys) has been reported to interfere with nucleocapsid formation which is associated with HBV capsid disassembly and replication during infection, raising the possibility that with the increased expression, it may play a functional role in HBV replication [1]. Previous studies have reported these spliced variants are not important for viral replication in transfection system. However, in our preliminary results from in vivo experiment indicated that splicing-deficient HBV shows impaired infectivity compared to HBV wild-type in humanized liver chimera mice. In this study, we sought to explore the functional role of the spliced RNA specifically the HBc-Cys protein encoded by the SP1 spliced RNA species. In addition, the capsid integrity of wild-type encoding the HBc-Cys is different from the A487C splicing-deficient mutant under treatment with β-mercaptoethanol. This result may suggest the cysteine-mediated disulfide bond linkages regulate the formation of the capsid integrity. Thus, the possibilities of which it may influence the steps of viral entry and uncoating process at the early phase of HBV infection in vitro. Our result thus indicates wild type HBV through capsid disassembly and DNA deproteinization, PF-rcDNA is detected in the cytoplasmic fraction with more amount compared to spliced-deficient mutant.