行政院國家科學委員會補助專題研究計畫進度報告
※ ※※※※※※※※※※※※※※※※※※※※※※※※ ※ ※※B 型肝炎病毒表面抗原變異種的分子遺傳及功能的分析
M o l e c u l a r g e n e t i c a n d f u n c t i o n a l a n a l y s i s o f
H B V S g e n e m u t a n t s ※
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計畫類別：X 個別型計畫 □整合型計畫
計畫編號：NSC 89－2320－B－006－131－
執行期間：89 年 8 月 1 日至 90 年 7 月 31 日
計畫主持人 ：呂政展成功大學醫學院病理科
計畫參與人員
：范育芬
本成果報告包括以下應繳交之附件：
□赴國外出差或研習心得報告一份
□赴大陸地區出差或研習心得報告一份
□出席國際學術會議心得報告及發表之論文各一份
□國際合作研究計畫國外研究報告書一份
執行單位：成功大學醫學院病理科, [email protected]
中 華 民 國 90 年 10 月 31 日
行政院國家科學委員會專題研究計畫進度報告
B 型肝炎病毒表面抗原變異種的分子遺傳及功能的分析
Molecular genetic and functional analysis of HBV S gene mutants
計畫編號：NSC 89－2320－B－006－131－
執行期限：89 年 8 月 1 日至 90 年 7 月 31 日
主持人：呂政展成功大學醫學院病理科
一、中文摘要 經由長期 B 型肝炎病毒（ HBV）感染，許多 逃避免疫偵測的突變種已經從帶原者的血 清及肝組織中被分離出來。有在這些突變 種中可以找到不同的表面抗原突變。在 HBV 自然感染的歷史中，經由表面抗原蛋 白的免疫組織化學染色，在所感染的肝組 織中可發現幾種染色特徵形式。這些特徵 性的染色形式顯示可能跟細胞內表面抗原 蛋白的異常調控與堆積有關。然而，如何 造成表面抗原蛋白不平衡合成的分子遺傳 機轉還不是很清楚。為了闡明是什麼機制 導致表面抗原蛋白異常表現，不同的表面 抗原基因突變種經由轉染到 HUH-7 細胞 中，細胞裡跟細胞外的蛋白表現型式將個 別經由酵素連結免疫分析及西方墨染法來 分析。從這些實驗的結果顯示這些不同的 突變對表面抗原蛋白的分泌有不同的影 響。例如，在核酸序例 3040 到 3111（△1） 進 行 刪 除 對 於 表 面 抗 原 的 分 泌 及 蛋 白 （gp27 和 p24）的表現並沒有顯著的影 響。在序列 2 到 55（△2）刪除明顯的增 加表面抗原蛋白胞內/胞外比例，經由西方 墨染也顯示減少表面抗原蛋白（gp27 和 p24）的表現，同樣的，在 3153 到 24（△ 3）的序列刪除顯著增加抗原蛋白胞內/胞 外的比例。同時，抗原蛋白表現幾乎看不 到。這些結果顯示除了某些已知的轉錄因 子影響表面抗原基因的轉錄，可能有一些 序列參與調控表面抗原基因的表現。 關 鍵 詞 : B 型肝炎病毒（HBV）， 突 變種，轉錄因子，免疫組織化學染色 A b s t r a c tDuring chronic HBV infection, various immune escape mutants have been isolated from sera and livers of carriers. Different surface mutations have been found in these naturally occuring mutants. During the natural history of chronic HBV infection, several characteritic immunohistochemical staining patterns of S Ag have been described from infected livers. These characteristical staining patterns have been postulated to be derived from aberrant regulation and accumulation of S Ag intracellularly. However, the molecular genetic mechanism(s) responsible for the unbalanced synthesis of SAg are virtually unknown. To elucidate the molecular mechanism governing the aberrant expression of S Ag, various SAg mutant constructs were transfected in Huh7 cells and intracelluar and extracellular levels as well as protein expression profils were analyzed by enzyme linked immunoassay and Western blot analysis, respectively. The results from these expreiments indicated that these different mutations have different effects on secretion of S Ag. For example, deletion of nt 3040 to 3111（∆1） has no significant effect on the secretion of SAg, as well as on expression of SAg (p24 and gp27). Deletion of nt from 2 to 55（∆2） has significantly increased the ratio of intracellular/extracellular level of S Ag. Also, SAg (p24 and gp27) levels is decreased as revealed by Western blot. Similarly, deletion of nt 3153-24（∆3）has significantly increased the ratio of intracellular/extracellular level of S Ag. Simultaneously, the level of S Ag was almostly undetectable. These results suggest that besides the transcriptional factors known to bind and affect the transcript levels of S Ag,
there may some elements involved in the controlling the expression of SAg.
K e y w o r d s : Hepatitis B virus,
immunohistochemical staining, transcriptional factor
二、緣由與目的
Chronic infection of hepatitis B virus is a public health concern and is closely associated with the development of cirrhosis and hepatocellular carcinoma. The hepatitis B virus (HBV) surface gene (reviewed in Tiollais, 1985; Yen, 1993) encodes three forms of the viral surface (envelope) protein.It is divided into three parts by two internal start codons: preS-S1 region, pre-S2 region and the S region. The combination of these three regionsforms the large surface proteins. The pre-S2 as well as the S region and the S region alone form the pre-S2 protein and S protein, respectively. These three proteins are translated from three corresponding mRNA. These groups of RNA are transcribed from two tandem promoters. The up-stream preS1 promoter gives rise to 2.6 kb transcripts that are translated into the large surface protein. The downstream S promoters gives rise to 5'-heterogeneous 2.2 kb transcripts. The upstream preS1 promoters contains ubiquitous transcriptional factors binding sites for Oct 1, Sp1, and liver-specific factors for HNF1, HNF3 (Raney et al., 1995; Raney & McLachlan, 1995; Chang et al., 1989; Zhou & Yen, 1991). The downstream promoter contains a few non hepatocye-specific transcription factor binding sites, ie, NF-Y and Sp1(Raney et al., 1992; Lu et al., 1996).These transcripts are translated into the middle and small (major) surface proteins, initiated from internal AUG codons. Before using as a template for reversed transcription for viral pregenome RNA, covalently closedly circular viral DNA is amplified in the nucleus, under the stringent control of the large surface proteins(Summers et
al., 1990). In both infected and transfected hepatocytes, the amount of preS1 transcripts is much lower than the amount of S transcripts (Yen, 1993; Ganem, 1996). Hence, the large surface proteins constitute only a few percent of the total surface proteins synthesized in the infected cell. This is an important aspect of the viral life cycle, since the large surface protein, unlike the other forms of the surface protein, is incompetent for secretion, and relative over-expression of the large surface protein leads to the intracellular retention of all forms of the surface protein, because of heteromultimer formation (Persing et al., 1986; Ou & Rutter, 1987; Bruss & Ganem 1991; Gilles et al., 1992). This retention not only blocks virion secretion (Bruss & Ganem,1991), but is also injurious to the host cell (Gilles et al., 1992).
Curiously, although the preS1 promoter appears to be much weaker than the S promoter in the context of either the entire viral genome or of a large sub-viral fragment, the preS1 promoter is actually somewhat stronger than the S promoter when they are individually tested in transfection studies with reporter plasmids (Antonucci & Rutter, 1989; Bulla & Siddiqui, 1989; Chang & Ting, 1989). Therefore, other cis-elements in the viral genome must differentially regulate the amount of preS1 and S transcripts. One factor that accounts for part of the difference is that the HBV enhancer II has a somewhat stronger effect on the S promoter than the preS1 promoter (Zhou & Yen,1990; Yuh & Ting, 1990). During chronic HBV infection, immune escape mutants have been frequently isolated from sera and infected livers. These mutants have been postulated to be associated with characteristic immunohistochemical staining patterns. In this study, we intend to examine those naturally occurring mutants to determine if those muations may have led to desregulation of S gene expression and aberrant accumulation of SAg intracellularly.
Construction of 6 representative SAg mutants used in transfection assay. Fig. B
showed the location of deletions in various contructs. The solid box designated the deleted region, and the number indicated the range of nucleotide sequence deleted.
Different mutations have variable effect on the major S Ag expression. Fig. A
showed that depending on the location of mutation, the HBsAg expression is afftected to the different extent. For example, the deletion of nt 2-55 (∆2) decreased the major SAg. In contrast, deletion of nt 3151-22(∆3) drastically reduced the SAg to the extent of being undetectably. Unexpectedly, mutation of
translation start codon (ATG→AGG) (∆7) is expected to abolish the expression of major SAg. However, unexpectedly it drastically increased the ratio of intracellular/extracellular SAg level.
Different mutations have variable effect on the S Ag secretion. Fig. C showed
the relative HBsAg secretion of 6 representative cloned mutants in transfected Huh 7 cells. Compared with the wild-type construct (p(3A)Saag). Except for the preS1 deletion (∆1), the remaining mutants showed retention of HBsAg in the cytoplasm, resulting in high ratio of intracellular/extracellular SAg levels.
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四、參考文獻
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