D型肝炎病毒(hepatitis delta virus, 簡稱HDV)為B型肝炎的衛星病毒，因為病毒的包裝需要HBV的外膜參與。HDV病毒顆粒含有1.7-kb RNA環狀分子和許多HDAg蛋白。而HDAg蛋白具有2種型式，分別為由195胺基酸(24 kDa)組成的S-HDAg (Small-HDAg)，其功能為參與HDV RNA的複製；而另一個則是由214胺基酸(27 kDa)組成的L-HDAg (Large-HDAg)，其功能為參與病毒的包裝和抑制HDV RNA的複製。相較於S-HDAg，L-HDAg在C端多了19個胺基酸，但二者調節病毒的功能是截然不同的。在較早的實驗都是使用HDV cDNA來轉染而得到對HDV的瞭解，但在HDV感染的細胞中RNA直接的複製仍不清楚。在本研究中，我們從表現S-HDAg的E. coli中純化出S-HDAg蛋白並以西方點墨法(Western blot)確認後，再將所純化的S-HDAg與HDV基因股RNA以RNP轉染方式到Cos7細胞中，並且使用西方墨點法與北方墨點法(Northern blot)觀察細胞內HDV的複製狀況。實驗結果証明，由表現S-HDAg的E. coli中所純化的S-HDAg具有啟動病毒複製的功能。接著我們再進一步分析HDV cDNA的轉染方式和RNP轉染方式中HDV在細胞的複製持續性，發現使用HDV cDNA轉染的方式中HDV在細胞中持續時間大於以RNP轉染的方式。最後以不同長度的HDV基因股RNA (1.7-kb、1.6-kb、1.2-kb) 以RNP轉染方式來觀察HDV RNA的複製能力是否會因為基因股板模的縮小而影響HDV RNA的複製，結果發現最短的1.2-kb之HDV RNA仍會持續的複製，這個結果將會對HDV複製中對RNA板模之要求產生極重要的討論。
Human hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV) and requires HBV envelope proteins for packaging. HDV particles contain a ribonucleoprotein core composed of the circular 1.7-kb RNA genome and multiple copies of the only HDV-encode protein, delta antigen. There are two forms of delta antigens. The small form is a 24 kDa (195 amino acids) of S-HDAg, which is essential for replication of RNA genome. The large form is L-HDAg, which is a 27 kDa (214 amino acids), essential for particle assembly and inhibition of HDV RNA replication. Although L-HDAg only contains an additional 19 amino acids at the C-terminus of S-HDAg, they regulate viral function differently. Early studies using HDV cDNA transfection have generated large amounts of information about RNA-dependent RNA replication but that in infected cells is still not clear. In this study, we showed the expression and purification of recombinant S-HDAg from E. coli by ion-exchange chromatography. To characterize the activity in the early stage of HDV replication, the in vitro synthesized HDV genomic RNA and purified S-HDAg was mixed as RNP complex and transfected into Cos7 cells. We demonstrated the ability of the RNP complex to initiate the replication of HDV by Western and Northern blot analysis. The persistence of HDV replication has been examined by using the cDNA and RNP transfection systems and both could maintain HDV replication up to 26 days although the cDNA system seem retained the replication activity longer than RNP system. Finally we used different lengths (1.7-kb、1.6-kb、1.2-kb) of HDV genomic RNA with purified recombinant S-HDAg to study the template requirement of HDV replication. Surprisingly, the data showed that all three different lengths of HDV genomic templates were able to initiate HDV replication. The ability of the 1.2-kb RNA template for replication will generate important discussion in the field of HDV replication.
