建立肝臟去氧核醣核酸病毒中共價閉合環狀去氧核醣 核酸的報告基因系統

Abstract

Hepadnavirus, including hepatitis B virus (HBV), possesses a partial double-strand DNA genome, also known as relaxed circular DNA (RC-DNA), which is converted to covalently closed circular DNA (cccDNA), a critical template for hepadnavirus replication, after entry to the hepatocytes. However, little is known about the detailed mechanisms regulating the conversion from RC-DNA to cccDNA. Detection of cccDNA usually requires Southern blotting, which is quite labor-intensive. Therefore, a convenient in vitro cccDNA reporter system should facilitate the research of cccDNA. However, in the present there is still no in vivo and in vitro system that can support efficient HBV infection and cccDNA formation. In contrast, duck hepatitis B virus (DHBV) can efficiently form cccDNA in human cell lines as long as its pregenomic DNA is made. In this study, we utilized DHBV system to construct the cccDNA reporter for ready detection of cccDNA formation. We first showed that trans-complementation of core and polymerase could rescue the replication cycle of core- and polymerase-deficient DHBV mutants. We then generated an array of deletion mutations that spanning the region between the start codons of core and polymerase genes. Interestingly, we found that the region between the poly A signal and the start codon of polymerase gene could be deleted without significant reduction of cccDNA formation. Thus, we inserted two reporter genes, the zeocin-resistant gene and a fluorescent protein into this region. Using Southern blotting, we demonstrated that they both maintain the ability to produce cccDNA and RC-DNA, indicating the feasibility of our reporter system. Further experiments are required to prove its utility in serving as a reporter of hepadnavirus cccDNA.