D型肝炎病毒mRNA體外轉錄系統製備：細胞RNA聚合脢之鑑定與病毒抗原的功能分析

Abstract

Hepatitis delta virus (HDV) is a single-stranded RNA virus that encodes two viral nucleocapsid proteins named small and large form hepatitis delta antigen (SHDAg and LHDAg). The SHDAg is essential for viral RNA replication while the LHDAg is required for viral assembly. We had identified HDAg as an acetylated protein and Lys-72 of SHDAg as one of the acetylation sites. Substitution of Lys-72 to Arg modulated HDAg subcellular localization and might participate in viral RNA nucleocytoplasmic shuttling and replication. In the following study, overexpression of SIRT, the type III histone deacetylase, translocated HDAg from mainly nucleolar distribution to nucleoplasmic or even cytoplasmic distribution. To elucidate the correlation between acetylation and nucleolar location of HDAg, putative acetylation motif (K/R)XKK of nuclear receptor and the nucleolar localization motif (R/K)(R/K)X(R/K) was found between 38KKKLKK43 of SHDAg. SHDAg mutant with Lys-to-Arg substitutions of 38KKKLKK43 (SHDAg-(K38-43R)) localized outside the nucleoli and could not facilitate the replication of HDV RNA. We added a heterologous nucleolar localization sequence to this mutant to restore its nucleolar localization. This will disclose the role of nucleolar localization for HDV replication. The functions and mechanisms of nucleolus-localization of SHDAg on HDV RNA replication are under investigation. In another part of our study, we also analyzed the cellular machinery for HDV RNA replication. By ultracentrifugation, nuclear extracts from HDV cDNA transfected cells were fractionated and the distribution of HDV RNA and HDAg were analyzed. RNA smearing in fraction 8-11 was observed, suggesting replication intermediates were produced during HDV replication cycle. These fractions were perforned in vitro transcription with 32P labeling and the products then hybridized with cold HDV genomic or antigenomic RNA probe in slot blot. Unfortunately, the signal in slot blot was not strong enough to show the replication activity in these fractions. Optimization of the system is proceeding. Keywords: HDV, nucleolus, NoLS, acetylation, in vitro transcription