Cloning, expression and functional study of human hepadnaviral polymerase.

摘要

Human hepatitis B virus (HBV), the prototype of the hepadnavirus family, is the most common cause in humans of acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Although an effective vaccine has been developed against HBV, more than 300 million chronic carriers worldwide cannot benefit from vaccination. Our laboratory is, therefore, engaged in the development of antiviral therapy. Since the pivotal role of viral polymerase in the viral replication cycle makes it an attractive target for antiviral agents, the work presented in this thesis was undertaken to investigate the molecular biology, synthesis and function of the viral polymerase.; HBV polymerase has been cloned and expressed in a rabbit reticulocyte lysate transcription-translation-coupled system. In vitro site-directed mutagenesis confirmed that the recombinant polymerase cDNA produces three translation products: a full-length (∼94 kDa), a truncated N-terminal protein (∼40 kDa) and an internally initiated protein (∼80 kDa). The recombinant polymerase possessed protein priming activity, as demonstrated by 32P-dGTP-labeling of the full size polymerase and the truncated N-terminal polymerase in an in vitro priming assay. The recombinant polymerase exhibited polymerization activity, as detected in an in vitro polymerase assay by incorporation of radionucleotides into acid-precipitable polynucleotides and by synthesis of HBV specific DNA in the range of 100–500 nucleotides in length. Both protein priming and polymerization activities of the recombinant polymerase were dependent on the RNA template bearing the HBV DR1 and epsilon stem-loop sequences.; Further studies showed that the recombinant HBV polymerase specifically inhibited the translation of HBV core mRNA in a rabbit reticulocyte lysate translation system. In addition, we demonstrated a cross-species translational regulation in the hepadnaviridae family. Previous studies by Anita Howe in our laboratory showed that DHBV core mRNA translation was inhibited by DHBV polymerase. I have further shown that DHBV polymerase inhibited the translation of HBV and WHV core mRNAs. Similarly, HBV polymerase inhibited DHBV and WHV core mRNA translation. Such a cross-species suppression of hepadnaviral core mRNA translation by the viral polymerase strongly suggests that this function is conserved. This translational regulation may provide a mechanism by which pregenomic RNA (pgRNA) is designated to function as either mRNA or a genomic element.; Previous deletion studies by Anita Howe showed that the 3′ periphery of DHBV core mRNA may be involved in the inhibitory effect of the polymerase. I have extended these studies utilizing an RNA folding computer program to analyze the 3′ periphery of HBV, DHBV or WHV core mRNAs, referred to as the trans repression element (TR), and identified a conserved epsilon stem-loop-like structure within this region. It is worthwhile to note that hepadnaviral polymerase interacts with a 5 ′ epsilon stem-loop structure (encapsidation signal) of pgRNA to initiate viral encapsidation. Other studies have indicated that an additional noncontiguous cis-element downstream from the encapsidation signal, which completely overlaps our proposed TR sequence, is also required for genomic encapsidation (Calvert et al. 1994; Hirsch et al. 1991). We hypothesized that hepadnaviral polymerase may interact with this conserved structure in the TR region for its translational inhibitory effect and for its involvement in viral encapsidation. The proposed role of the TR region was further examined by cloning the DHBV-TR sequence into a chloramphenicol acetyl transferase (CAT) expression plasmid downstream from CAT ORF. The in vitro translation as well as the CAT activity of the translational product of the chimeric mRNA was 90&nd