In this proposal, we wish to characterize the structure and function of the polymerase gene product of human hepatitis B virus. Using site- directed mutagenesis, gene transfer and various kinds of assays, three major projects will be pursued: 1. To study the strategy of pol gene expression from the 3.5 kb transcript: Three different hypotheses of the pol ene expression strategy will be tested (ribosomal frame- shifting, termination-backward scanning-reinitiation as well as direct internal initiation). a. To continue and complete our initial characterizations of mutants designed to study the translational control of pol gene. b. To attenuate the translational efficiency of the upstream core ORF by changing the sequence context around the ATG initiation codon or by introducing an out-of-frame, upstream ATG inhibitory codon. c. To affect the translational efficiency of the downstream pol ORF by varying the length of c/pol overlap. d. To affect the expression of the upstream ORF by polio virus infection or transfection. e. To attenuate the translational efficiency of the upstream core ORF via hybrid-arrest in vitro translation. 2. To study the functional significance of a newly discovered, spliced transcript (2.3 kb): a. To disrupt spicing by mutating the splice donor and the acceptor site; b. To look for other unknown transcript via in vitro PCR amplification, followed by cDNA cloning. c. functional characterization of the cDNA clones deriving from the spliced transcript. 3. Domain definition of the pol ORF: Three predicted functional domains of pol include terminal protein, reverse transcriptase and RNase H. About a dozen different domain mutants will be characterized by various functional assays.
