The recent development of two systems for expressing function DHBV reverse transcriptase outside virions has made possible an array of experiments to characterize the unique features of this enzyme. Both expression systems and native P from virions will be used to probe the polymerase's activities. The experiments are divided into 4 aims: (1) The physical interaction of P and SL will be explored through binding assays and partial proteolysis of P. These experiments will test the hypothesis that an isomerization of P induced by binding to a functional SL is essential for packaging the viral RNA and priming reverse transcription. (2) The reverse transcriptase activity will be characterized to provide basic biochemical information about the enzymatic activity and to furnish a baseline for interpreting the activity of the mutant polymerases to be produced in Aim 4. (3) The RNaseH activity of P will be investigated through expression of the RNaseH domain in the absence of the rest of P and through attempts to alleviate the template commitment of the enzyme. If successful, these experiments will provide the first detailed examination on this essential activity. (4) P will be extensively mutagenized, concentrating on 9 regions of high homology between DHBV and HBV enzymes. Each of these mutant enzymes will be will be analyzed in a battery of 5 activities. The analysis if these mutants will increase our understanding of the molecular mechanisms employed by the multiple activities of P and will begin to define their interaction(s) with each other. The results from the experiments proposed will provide fundamental information about the hepadnaviral polymerase that was previously unavailable. This information is intended to increase our understanding of the mechanism of action of this intriguing enzyme and to provide direction for future development of novel inhibitors directed against not only the polymerase activity, but against other enzymatic functions as well.
| Badtke, Matthew P; Khan, Irfan; Cao, Feng et al. (2009) An interdomain RNA binding site on the hepadnaviral polymerase that is essential for reverse transcription. Virology 390:130-8 |
