Hepatitis B virus (HBV) is a hepatotropic DNA virus that replicates by reverse transcription. It chronically infects up to 350 million people and kills >600,000 annually. Therapy primarily employs nucleos(t)ide analog drugs that drive HBV to near or below the clinical limit of detection. However, viral replication is not eliminated and virl replication resurges if drug is withdrawn. Greater suppression of HBV will require new drugs to be used in combination with the nucleos(t)ide analogs. Reverse transcription requires two viral enzymatic activities encoded on separate domains of the viral polymerase protein. The reverse transcriptase synthesizes new DNA and the ribonuclease H (RNaseH) destroys the RNA after conversion into DNA. Blocking either activity prevents synthesis of the HBV genome. Drugs have not been designed against the RNaseH because enzyme suitable for drug screening could not be made. We recently produced recombinant HBV RNaseH suitable for drug discovery and structural studies, and found all 25 known inhibitors of the RNaseH. The inhibitors had IC50 values as low as 2.3 M, EC50 values against viral replication as low as 0.34 M, and therapeutic indexes as high as 94. This is an outstanding starting point for development of anti-RNaseH drugs. However, difficulties in expressing the RT and RNaseH domains have prevented structural analysis of either domain, although reliable computational models exist for the RT. The lack of structural information for the HBV RNaseH is a major barrier to drug development because it precludes structure-based design of RNaseH inhibitors. This collaborative R03 project between Dr. Tavis (virologist) and Dr. Korolev (structural biologist) seeks to remove this block. The R03 mechanism was chosen to provide key pilot data for a future structure-function analysis of the enzyme that would not be competitive without preliminary crystallization data. Aim 1. Initiate structural evaluation of the HBV RNaseH domain. Enzymatically active variants of the HBV RNaseH will be systematically subjected to extensive crystallization trials. Initial efforts to solve the structure of the RNaseH will be determined usig the best crystals as time permits. Aim 2. Integrate the HBV RNaseH structure with the RT domain model. Our predicted RNaseH structure and a published predicted model will be computationally combined with the accepted model of the RT domain to generate the first 2-domain model for the HBV polymerase. This R03 project will begin deriving the first experimentally-determined structural information for any part of the multi-functional HBV polymerase protein. Combining the RNaseH domain structure with the existing validated models of the RT domain will yield the first view of the catalytic engine of HBV replication. This 2-domain model will be invaluable for both translational studies seeking to improve anti-HBV therapies, and also greatly advance our understanding of the HBV replication mechanism.