RNASE H ACTIVITY ENCODED BY HEPADNAVIRUSES

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The primary long-term objective of this application is to provide the applicant trainee (Michael Tencza, D.D.S.) with an excellent research-based laboratory training in basic molecular virology that would lead to is obtaining the Ph.D. degree from the Department of Microbiology and Immunology at the University of North Carolina at Chapel Hill. The focus for the research component of the proposed training program will be the hepadnavirus group headed by Dr. John Newbold. The proposed research will attempt to identify a viral encoded RNaseH enzyme activity that is believed to be active within the immature core particle, a subviral nucleocapsid that is a precursor to infectious progeny virions and which is abundant in the cytoplasm of infected liver cells. Identification of the RNaseH will be by in situ gel assay (activity gels) using acrylamide gels which are cast to contain the enzyme substrate (a DNA.RNA hybrid) radiolabeled with P32 in the RNA strand. The studies will use partially and highly purified preparations of DHBV core particles isolated from the livers of congenitally infected ducks. The RNaseH is widely believed to be essential for a crucial step in the replication of the hepadnavirus genome and which occurs within the cores subsequent to reverse transcription. Protein sequence analyses of hepadnaviruses by computer programs have identified an RNaseH-like domain at the 3' end of the hepadnaviral POL ORF, but direct identification remains lacking. In the activity gel assay the concentrated DHBV core particles are disrupted by SDS, electrophoresed in SDS-PAGE gels, rinsed several times in a buffer that dilutes away the SDS and allows the proteins to reform their native conformation within the gel matrix. RNaseH proteins, during the rinsing, begin to degrade the template in situ and produce a band depleted for the radiolabeled substrate. This method has been used successfully to assay the RNaseH of MoMLV. The studies will then exploit a panel of antipeptide antibodies specific for DHBV POL sequences. Western blot and immunoprecipitation analysis coupled with the RNaseH activity gel assay could associate the enzyme with a domain of the POL ORF. Finally this putative RNaseH domain will be subcloned into an E. coli expression vector (M13 mptac18) and the enzyme detected in E. coli by the same gel assay. The subcloning will be extended to the corresponding domain for HBV POL, thus making this enzyme available for study and evaluation as an antiviral target for drug therapy. The subclones of DHBV and HBV RNaseH will be subjected to directed mutagenesis analysis to identify the critical amino acids in this viral enzyme.