ABSTRACT
Replication of the hepadnavirus genome is catalyzed by a multifunctional reverse transcriptase (the pol protein) that exhibits DNA polymerase and DNA priming activities and has the ability to transfer RNA and DNA strands across the viral genome. A salient feature of this enzyme is the ability to prime RNA-directed DNA synthesis with protein rather than with RNA. This is reflected in its unique physical make up, which includes an amino-terminal (TP) domain that is separated by a spacer from the reverse transcriptase (RT) domain. To establish a structure function relationship for the pol protein, we examined 52 mutants for their ability to replicate viral DNA in vitro and in cultured cells. We demonstrated that the role of the TP domain is limited to the early steps of viral DNA synthesis including RNA packaging and protein priming. Both the TP and the RT domains are required for the interaction with epsilon RNA, which is the template for the protein-priming reaction and serves as the RNA packaging signal. In addition, we report the isolation of a thermosensitive variant of a hepadnavirus that will permit investigations of individual steps of the viral replication cycle under synchronized conditions.
Subject(s)
Genes, pol , Hepatitis B virus/enzymology , RNA-Directed DNA Polymerase/genetics , Animals , Binding Sites , Chickens , Hepatitis B virus/genetics , Hepatitis B virus/physiology , Humans , Mutagenesis , Temperature , Tumor Cells, Cultured , Viral Proteins/metabolism , Virus ReplicationABSTRACT
The hepadnavirus reverse transcriptase is a multifunction enzyme. In addition to its role in DNA synthesis, the polymerase is required for RNA packaging and also functions as the primer for minus-strand DNA synthesis. Previously, we demonstrated that the protein-priming activity of the polymerase requires a viral RNA segment, termed epsilon, which serves as a template for the synthesis of a short DNA oligomer that is covalently attached to the reverse transcriptase (G.-H. Wang and C. Seeger, J. Virol. 67:6507-6512, 1993). We now report that epsilon is sufficient for activation of the reverse transcriptase to prime DNA synthesis through the formation of a stable RNA-protein (RNP) complex. We also demonstrate that the binding reaction depends on sequence-specific determinants on epsilon. Moreover, our results indicate that two genetically separated domains of the reverse transcriptase are required for formation of the RNP complex. Finally, we show that the polymerase has a DNA polymerase activity in the absence of epsilon which does not depend on the protein-priming mechanism.
