Mutagenesis of a Hepatitis B Virus Reverse Transcriptase Yields Temperature-Sensitive Virus

AbstractReplication of the hepadnavirus genome is catalyzed by a multifunctional reverse transcriptase (thepolprotein) 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 thepolprotein, we examined 52 mutants for their ability to replicate viral DNAin vitroand 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