MUTAGENESIS OF A HEPATITIS-B VIRUS REVERSE-TRANSCRIPTASE YIELDS TEMPERATURE-SENSITIVE VIRUS

Replication of the hepadnavirus genome is catalyzed by a multifunction al reverse transcriptase (the pol protein) that exhibits DNA polymeras e and DNA priming activities and has the ability to transfer RNA and D NA strands across the viral genome. A salient feature of this enzyme i s 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-f unction relationship for the pol protein, we examined 52 mutants for t heir 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 s teps of viral DNA synthesis including RNA packaging and protein primin g. Both the TP and the RT domains are required for the interaction wit h epsilon RNA, which is the template for the protein-priming reaction and serves as the RNA packaging signal. In addition, we report the iso lation of a thermosensitive variant of a hepadnavirus that will permit investigations of individual steps of the viral replication cycle und er synchronized conditions. (C) 1996 Academic Press, Inc.