Abstract
Hepadnaviruses replicate their circular DNA genomes via reverse transcription of an RNA intermediate. The initial product of reverse transcription, minus-strand DNA, contains two copies of a short direct repeat (DR) sequence, termed DR1 and DR2. Plus-strand DNA synthesis initiates at DR2 on minus-strand DNA, using as a primer a short, DR1-containing oligoribonucleotide derived by cleavage and translocation from the 5' end of pregenomic RNA. To clarify the sequence requirements for plus-strand primer cleavage and translocation, we have constructed mutants of the duck hepatitis B virus bearing base changes in or around the DR1 sequence in the primer. A point mutation at the terminal nucleotide of DR1 has a striking phenotype: normal levels of duplex viral DNA are produced, but nearly all of the DNA is linear rather than circular. Mapping of the 5' end of plus-strand DNA reveals that primer cleavage occurs with normal efficiency and accuracy, but the primer is not translocated to DR2; rather, it is extended in situ to generate duplex linear DNA. Other mutations just 3' to DR1 similarly affect primer translocation, although with differing efficiencies. Linear DNA found in wild-type virus preparations has the same fine structure as the mutant linears described above. These results indicate that (i) plus-strand primer cleavage and translocation are distinct steps that can be dissociated by mutation, (ii) lesions in sequences not included in the primer can severely inhibit primer translocation, and (iii) elongation of such untranslocated primers is responsible for the variable quantities of linear DNA that are found in all hepadnaviral stocks.