Mutations within DR2 independently reduce the amount of both minus- and plus-strand DNA synthesized during duck hepatitis B virus replication

Abstract

The initial aim of this study was to examine the role of complementarity between the plus-strand primer and the minus-strand DNA template for translocation of the plus-strand primer in hepadnaviral replication. We show that when a 5-nucleotide substitution was placed in either DR1 or DR2, translocation of the primer at a detectable level did not occur. Placing the mutation in both DR1 and DR2 did not restore primer translocation, which indicates that complementarity is not the sole determinant for primer translocation. These mutants, in which primer translocation has been inhibited, have been additionally informative. The mutation in DR1 led to efficient synthesis of plus-strand DNA, albeit primed in situ. In contrast, the mutation in DR2 resulted in a reduction in the amount of plus-strand DNA synthesized per unit of minus-strand DNA. These findings were interpreted as indicating that a mutation at DR2, the primer acceptor site, can inhibit both primer translocation and in situ priming. Lastly, we show that mutations within DR2 can result in a reduction in the synthesis of minus-strand DNA and that this reduction is occurring at an early phase of the process. We speculate that this reduction in the amount of minus-strand DNA synthesized could be due to an inhibition of the template switch during minus-strand DNA synthesis.

Complementarity between epsilon and phi sequences in pregenomic RNA influences hepatitis B virus replication efficiency

“…As a result of the RNaseH activity present in the carboxy-terminal domain of the HBV polymerase, the pregenomic RNA is degraded generating an approximately 17 nucleotide primer derived from the 5′-end of the pregenomic RNA (Lien et al, 1986;Will et al, 1987;Loeb et al, 1991). This sequence includes the 5′-end copy of the DR1 sequence which is translocated to the complementary DR2 sequence within the newly synthesized HBV minusstrand DNA (Will et al, 1987;Seeger et al, 1986;Staprans et al, 1991;Loeb et al, 1996;Seeger and Maragos, 1989). This RNA primer serves to initiate plus-strand DNA synthesis leading ultimately to the production of the relaxed-circular 3.2-kb DNA present in the mature virions (Will et al, 1987;Loeb et al, 1997;Lien et al, 1987;Havert and Loeb, 1997).…”

cis-Acting sequences in addition to donor and acceptor sites are required for template switching during synthesis of plus-strand DNA for duck hepatitis B virus

“…The locations of the deleted sequences in mutants 2549-2561, 723-833, and 2205-2462 that disrupted relaxed-circular DNA formation (Fig. 2), are distinct from the DR1/DR2 and the r sequences, which have been shown previously to be involved in the synthesis of relaxed-circular DNA (7,15,18,25).…”