Template Switches during Plus-Strand DNA Synthesis of Duck Hepatitis B Virus Are Influenced by the Base Composition of the Minus-Strand Terminal Redundancy

Abstract

Two template switches are necessary during plus-strand DNA synthesis of the relaxed circular (RC) form of the hepadnavirus genome. The 3 end of the minus-strand DNA makes important contributions to both of these template switches. It acts as the donor site for the first template switch, called primer translocation, and subsequently acts as the acceptor site for the second template switch, termed circularization. Circularization involves transfer of the nascent 3 end of the plus strand from the 5 end of the minus-strand DNA to the 3 end, where further elongation can lead to production of RC DNA. In duck hepatitis B virus (DHBV), a small terminal redundancy (5r and 3r) on the ends of the minus-strand DNA has been shown to be important, but not sufficient, for circularization. We investigated what contribution, if any, the base composition of the terminal redundancy made to the circularization process. Using a genetic approach, we found a strong positive correlation between the fraction of A and T residues within the terminal redundancy and the efficiency of the circularization process in those variants. Additionally, we found that the level of in situ priming increases, at the expense of primer translocation, as the fraction of A and T residues in the 3r decreases. Thus, a terminal redundancy rich in A and T residues is important for both plus-strand template switches in DHBV.The hepadnaviruses are a family of DNA viruses constrained by a small genome size of ϳ3 kbp. Members of this family include the human pathogen hepatitis B virus and the avian duck hepatitis B virus (DHBV). DHBV has served as a useful model for understanding many aspects of hepadnavirus biology, including replication (6). Hepatitis B virus and DHBV share very little, if any, nucleotide sequence identity; yet, they maintain a very similar genomic organization and basic replication strategy (6). Their lack of nucleotide conservation is intriguing in light of the important contributions made by the nucleic acid template to its own replication. cis-acting elements are prevalent in these viruses and have been shown to be important for such diverse processes as transcriptional initiation and elongation, regulation of splicing and transport, selective encapsidation, and the template switches that occur during replication (6,8,9,16,18). Viral replication is necessary for maintenance of the chronic carrier state; understanding this replication is critical for limiting disease in carriers.The infection process leads to the formation of a genomiclength covalently closed circular (ccc) DNA species, which is maintained as a plasmid in the nucleus (29). The cccDNA species serves as the transcriptional template for production of all viral RNA species, including the pregenomic (pg) RNA (Fig. 1A). The pgRNA is a longer-than-genome-length mRNA, which is converted into either a relaxed circular (RC) or duplex linear (DL) form of the DNA genome via reverse transcription (Fig. 1B to I).Synthesis of RC DNA requires two template switches during plus-strand replicati...

The Sequence of the RNA Primer and the DNA Template Influence the Initiation of Plus-strand DNA Synthesis in Hepatitis B Virus

“…Additionally, previous studies in DHBV showed a direct relationship between the number of G and C nucleotides adjacent to DR1 and the level of priming at DR1 and DR2. 16 The greater the number of G and C nucleotides, the higher the level of priming at DR1 and the lower the level of priming at DR2. Our HBV analyses with variants SubA, SubB, and SubC did not show a relationship between the number of G and C nucleotides and the level of priming at DR1 and/or DR2 (see Figure 6).…”

“…In DHBV, sequence identity between the donor and acceptor sites for primer translocation is necessary for efficient priming at DR2. 15 Additionally, the nucleotide composition of the 5′ end of the pgRNA influences priming at DR1 and 16 Analogous studies of the effect of the sequence at the primer translocation donor (DR1) and acceptor (DR2) site on in situ priming and primer translocation have not been reported for HBV.…”

Hepatitis B virus replication

“…Using quantitative genetic techniques, the Loeb laboratory has defined, mostly in DHBV, several such cis-elements on the (-)-DNA [144,[156][157][158][159][160][161] , e.g. 3E, M, and 5E, which are located at both termini and in the middle of pgRNA.…”