The Conformation of the 3′ End of the Minus-Strand DNA Makes Multiple Contributions to Template Switches during Plus-Strand DNA Synthesis of Duck Hepatitis B Virus

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. A small DNA hairpin has been shown to form near the 3 end of the minus-strand DNA overlapping the direct repeat 1 in avihepadnaviruses. Previously we showed that this hairpin is involved in discriminating between two mutually exclusive pathways for the initiation of plus-strand DNA synthesis. In its absence, the pathway leading to production of duplex linear DNA is favored, whereas primer translocation is favored in its presence, apparently through the inhibition of in situ priming. Circularization involves transfer of the nascent 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. Using both genetic and biochemical approaches, we now have found that the small DNA hairpin in the duck hepatitis B virus (DHBV) makes a positive contribution to circularization. The contribution appears to be through its impact on the conformation of the acceptor site. We also identified a unique DHBV variant that can synthesize RC DNA well in the absence of the hairpin. The behavior of this variant could serve as a model for understanding the mammalian hepadnaviruses, in which an analogous hairpin does not appear to exist.Hepadnaviruses are a family of small, enveloped DNA viruses that display a narrow host range, a tropism for the liver, and are capable of both acute and chronic infections in their hosts. The human hepatitis B virus, a member of the family, is a major worldwide health problem exposing ca. 350 million chronic carriers to an increased risk of developing hepatocellular carcinoma (11). Viral replication is necessary for maintenance of the chronic carrier state; understanding this replication is critical for limiting disease in carriers. The duck hepatitis B virus (DHBV) has proven a powerful model for studying many aspects of hepadnaviral biology, including replication (reviewed in reference 3). Although divergent at the level of nucleic acid sequence, all hepadnaviruses share a similar genetic organization and general strategy of replication.Three template switches are integral to viral replication: one during synthesis of the minus-strand DNA and two during synthesis of plus-strand DNA during the production of the relaxed circular (RC) DNA genome. Minus-strand DNA synthesis occurs via reverse transcription of an RNA intermediate, the pregenomic RNA (23). Upon completion of the minusstrand DNA, there are two mutually exclusive pathways for plus-strand DNA synthesis. The predominant pathway gives rise to RC DNA (Fig. 1A, panels a to d), while the alternative pathway yields duplex linear (DL) DNA (Fig. 1A, panel e). Both pathways use the sam...

A High Level of Mutation Tolerance in the Multifunctional Sequence Encoding the RNA Encapsidation Signal of an Avian Hepatitis B Virus and Slow Evolution Rate Revealed by In Vivo Infection

“…Perhaps more importantly, in cell culture various mutations within cis elements had gradual rather than massive effects, uncovered only by thorough quantitative assessment (21,33,34,44). The drastic impact of the pet element on pgRNA accumulation was discovered using large deletions (26).…”

“…Partial overlaps exist with other cis elements, including region A, involved in splicing regulation by base pairing with downstream region B (34), and region 3E, proposed to facilitate proper (ϩ)DNA synthesis and rcDNA formation by base pairing, on minus-strand DNA, with region M3 (33). Immediately neighboring Dε are the translational start of the core protein and a small hairpin-forming sequence that promotes proper (ϩ)DNA formation (21). Impairment of any of these functions by mutations in the Dε sequence may reduce if not Select cis elements are indicated; ε, RNA stem-loop; εII, region II, corequired for pgRNA packaging; DR, direct repeat; 3E, 5E, M3, and M5, elements on minus-strand DNA proposed to promote rcDNA formation by base pairing (33); white arrow, heterologous promoter controlling pgRNA transcription; gray arrow with dashed outline, endogenous core promoter controlling pgRNA and precore RNA transcription on authentic cccDNA.…”

Hepatitis B virus replication

“…Why the RNA primer predominantly jumps to DR2 although its complementarity to the initial site is larger (more than 15 versus 11 or 12 nt) is not obvious. An interesting explanation has been proposed for DHBV, according to which burying part of the 5´ DR1 sequence in a competing intramolecular hairpin structure effectively shortens the sequence available for hybridization with the RNA primer [156] . From its new location on DR2 the RNA primer is extended towards the P bound 5´ end of the (-)-DNA, including the 5´ r redundancy.…”

“…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.…”

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

“…Interactions between two sets of distally located cis-acting elements in the minusstrand DNA (3E-M3 and 5E-M5) have been shown to contribute to both processes, presumably by base pairing to juxtapose the donor and acceptor sites for the two template switches (9,15). Additionally, a small DNA hairpin near the 3Ј end of the minus-strand DNA has been shown to promote primer translocation by inhibiting in situ priming (8), as well as contributing to the circularization process (7). In this study, we provide evidence of another example of a cis-acting element that contributes to both plus-strand template switches.…”