A Novel cis -Acting Element Facilitates Minus-Strand DNA Synthesis during Reverse Transcription of the Hepatitis B Virus Genome

Abstract

Hepadnaviruses replicate through reverse transcription of an RNA pregenome, resulting in a relaxed circular DNA genome. The first 3 or 4 nucleotides (nt) of minus-strand DNA are synthesized by the use of a bulge in a stem-loop structure near the 5 end of the pregenome as a template. This primer is then transferred to a complementary UUCA motif, termed an acceptor, within DR1* near the 3 end of the viral pregenome via 4-nt homology, and it resumes minus-strand DNA synthesis: this process is termed minus-strand transfer or primer translocation. Aside from the sequence identity of the donor and acceptor, little is known about the sequence elements contributing to minus-strand transfer. Here we report a novel cis-acting element, termed the ␤5 region (28 nt in length), located 20 nt upstream of DR1*, that facilitates minus-strand DNA synthesis. The deletion or inversion of the sequence including the ␤5 region diminished minus-strand DNA synthesis initiated at DR1*. Furthermore, the insertion of the ␤5 region into its own position in a mutant in which the sequences including the ␤5 region were replaced restored minus-strand DNA synthesis at DR1*. We speculate that the ␤5 region facilitates minus-strand transfer, possibly by bringing the acceptor site in proximity to the donor site via base pairing or by interacting with protein factors involved in this process.Hepadnaviruses infect the liver tissue of their mammalian and avian hosts, resulting in acute and chronic liver diseases such as hepatitis, cirrhosis, and hepatocellular carcinoma (5). Prototypic members of the family include human hepatitis B virus (HBV), woodchuck hepatitis virus, and duck hepatitis B virus (DHBV). Hepadnaviruses have a DNA genome which replicates through an RNA intermediate via reverse transcription (5, 20). Genome replication of hepadnaviruses, catalyzed by a viral reverse transcriptase, involves the conversion of the single-stranded RNA genome into double-stranded DNA through a complex series of reactions. The strategy of hepadnaviral reverse transcription parallels that of retroviruses in many respects (30). For instance, both of its reverse transcription reactions require multiple template switching events for the completion of viral genome replication. These template switching events are mediated primarily through complementarity between donor sites and acceptor sites present in the terminal redundancy region of the RNA genomes (2,19,29).Despite its fundamental similarity to retroviral reverse transcription, many features of hepadnaviral reverse transcription are distinct. Hepadnaviral reverse transcription occurs inside nucleocapsids after encapsidation of the pregenomic RNA (pgRNA) (5, 20). A stem-loop structure (i.e., ε) near the 5Ј end of the pgRNA serves not only as an encapsidation signal (6, 10, 11, 21) but also as the initiation site for minus-strand DNA synthesis (19,23,29,32). Consequently, unlike the case for retroviruses, the initiation of minus-strand DNA synthesis is mechanistically coupled to encapsidation of the pgRNA. Fu...

Hepatitis B virus replication

“…A general mechanism would be closed-loop formation of the pgRNA via cellular proteins such as elongation initiation factor 4G (eIF-4G) which links 5´ cap and 3´ poly-A binding proteins [80] . A recent more specific model is a longrange RNA interaction between ε and a new cis-element ("φ" or "β5") slightly upstream of DR1* that is involved in proper (-)-DNA synthesis [144][145][146] ; it contains a sequence that DNA primer translocation (first template switch). P copies 3 to 4 nt from the 5´ ε bulge, yielding the TP-linked DNA oligonucleotide which is translocated to the complementary motif in the 3´ proximal DR1*.…”

DDX3 DEAD-Box RNA Helicase Inhibits Hepatitis B Virus Reverse Transcription by Incorporation into Nucleocapsids

“…In addition, the interaction between DDX3 and the HCV core protein was shown to be required for HCV genome replication (4,29). Despite the common use of DDX3 by HIV and HCV, these viruses employ distinct mechanisms to subvert DDX3 for their own RNA metabolism needs.Extensive genetic analysis has provided many mechanistic details of hepadnaviral reverse transcription (1,2,24,25,27,35,36). However, little is known about host factors that contribute to viral genome replication.…”

Base Pairing between the 5′ Half of ε and a cis -Acting Sequence, Φ, Makes a Contribution to the Synthesis of Minus-Strand DNA for Human Hepatitis B Virus

“…Recently, through deletion analysis, a cis-acting sequence that lies between DR2 and the 3Ј copy of DR1 on pgRNA was uncovered that was postulated to be involved in minus-strand DNA synthesis (11,21). Nucleotides within this region (termed ⌽) have been hypothesized to base pair with the 5Ј half of the encapsidation signal, ε (Fig.…”