No abstract

Mutagenesis by the overlap extension PCR has become a standard method of creating mutations including substitutions, insertions, and deletions. Nonetheless, the established overlap PCR mutagenesis is limited in many respects. In particular, it has been difficult to make an insertion larger than 30 nt, since all sequence alterations must be embedded within the primer. Here, we describe a rapid and efficient method for creating insertions or deletions of any length at any position in a DNA molecule. This method is generally applicable, and therefore represents a significant improvement to the now widely used overlap extension PCR method.

Hepadnaviruses replicate via reverse transcription of an RNA template, the pregenomic RNA (pgRNA). Although hepadnaviral polymerase (Pol) and retroviral reverse transcriptase are distantly related, some of their features are distinct. In particular, Pol contains two additional N-terminal subdomains, the terminal protein and spacer subdomains. Since much of the spacer subdomain can be deleted without detrimental effects to hepatitis B virus (HBV) replication, this subdomain was previously thought to serve only as a spacer that links the terminal protein and reverse transcriptase subdomains. Unexpectedly, we found that the C terminus of the spacer subdomain is indispensable for the encapsidation of pgRNA. Alanine-scanning mutagenesis revealed that four conserved cysteine residues, three at the C terminus of the spacer subdomain and one at the N terminus of the reverse transcriptase subdomain, are critical for encapsidation. The inability of the mutant Pol proteins to incorporate into nucleocapsid particles, together with other evidence, argued that the four conserved cysteine residues are critical for RNA binding. One implication is that these four cysteine residues might form a putative zinc finger motif. Based on these findings, we speculate that the RNA binding activity of HBV Pol may be mediated by this newly identified putative zinc finger motif.

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...

Synthesis of the relaxed-circular (RC) DNA genomes of hepadnaviruses by reverse transcriptase involves two template switches during plus-strand DNA synthesis. These template switches require repeat sequences (so-called donor and acceptor sites) between which a complementary strand of nucleic acid is transferred. To determine cis-acting elements apart from the donor and acceptor sites that are required for plus-strand RC DNA synthesis by hepatitis B virus (HBV), a series of mutants bearing a small deletion were made and analyzed for their impact on the viral genome synthesis. We found three novel cis-acting elements in the HBV genome: one element, located in the middle of the minus strand, is indispensable, whereas the other two elements, located near either end of the minus strand, contribute modestly to the plus-strand RC DNA synthesis. The data indicated that the first element facilitates plus-strand RNA primer translocation or subsequent elongation during plus-strand RC DNA synthesis, while the last two elements, although distantly located on the minus strand, act at multiple steps to promote plus-strand RC DNA synthesis. The necessity of multiple cis-acting elements on the minus-strand template reflects the complex nature of hepadnavirus reverse transcription.Hepadnaviruses are small (ca. 3.2-kb), double-stranded DNA viruses that replicate through an RNA intermediate via reverse transcription. Prototypical members of the family include human hepatitis B virus (HBV), woodchuck hepatitis virus, and duck hepatitis B virus (DHBV) (4). Retroid elements, including retroviruses and hepadnaviruses, replicate by converting their single-stranded RNA templates into doublestranded DNAs via reverse transcription. During reverse transcription, a process described as strand transfer or template switching is required for the successful synthesis of a doublestranded DNA genome (5). Template switching, in which the DNA strand undergoing synthesis switches from one template to another, is mediated by complementarity between a donor and an acceptor site. For retroviruses, two template switches are required to generate a linear double-stranded DNA: one for minus-strand DNA synthesis and another for plus-strand DNA synthesis. In hepadnaviruses, in addition to these two template switches, a third template switch, termed circularization, is required to generate a relaxed-circular (RC) DNA genome (4).Reverse transcription of hepadnaviruses takes place within the viral capsid in the cytoplasm of infected cells (Fig. 1). The first template switch required for the synthesis of the RC DNA genome occurs shortly after the initiation of minus-strand DNA synthesis. Recognition of a stem-loop structure (an encapsidation signal designated ε near the 5Ј end of the pregenomic RNA [pgRNA]) by P (polymerase) protein directs encapsidation of the pgRNA and P protein into a nascent capsid particle (8, 10, 11). Minus-strand DNA synthesis is initiated by protein priming with P protein as a primer and the bulge region of ε as a template (26,27). Follow...