End Of Minus-strand DNA Research Articles

Mechanism for CCC DNA Synthesis in Hepadnaviruses

Hepadnavirus replication requires the synthesis of a covalently closed circular (CCC) DNA from the relaxed circular (RC) viral genome by an unknown mechanism. CCC DNA formation could require enzymatic activities of the viral reverse transcriptase (RT), or cellular DNA repair enzymes, or both. Physical mapping of the 5′ and 3′ ends of RC DNA and sequence analysis of CCC DNA revealed that CCC DNA synthesis requires the removal of the RT and an RNA oligomer from the 5′ ends of minus and plus strand DNA, respectively, removal of sequences from the terminally redundant minus strand, completion of the less than full-length plus strand, and ligation of the ends. Two models have been proposed that could explain CCC DNA formation. The first (model 1) invokes a role for the RT to catalyze a cleavage-ligation reaction leading to the formation of a unit length minus strand in CCC DNA and a DNA repair reaction for the completion and ligation of plus strand DNA; the second (model 2) predicts that CCC DNA formation depends entirely on cellular DNA repair enzymes. To determine which mechanism is utilized, we developed cell lines expressing duck hepatitis B virus genomes carrying mutations permitting us to follow the fate of viral DNA sequences during their conversion from RC to CCC DNA. Our results demonstrated that the oligomer at the 5′ end of minus strand DNA is completely or at least partially removed prior to CCC DNA synthesis. The results indicated that both RC DNA strands undergo DNA repair reactions carried out by the cellular DNA repair machinery as predicted by model 2. Thus, our study provided the basis for the identification of the cellular components required for CCC DNA formation.

Analysis of duck hepatitis B virus reverse transcription indicates a common mechanism for the two template switches during plus-strand DNA synthesis.

The synthesis of the hepadnavirus relaxed circular DNA genome requires two template switches, primer translocation and circularization, during plus-strand DNA synthesis. Repeated sequences serve as donor and acceptor templates for these template switches, with direct repeat 1 (DR1) and DR2 for primer translocation and 5'r and 3'r for circularization. These donor and acceptor sequences are at, or near, the ends of the minus-strand DNA. Analysis of plus-strand DNA synthesis of duck hepatitis B virus (DHBV) has indicated that there are at least three other cis-acting sequences that make contributions during the synthesis of relaxed circular DNA. These sequences, 5E, M, and 3E, are located near the 5' end, the middle, and the 3' end of minus-strand DNA, respectively. The mechanism by which these sequences contribute to the synthesis of plus-strand DNA was unclear. Our aim was to better understand the mechanism by which 5E and M act. We localized the DHBV 5E element to a short sequence of approximately 30 nucleotides that is 100 nucleotides 3' of DR2 on minus-strand DNA. We found that the new 5E mutants were partially defective for primer translocation/utilization at DR2. They were also invariably defective for circularization. In addition, examination of several new DHBV M variants indicated that they too were defective for primer translocation/utilization and circularization. Thus, this analysis indicated that 5E and M play roles in both primer translocation/utilization and circularization. In conjunction with earlier findings that 3E functions in both template switches, our findings indicate that the processes of primer translocation and circularization share a common underlying mechanism.

Mutations that increase in situ priming also decrease circularization for duck hepatitis B virus.

The process of hepadnavirus reverse transcription involves two template switches during the synthesis of plus-strand DNA. The first involves translocation of the plus-strand primer from its site of generation, the 3' end of minus-strand DNA, to the complementary sequence DR2, located near the 5' end of the minus-strand DNA. Plus strands initiated from DR2 are extended to the 5' end of the minus-strand DNA. At this point, the 3' end of the minus strand becomes the template via the second template switch, a process called circularization. Elongation of circularized plus-strand DNA generates relaxed circular DNA. Although most virions contain relaxed circular DNA, some contain duplex linear DNA. Duplex linear genomes are synthesized when the plus-strand primer is used at the site of its generation, the 3' end of the minus-strand template. This type of synthesis is called in situ priming. Although in situ priming is normally low, in some duck hepatitis B virus mutants this type of priming is elevated. For example, mutations within the 3' end of the minus-strand DNA can lead to increased levels of in situ priming. We report here that these same mutations result in a second defect, a less efficient template switch that circularizes the genome. Although it is not clear how these mutations affect both steps in DNA replication, our findings suggest a commonality in the mechanism of initiation of plus-strand synthesis and the template switch that circularizes the genome.

Cleavage Specificities of Moloney Murine Leukemia Virus RNase H Implicated in the Second Strand Transfer During Reverse Transcription

Reverse transcription of a retroviral RNA genome requires two template jumps to generate the linear double-stranded DNA required for integration. The RNase H activity of reverse transcriptase has several roles during this process. We have examined RNase H cleavages that define the maximal 3' and 5' ends of Moloney murine leukemia virus minus strand DNA prior to the second template jump. In both the endogenous reaction and on model substrates in vitro, RNase H cleaves the genomic RNA template between the second and third ribonucleotides 5' of the U5/PBS junction, but other minor cleavages between 1 and 10 nucleotides 5' of this junction are also observed. Similar experiments examining the specificity of RNase H for tRNA primer removal revealed that cleavage generally leaves a ribo A residue at the 5' end of minus strand DNA. These observations suggest that three bases are typically duplicated on the ends of the minus strands, leading to an intermediate following the second jump which contains unpaired nucleotides. Model substrates mimicking the structure of this intermediate demonstrate that reverse transcriptase has little difficulty in utilizing such a branched structure for the initiation of displacement synthesis.

RNA sequences controlling the initiation and transfer of duck hepatitis B virus minus-strand DNA.

Hepadnaviruses replicate by reverse transcription of an RNA pregenome. Reverse transcription initiates within the stem-loop (SL) of the epsilon RNA packaging signal and is discontinuous: the nascent minus-polarity DNA is transferred to direct repeat 1 (DR1) at the 3' end of the pregenomic RNA prior to extensive elongation. In this study we analyzed the initiation and transfer of duck hepatitis B virus minus-strand DNA by using functional viral polymerase expressed in yeast cells. We extensively mutagenized both DR1 and the SL and observed the effects on reverse transcription initiation and on the transfer and subsequent extension of minus-strand DNA. Our results indicate that sequences throughout the SL affect initiation and that minus-strand DNAs initiated at three locations within the SL are competent for transfer to DR1. A short region of homology between the 5' end of minus-strand DNA and DR1 was necessary but not sufficient to direct the transfer and subsequent extension reactions. This homology was tolerant of minor substitutions, and 2 nucleotides of homology mediated transfer accurately. Mutations had greater detrimental effects on transfer and subsequent extension of minus-strand DNA when they were placed in DR1 than when they were placed in the SL. Efficient transfer of minus-strand DNA from a mutant SL to DR2 was observed in the yeast system. The hexanucleotide AAUUAC was identified as the primary cis element of the transfer acceptor, but this element was also insufficient to independently specify the acceptor location. Therefore, additional information, possibly positional context or unrecognized RNA secondary structure, is required.

Identification of a signal necessary for initiation of reverse transcription of the hepadnavirus genome

Reverse transcription of the hepadnavirus genome initiates near the 3' end of the RNA template and has previously been shown to depend on sequences flanking the initiation site for DNA synthesis (C. Seeger and J. Maragos, J. Virol. 64:16-23, 1990). DNA synthesis leads to the covalent attachment of a protein to the 5' end of minus-strand DNA, and it is generally believed that this protein serves as the primer for reverse transcription. To examine priming in more detail, we have carried out a detailed genetic analysis of the nucleotide sequences at the origin of minus-strand DNA synthesis characterized in our earlier study. This mutational analysis has led to the identification of a short, four-nucleotide-long sequence as the signal for initiation of reverse transcription. This signal is a UUUC sequence motif flanking the position of the 5' end of minus-strand DNA, which alone is not sufficient for DNA synthesis, indicating that positional effects are also important to specify the origin of DNA synthesis.

Identification and characterization of the woodchuck hepatitis virus origin of DNA replication

Replication of the woodchuck hepatitis virus (WHV) genome requires the synthesis of minus-strand DNA from an RNA template, the pregenome, by reverse transcription. During this reaction, the 5' end of minus-strand DNA becomes covalently linked to a protein. The position of the 5' end of minus-strand DNA was identified previously, but the initiation site for DNA synthesis on pregenomic RNA remained ambiguous because of a sequence repetition at the termini of the RNA template for reverse transcription. Employing a recently designed expression vector for the production of infectious WHV, we localized the origin of minus-strand DNA synthesis to the 3' end of pregenomic RNA. In addition, we identified the nucleotide sequences on pregenomic RNA that provide the signal for the initiation of reverse transcription. Removal of this signal sequence from pregenomic RNA abolished minus-strand DNA synthesis. Insertion of a DNA oligomer bearing this signal sequence at the 3' end of pregenomic RNA restored the production of minus-strand DNA joined to protein. Our results support a model in which protein is the primer for reverse transcription of minus-strand DNA of WHV.

Reverse transcription of avian myeloblastosis virus 35S RNA. Early synthesis of plus strand DNA of discrete size in reconstructed reactions.

The early DNa products of reverse transcription have been analyzed from reconstructed reactions containing avian myeloblastosis virus 35S RNA . tRNAtrp complex and highly purified reverse transcriptase. We describe conditions for the synthesis of genome-length complementary DNA and two discrete species of plus strand DNA (the same chemical polarity as the viral RNA genome) about 300 and 400 nucleotides in length. Plus DNA400 and plus DNA300 were detected by molecular hybridization with DNA probes complementary to sequences from both the 3'- and 5'-ends of the viral RNA. Both species appear to be copied from the 5'-end of minus strand DNA by their hybridization properties and their early synthesis when only the 5'-end of minus strand DNA is available as template. Restriction endonuclease mapping of plus DNA400 and plus DNA300 rules out a precursor-product relationship between the two. Rather the results suggest a unique initiation site for both species, with plus DNA400 containing internal sequences not present in plus DNA300. Plus DNA400 and plus DNA300 appear to be analogous to early plus DNA species detected in cells early after retrovirus infection. Thus, purified reverse transcriptase appears to be enzymatically sufficient for synthesis of genome-length complementary DNA and initiation and synthesis of early plus strand DNA as observed in infected cells.