cis-Acting sequences 5E, M, and 3E interact to contribute to primer translocation and circularization during reverse transcription of avian hepadnavirus DNA.
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Hepadnaviral reverse transcription requires template switches for the genesis of relaxed circular (RC) DNA, the major genomic form in virions. Two template switches, primer translocation and circularization, are required during the synthesis of the second, or plus, strand of DNA. Studies of duck hepatitis B virus (DHBV) indicate that in addition to the requirement for repeated sequences at the donor and acceptor sites, template switching requires at least three other cis-acting sequences, 5E, M, and 3E. In this study we analyzed a series of variant heron hepatitis B viruses (HHBV) in which the regions of the genome that would be expected to contain 5E, M, and 3E were replaced with DHBV sequence. We found that all single and double chimeras were partially defective in the synthesis of RC DNA. In contrast, the triple chimera was able to synthesize RC DNA at a level comparable to that of unchanged HHBV. These results indicate that the three cis-acting sequences, 5E, M, and 3E, need to be compatible to contribute to RC DNA synthesis, suggesting that these sequences interact during plus-strand synthesis. Second, we found that the defect in RC DNA synthesis for several of the single and double chimeric viruses resulted from a partial defect in primer translocation/utilization and a partial defect in circularization. These findings indicate that the processes of primer translocation and circularization share a mechanism during which 5E, M, and 3E interact. (+info)
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.
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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. (+info)
Template switches during plus-strand DNA synthesis of duck hepatitis B virus are influenced by the base composition of the minus-strand terminal redundancy.
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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 (5'r and 3'r) 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 3'r decreases. Thus, a terminal redundancy rich in A and T residues is important for both plus-strand template switches in DHBV. (+info)
Age-related differences in amplification of covalently closed circular DNA at early times after duck hepatitis B virus infection of ducks.
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Inoculation of 3-day-old (3D) or 3-week-old (3W) ducklings with duck hepatitis B virus results in chronic or transient infection, respectively. We previously showed that rapid production of neutralizing antibody following inoculation of 3W ducklings prevents virus from spreading in the liver and leads to a transient infection (Y.-Y. Zhang and J. Summers, J. Virol. 78:1195-1201, 2004). In this study we further investigated early events of viral infection in both 3D and 3W ducks. We present evidence that a lower level of virus replication in the hepatocytes of 3W birds is an additional factor that probably favors transient infection. We suggest that lower virus replication is due to a less rapid covalently closed circular DNA amplification, leading to lower viremias and a slower spread of infection in the liver, and that the slower spread of infection in 3W ducks makes the infection more sensitive to interruption by the host immune responses. (+info)
Molecular analysis of duck hepatitis virus type 1 reveals a novel lineage close to the genus Parechovirus in the family Picornaviridae.
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Duck hepatitis virus type 1 (DHV-1) was previously classified as an enterovirus, based primarily on observed morphology and physicochemical properties of the virion. The complete nucleotide sequences of two strains (DRL-62 and R85952) of DHV-1 have been determined. Excluding the poly(A) tail, the genomes are 7691 and 7690 nt, respectively, and contain a single, large open reading frame encoding a polyprotein of 2249 aa. The genome of DHV-1 is organized as are those of members of the family Picornaviridae: 5' untranslated region (UTR)-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3' UTR. Analysis of the genomic and predicted polyprotein sequences revealed several unusual features, including the absence of a predicted maturation cleavage of VP0, the presence of two unrelated 2A protein motifs and a 3' UTR extended markedly compared with that of any other picornavirus. The 2A1 protein motif is related to the 2A protein type of the genus Aphthovirus and the adjacent 2A2 protein is related to the 2A protein type present in the genus Parechovirus. Phylogenetic analysis using the 3D protein sequence shows that the two DHV-1 strains are related more closely to members of the genus Parechovirus than to other picornaviruses. However, the two DHV-1 strains form a monophyletic group, clearly distinct from members of the genus Parechovirus. (+info)
Molecular analysis of duck hepatitis virus type 1.
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The genome sequence of a duck hepatitis virus type 1 (DHV-1) strain was determined. Comparative sequence analysis showed that the genome possesses a typical picornarivus organization and also exhibits several unique features, such as the similarity of internal ribosome entry site to that of Porcine teschovirus 1 and Hepatitis C virus, the presence of a longest 3' untranslated region and a shorter leader protein in the Picornaviridae, the absence of a predicted maturation cleavage of VP0, the association of an aphthovirus-like 2A1 and parechovirus-like 2A2, and the unprecedented presence of an AIG1 domain in the N-terminus of 2A2. It is concluded that DHV-1 belongs to a new group of the family Picornaviridae that may form a separate genus most closely related to the genus Parechovirus. (+info)
AIM: To present an approach for selectively killing retrovirus-infected cells that combines the toxicity of Pseudomonas exotoxin (PE) and the presence of reverse transcriptase (RT) in infected cells. METHODS: PE antisense toxin RNA has palindromic stem loops at its 5' and 3' ends enabling self-primed generation of cDNA in the presence of RT. The RT activity expressed in retrovirus-infected cells converts "antisense-toxin-RNA" into a lethal toxin gene exclusively in these cells. RESULTS: Using cotransfection studies with PE-expressing RNAs and beta-gal expressing reporter plasmids, we show that, in HepG2 and HepG2.2.15 hepatoma cells as well as in duck hepatitis B virus (DHBV) infected cells, HBV or DHBV-polymerase reverse transcribe a lethal cDNA copy of an antisense toxin RNA, which is composed of sequences complementary to a PE gene and eukaryotic transcription and translation signals. CONCLUSION: This finding may have important implications as a novel therapeutic strategy aimed at the elimination of HBV infection. (+info)
The duck hepatitis virus 5'-UTR possesses HCV-like IRES activity that is independent of eIF4F complex and modulated by downstream coding sequences.
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