Synthesis of the relaxed-circular (RC) genome of hepadnaviruses is a multistep process that requires template switching during reverse transcription. Studies of duck hepatitis B virus indicated the presence of cis-acting sequences, distinct from the donor and acceptor sequences for the template switches, which contribute to the synthesis of RC DNA. However, knowledge about cis-acting requirements distinct from the donor and acceptor sites for human hepatitis B virus (HBV) was lacking. In this study, we searched for cis-acting sequences for synthesis of HBV RC DNA by analyzing a set of deletion variants that collectively represent most of the HBV genome. Sequences of epsilon, DR1, DR2, 5r, and 3r were not analyzed in the study. Results from Southern blotting showed that multiple cis-acting sequences were involved in the synthesis of HBV RC DNA. Analysis of several HBV/woodchuck hepatitis virus chimeras corroborated the findings from the analysis of deletion variants. This study represents a comprehensive and quantitative analysis of cis-acting sequences that contribute to the synthesis of HBV RC DNA.Hepadnaviruses are a family of enveloped viruses with circular double-stranded DNA genomes of 3.0 to 3.3 kb. Members of the family include human hepatitis B virus (HBV), woodchuck hepatitis virus (WHV), ground squirrel hepatitis virus, and duck hepatitis B virus (DHBV) (for reviews, see references 4 and 27). Hepadnaviruses infect the livers of their hosts and can cause acute and chronic liver diseases. HBV is the leading cause of primary hepatocellular carcinoma. More than 350 million people are chronically infected with HBV worldwide (10). Chronic HBV infection increases the risk for hepatocellular carcinoma 100-fold (3). Since viral replication is necessary for maintaining viral persistence, the study of HBV DNA replication is crucial for understanding and ultimately controlling HBV pathogenesis.Hepadnaviruses replicate their genomes by reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA) (for a review, see reference 4). Reverse transcription is a complex multistep process. DHBV is an invaluable model in elucidating the mechanism by which hepadnaviruses replicate. DHBV and HBV share little, if any, nucleotide sequence similarity; however, they have similar genome organizations and are believed to use similar mechanisms for reverse transcription. Our present understanding of hepadnavirus reverse transcription is derived largely from the study of DHBV.Reverse transcription of hepadnaviruses takes place in the cytoplasmic nucleocapsids of infected cells (29). The first two steps are encapsidation of pgRNA and initiation of minusstrand DNA synthesis. Hepadnavirus P protein binds an RNA stem-loop at the 5Ј end of the pgRNA, termed epsilon, to initiate encapsidation of pgRNA (1, 2, 8, 24). P protein, acting as primer and reverse transcriptase, initiates minus-strand DNA synthesis using nucleotides within epsilon as a template (Fig. 1A) (24,(33)(34)(35). After the synthesis of three or four nucleotides ...

A ferric leghemoglobin reductase from the cytosol of soybean (Glyclne max) root nodules was purified to homogeneity and partlafly characterized. The enzyme is a flavoprotein with flavin adenine dinuclotide as the prosthetic group and consists of two identical subunits, each having a molecular mass of 54 kilodaltons. The pure enzyme shows a high activity for ferric leghemoglobin reduction with NADH as the reductant in the absence of any exogenous mediators. The enzyme also exhibits NADH-dependent 2,6-dichloroindophenol reductase activity. A sequence of the first 50 N-terminal amino acids of the purified protein was obtained. Comparisons with known protein sequences have shown that the sequence of the ferric leghemoglobin reductase is highly related to those of the flavin-nucleotide disulfide oxidoreductases, especially dihydrolipoamide dehydrogenase of the pyruvate dehydrogenase complex.proposed to be flavoproteins or metalloflavoproteins. All have low affinity for Lb"3, low Lb"3 reductase specific activity, and usually require for activity some exogenous electron carriers such as DCIP, methylene blue, or Cyt c. Their ability to catalyze Lb"3 reduction in vivo is doubtful. Klucas and colleagues (9,16,17) identified a protein with FLbR activity from soybean nodule cytosol. This enzyme showed a high activity for Lb"3 reduction with NAD(P)H as reductant in the absence of exogenous electron mediators. In this study the FLbR was purified to homogeneity. The enzyme was partially characterized and the sequence of N-terminal amino acids was determined. Based on the amount enzymatic activity and the apparent high affinity for Lb"3 of the purified enzyme, it seems likely that this enzyme may play a physiological role in maintaining Lb in the functional ferrous state in soybean nodules. MATERIALS AND METHODSLb3 plays an important role in symbiotic nitrogen fixation because it facilitates diffusion of02 to the N2-fixing bacteroids and buffers 02 concentration within the infected cells of nodules (2,

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Abstract. Both iodic acid (HIO3, IA) and methanesulfonic acid (CH3S(O)2OH, MSA) have been identified by field studies as important precursors of new particle formation (NPF) in marine areas. However, the mechanism of NPF in which IA and MSA are jointly involved is still unclear. Hence, we investigated the IA-MSA nucleation system under different atmospheric conditions and uncovered the corresponding nucleating mechanism at the molecular level for the first time, using a quantum chemical approach and Atmospheric Cluster Dynamics Code (ACDC). The findings show that the pure-IA nucleation rate was much lower than the results of CLOUD (Cosmics Leaving Outdoor Droplets) experiments. MSA can promote IA cluster formation through stabilizing IA via both hydrogen and halogen bonds, especially under conditions with lower temperatures, sparse IA, and rich MSA. However, the nucleation rate of the IA-MSA mechanism is much lower than that of field observations, indicating that the effect of additional nucleation precursors needs to be considered (e.g., H2SO4, HIO2, NH3, and amines). The IA-MSA nucleation mechanism revealed in this study may help to gain insight into the joint effect of marine sulfur- and iodine-containing components on marine NPF.

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 5r and 3r 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.Hepadnaviruses are a family of DNA viruses whose primary site of replication is the liver (for reviews, see references 3 and 13). Each family member displays a narrow host range. Infections can be either acute or chronic. Liver disease is a common, but not obligatory, consequence of infection. It is thought that the host's immune response to the infection is a major contributor to the development of liver disease. Humans that are chronically infected with hepatitis B virus (HBV) are at an increased risk for the development of primary liver cancer, making HBV one of the leading causes of this malignancy worldwide.Hepadnaviruses replicate their genomes through reverse transcription of an RNA precursor (16). DNA replication occurs within the viral capsid in the cytoplasm of the infected cell. Only after synthesis of a significant portion of plus-strand DNA are capsids able efficiently to leave the cell as enveloped virions. Capsids with immature genomes inefficiently exit the cell as enveloped virions. Therefore, virus production is dependent on correct and efficient execution of each of the steps of reverse transcription within the infected cell. As with other reverse transcription schemes, template switching is integral to hepadnavirus DNA synthesis (5, 17). Template switching is the process in which the DNA strand undergoing synthesi...