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Hepatitis B virus (HBV) capsid assembly is traditionally thought to occur predominantly in the cytoplasm, where the virus gains access to the virion egress pathway. To better define sites of HBV capsid assembly, we carried out single cell imaging of HBV Core protein (Cp) subcellular trafficking over time under conditions supporting genome packaging and reverse transcription in Huh7 hepatocellular carcinoma cells. Time-course analyses including live cell imaging of fluorescently tagged Cp derivatives showed Cp to accumulate in the nucleus at early time points (~24 h), followed by a marked re-distribution to the cytoplasm at 48 to 72 h. Nucleus-associated Cp was confirmed to be capsid and/or high-order assemblages using a novel dual label immunofluorescence strategy. Nuclear-to-cytoplasmic re-localization of Cp occurred predominantly during nuclear envelope breakdown in conjunction with cell division, followed by strong cytoplasmic retention of Cp. Blocking cell division resulted in strong nuclear entrapment of high-order assemblages. A Cp mutant, Cp-V124W, predicted to exhibit enhanced assembly kinetics, also first trafficked to the nucleus to accumulate at nucleoli, consistent with the hypothesis that Cp's transit to the nucleus is a strong and constitutive process. Taken together, these results provide support for the nucleus as an early-stage site of HBV capsid assembly, and provide the first dynamic evidence of cytoplasmic retention after cell division as a mechanism underpinning capsid nucleus-to-cytoplasm relocalization. IMPORTANCE Hepatitis B virus (HBV) is an enveloped, reverse-transcribing DNA virus that is a major cause of liver disease and hepatocellular carcinoma. Subcellular trafficking events underpinning HBV capsid assembly and virion egress remain poorly characterized. Here, we developed a combination of fixed and long-term (>24 h) live cell imaging technologies to study the single cell trafficking dynamics of the HBV Core Protein (Cp). We demonstrate that Cp first accumulates in the nucleus, and forms high-order structures consistent with capsids, with the predominant route of nuclear egress being relocalization to the cytoplasm during cell division in conjunction with nuclear membrane breakdown. Single cell video microscopy demonstrated unequivocally that Cp's localization to the nucleus is constitutive. This study represents a pioneering application of live cell imaging to study HBV subcellular transport, and demonstrates links between HBV Cp and the cell cycle.

Hepatitis B virus (HBV) is a worldwide health problem without curative treatments. Investigation of the regulation of HBV biosynthesis by class I and II histone deacetylases (HDACs) demonstrated that catalytically active HDAC5 upregulates HBV biosynthesis. HDAC5 expression increased both the stability and splicing of the HBV 3.5 kb RNA without altering the translational efficiency of the viral pregenomic or spliced 2.2 kb RNAs. Together, these observations point to a broader role of HDAC5 in regulating RNA splicing and transcript stability while specifically identifying a potentially novel approach toward antiviral HBV therapeutic development., Competing Interests: The authors have declared that no competing interests exist.

Unlabelled: To understand subcellular sites of hepatitis B virus (HBV) replication, we visualized core (Cp), polymerase (Pol), and pregenomic RNA (pgRNA) in infected cells. Interestingly, we found that the majority of Pol localized to the mitochondria in cells undergoing viral replication. The mitochondrial localization of Pol was independent of both the cell type and other viral components, indicating that Pol contains an intrinsic mitochondrial targeting signal (MTS). Neither Cp nor pgRNA localized to the mitochondria during active replication, suggesting a role other than DNA synthesis for Pol at the mitochondria. The Pol of duck hepatitis B virus (DHBV) also localized to the mitochondria. This result indicates that localization of Pol to mitochondria is likely a feature of all hepadnaviruses. To map the MTS within HBV Pol, we generated a series of Pol-green fluorescent protein (Pol-GFP) fusions and found that a stretch spanning amino acids (aa) 141 to 160 of Pol was sufficient to target GFP to the mitochondria. Surprisingly, deleting aa 141 to 160 in full-length Pol did not fully ablate Pol's mitochondrial localization, suggesting that additional sequences are involved in mitochondrial targeting. Only by deleting the N-terminal 160 amino acids in full-length Pol was mitochondrial localization ablated. Crucial residues for pgRNA packaging are contained within aa 141 to 160, indicating a multifunctional role of this region of Pol in the viral life cycle. Our studies show an unexpected Pol trafficking behavior that is uncoupled from its role in viral DNA synthesis., Importance: Chronic infection by HBV is a serious health concern. Existing therapies for chronically infected individuals are not curative, underscoring the need for a better understanding of the viral life cycle to develop better antiviral therapies. To date, the most thoroughly studied function of Pol is to package the pgRNA and reverse transcribe it to double-stranded DNA within capsids. This study provides evidence for mitochondrial localization of Pol and defines the MTS. Recent findings have implicated a non-reverse transcription role for Pol in evading host innate immune responses. Mitochondria play an important role in controlling cellular metabolism, apoptosis, and innate immunity. Pol may alter one or more of these host mitochondrial functions to gain a replicative advantage and persist in chronically infected individuals., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Hepatitis B virus (HBV) capsid proteins (Cps) assemble around the pregenomic RNA (pgRNA) and viral reverse transcriptase (P). pgRNA is then reverse transcribed to double-stranded DNA (dsDNA) within the capsid. The Cp assembly domain, which forms the shell of the capsid, regulates assembly kinetics and capsid stability. The Cp, via its nucleic acid-binding C-terminal domain, also affects nucleic acid organization. We hypothesize that the structure of the capsid may also have a direct effect on nucleic acid processing. Using structure-guided design, we made a series of mutations at the interface between Cp subunits that change capsid assembly kinetics and thermodynamics in a predictable manner. Assembly in cell culture mirrored in vitro activity. However, all of these mutations led to defects in pgRNA packaging. The amount of first-strand DNA synthesized was roughly proportional to the amount of RNA packaged. However, the synthesis of second-strand DNA, which requires two template switches, was not supported by any of the substitutions. These data demonstrate that the HBV capsid is far more than an inert container, as mutations in the assembly domain, distant from packaged nucleic acid, affect reverse transcription. We suggest that capsid molecular motion plays a role in regulating genome replication., Importance: The hepatitis B virus (HBV) capsid plays a central role in the virus life cycle and has been studied as a potential antiviral target. The capsid protein (Cp) packages the viral pregenomic RNA (pgRNA) and polymerase to form the HBV core. The role of the capsid in subsequent nucleic acid metabolism is unknown. Here, guided by the structure of the capsid with bound antiviral molecules, we designed Cp mutants that enhanced or attenuated the assembly of purified Cp in vitro. In cell culture, assembly of mutants was consistent with their in vitro biophysical properties. However, all of these mutations inhibited HBV replication. Specifically, changing the biophysical chemistry of Cp caused defects in pgRNA packaging and synthesis of the second strand of DNA. These results suggest that the HBV Cp assembly domain potentially regulates reverse transcription, extending the activities of the capsid protein beyond its presumed role as an inert compartment., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Hepadnaviruses selectively package capsids containing mature double-stranded DNA (dsDNA) genomes in virions. Snow goose hepatitis B virus (SGHBV) is the only known hepadnavirus that packages capsids containing single-stranded DNA (ssDNA) in virions. We found that cells replicating SGHBV produce virions containing ssDNA as efficiently as virions containing mature dsDNA. We determined that SGHBV capsid and envelope proteins independently contribute to the production of virions containing ssDNA, with the capsid protein (Cp) making a larger contribution. We identified that amino acid residues 74 and 107 of SGHBV Cp contribute to this feature of SGHBV. When we changed these residues in duck hepatitis B virus (DHBV) Cp, capsids containing immature ssDNA were packaged in virions. This result suggests that residues 74 and 107 contribute to the appearance of the "capsid packaging signal" on the surface of capsids and interact with the envelope proteins during virion formation. We also found that cells replicating SGHBV package a larger fraction of the total dsDNA they synthesize into virions than do those replicating DHBV. We determined that the SGHBV envelope proteins are responsible for this property of SGHBV. Determining if the ability of SGHBV envelope proteins to cause the formation of virions containing ssDNA is related to its ability to support high levels of virion production or if these two properties are mechanistically distinct will provide insights into virion morphogenesis., Importance: Cells replicating hepadnaviruses contain cytoplasmic capsids that contain mature and immature genomes. However, only capsids containing mature dsDNA genomes are packaged in virions. A mechanistic understanding of this phenomenon, which is currently lacking, is critical to understanding the process of hepadnaviral virion morphogenesis. In this study, we determined that the envelope proteins contribute to the ability of hepadnaviruses to selectively produce virions containing mature dsDNA genomes. Our finding sheds new light on the mechanisms underlying virion morphogenesis and challenges the dogma that "capsid maturation," and therefore the capsid protein (Cp), is solely responsible for the selective production of virions containing mature dsDNA genomes. Further, we identified amino acid residues of Cp that contribute to its ability to cause the selective production of virions containing mature dsDNA genomes. Future studies on the role of these residues in selective secretion will broaden our understanding of this poorly understood aspect of virus replication., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Assembly of a hepatitis B virus (HBV) virion begins with the formation of an RNA-filled core composed of a symmetrical capsid (built of core protein), viral pregenomic RNA, and viral reverse transcriptase. To generate the circular dsDNA genome of HBV, reverse transcription requires multiple template switches within the confines of the capsid. To date, most anti-HBV therapeutics target this reverse transcription process. The detailed molecular mechanisms of this crucial process are poorly understood because of the lack of structural information. We hypothesized that capsid, RNA, and viral reverse transcriptase would need a precise geometric organization to accomplish reverse transcription. Here we present the asymmetric structure of authentic RNA-filled cores, determined to 14.5-Å resolution from cryo-EM data. Capsid and RNA are concentric. On the interior of the RNA, we see a distinct donut-like density, assigned to viral reverse transcriptase, which pins the viral pregenomic RNA to the capsid inner surface. The observation of a unique ordered structure inside the core suggests that assembly and the first steps of reverse transcription follow a single, determinate pathway and strongly suggests that all subsequent steps in DNA synthesis do as well.

Capsid (core) assembly is essential for hepatitis B virus (HBV) replication. We hypothesize that assembly kinetics and stability are tuned for optimal viral replication, not maximal assembly. Assembly effectors (AEfs) are small molecules proposed to disrupt this balance by inappropriately enhancing core assembly. Guided by the structure of an AEf-bound core, we designed a structural mimic of AEf-bound core protein, the V124W mutant. In biochemical studies, the V124W mutant recapitulated the effects of AEfs, with fast assembly kinetics and a strong protein-protein association energy. Also, the mutant was resistant to exogenous AEfs. In cell culture, the V124W mutant behaved like a potent AEf: expression of HBV carrying the V124W mutant was defective for genome replication. Critically, the V124W mutant interfered with replication of wild-type HBV in a dose-dependent manner, mimicking AEf activity. In addition, the V124W mutant was shown to adopt a more compact conformation than that of the wild type, confirming the allosteric regulation in capsid assembly. These studies show that the heteroaryldihydropyrimidine (HAP) binding pocket is a promiscuous target for inducing assembly. Suppression of viral replication by the V124W mutant suggests that mutations that fill the HAP site are not a path for HBV to escape from AEfs.

Covalently closed circular DNA (cccDNA), the nuclear form of hepatitis B virus (HBV), is synthesized by repair of the relaxed circular (RC) DNA genome. Initially, cccDNA is derived from RC DNA from the infecting virion, but additional copies of cccDNA are derived from newly synthesized RC DNA molecules in a process termed intracellular amplification. It has been shown that the large viral envelope protein limits the intracellular amplification of cccDNA for duck hepatitis B virus. The role of the envelope proteins in regulating the amplification of cccDNA in HBV is not well characterized. The present report demonstrates regulation of synthesis of cccDNA by the envelope proteins of HBV. Ablation of expression of the envelope proteins led to an increase (>6-fold) in the level of cccDNA. Subsequent restoration of envelope protein expression led to a decrease (>50%) in the level of cccDNA, which inversely correlated with the level of the envelope proteins. We found that the expression of L protein alone or in combination with M and/or S proteins led to a decrease in cccDNA levels, indicating that L contributes to the regulation of cccDNA. Coexpression of L and M led to greater regulation than either L alone or L and S. Coexpression of all three envelope proteins was also found to limit completion of plus-strand DNA synthesis, and the degree of this effect correlated with the level of the proteins and virion secretion.

The core protein of hepatitis B virus can be phosphorylated at serines 155, 162, and 170. The contribution of these serine residues to DNA synthesis was investigated. Core protein mutants were generated in which each serine was replaced with either alanine or aspartate. Aspartates can mimic constitutively phosphorylated serines while alanines can mimic constitutively dephosphorylated serines. The ability of these mutants to carry out each step of DNA synthesis was determined. Alanine substitutions decreased the efficiency of minus-strand DNA elongation, primer translocation, circularization, and plus-strand DNA elongation. Aspartate substitutions also reduced the efficiency of these steps, but the magnitude of the reduction was less. Our findings suggest that phosphorylated serines are required for multiple steps during DNA synthesis. It has been proposed that generation of mature DNA requires serine dephosphorylation. Our results suggest that completion of rcDNA synthesis requires phosphorylated serines.

The carboxy-terminal domain (CTD) of the core protein of hepatitis B virus is not necessary for capsid assembly. However, the CTD does contribute to encapsidation of pregenomic RNA (pgRNA). The contribution of the CTD to DNA synthesis is less clear. This is the case because some mutations within the CTD increase the proportion of spliced RNA to pgRNA that are encapsidated and reverse transcribed. The CTD contains four clusters of consecutive arginine residues. The contributions of the individual arginine clusters to genome replication are unknown. We analyzed core protein variants in which the individual arginine clusters were substituted with either alanine or lysine residues. We developed assays to analyze these variants at specific steps throughout genome replication. We used a replication template that was not spliced in order to study the replication of only pgRNA. We found that alanine substitutions caused defects at both early and late steps in genome replication. Lysine substitutions also caused defects, but primarily during later steps. These findings demonstrate that the CTD contributes to DNA synthesis pleiotropically and that preserving the charge within the CTD is not sufficient to preserve function.

Hepadnaviruses are DNA viruses that are found in several mammalian and avian species. These viruses replicate their genome through reverse transcription of an RNA intermediate termed pregenomic RNA (pgRNA). pgRNA is reverse transcribed by the viral polymerase into a minus-strand DNA, followed by synthesis of the plus-strand DNA. There are multiple cis-acting sequences that contribute to the synthesis of minus-strand DNA for human hepatitis B virus (HBV). Less is known about the cis-acting sequences of avian hepadnaviruses that contribute to synthesis of minus-strand DNA. To identify cis-acting sequences of duck hepatitis B virus (DHBV) and heron hepatitis B virus (HHBV), we analyzed variants containing 200-nucleotide (nt) deletions. Most variants of DHBV synthesized minus-strand DNA to 50 to 100% of the wild-type (WT) level, while two variants synthesized less than 50%. For HHBV, most variants synthesized minus-strand DNA to less than 50% the WT level. These results differ from those for HBV, where most of the genome can be removed with little consequence. HBV contains a sequence, φ, that contributes to the synthesis of minus-strand DNA. It has been proposed that DHBV has an analogous sequence. We determined that the proposed φ sequence of DHBV does not contribute to the synthesis of minus-strand DNA. Finally, we found that the DR2 sequence present in all hepadnaviruses is important for synthesis of minus-strand DNA in both DHBV and HHBV but not in HBV. These differences in cis-acting sequences suggest that the individual hepadnaviruses have evolved differences in their mechanisms for synthesizing minus-strand DNA, more so than for other steps in replication.

Establishment of an infection with hepatitis B virus (HBV) requires synthesis and maintenance of a covalently closed circular DNA (cccDNA) form of the viral genome in the nucleus of host cells. To facilitate the investigation of the synthesis of cccDNA, cell cultures were developed that express HBV to high levels. Cell lines derived from hepatoma cells Huh7 and HepG2 were created that express Epstein-Barr virus (EBV) nuclear antigen-1 and a fusion protein of the Tet repressor and Kox1 transcriptional repression domain stably. Transfection of these cell lines with an expression plasmid for HBV that contains the origin of plasmid replication of EBV (oriP) led to increases in the intracellular levels of HBV core protein ( approximately 8- to 51-fold) and encapsidated HBV DNA ( approximately 3- to 12-fold) in comparison to Huh7 and HepG2 cells. Virion production was also increased ( approximately 3- to 12-fold) in these cell cultures and an increase in the level of cccDNA ( approximately 3-fold) was observed in the Huh7-derived cell lines. In addition, these cell lines maintained the HBV expression plasmid upon selection and expressed HBV conditionally. Thus, these cell cultures exhibit several features that facilitate study of the synthesis of cccDNA and other aspects of replication of HBV., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

A critical feature of a viral life cycle is the ability to selectively package the viral genome. In vivo, phosphorylated hepatitis B virus (HBV) core protein specifically encapsidates a complex of pregenomic RNA (pgRNA) and viral polymerase; it has been suggested that packaging is specific for the complex. Here, we test the hypothesis that core protein has intrinsic specificity for pgRNA, independent of the polymerase. For these studies, we also evaluated the effect of core protein phosphorylation on assembly and RNA binding, using phosphorylated core protein and a phosphorylation mimic in which S155, S162, and S170 were mutated to glutamic acid. We have developed an in vitro system where capsids are disassembled and assembly-active core protein dimer is purified. With this protein, we have reassembled empty capsids and RNA-filled capsids. We found that core protein dimer bound and encapsidated both the HBV pregenomic RNA and heterologous RNA with high levels of cooperativity, irrespective of phosphorylation. In direct competition assays, no specificity for pregenomic RNA was observed. This suggests that another factor, such as the viral polymerase, is required for specific packaging. These results also beg the question of what prevents HBV core protein from assembling on nonviral RNA, preserving the protein for virus production.

Hepatitis B virus synthesizes multiple spliced RNAs that can be reverse transcribed into viral DNA. We thoroughly characterized the contribution of spliced RNAs to DNA synthesis in transfected cultures of Huh7 and HepG2 cells. We found that up to 50% of DNA within intracellular capsids is derived from five spliced RNAs. Expressing HBV P protein and pgRNA from separate plasmids and the use of the CMV-IE promoter contributes to these high levels of encapsidated DNA derived from spliced RNA. A spliced RNA called Sp1 was the predominant species expressed in both cell lines. All spliced RNAs support the synthesis minus-strand DNA and duplex linear DNA. Only one of the spliced RNAs, Sp14, supported the synthesis of relaxed circular DNA because splicing removed an important cis-acting sequence (hM) in the other four RNAs. Additionally, we created a variant that was deficient in the synthesis of spliced RNA and supported DNA synthesis at wild-type levels. Our results reinforce and extend the idea that a significant fraction of HBV DNA synthesized under common experimental conditions is derived from spliced RNA. It is important that their presence be considered when analyzing HBV DNA replication in transfected cell cultures.

Previous analysis of hepatitis B virus (HBV) indicated base pairing between two cis-acting sequences, the 5' half of the upper stem of epsilon and phi, contributes to the synthesis of minus-strand DNA. Our goal was to identify other cis-acting sequences on the pregenomic RNA (pgRNA) involved in the synthesis of minus-strand DNA. We found that large portions of the pgRNA could be deleted or substituted without an appreciable decrease in the level of minus-strand DNA synthesized, indicating that most of the pgRNA is dispensable and that a specific size of the pgRNA is not required for this process. Our results indicated that the cis-acting sequences for the synthesis of minus-strand DNA are present near the 5' and 3' ends of the pgRNA. In addition, we found that the first-strand template switch could be directed to a new location when a 72-nucleotide (nt) fragment, which contained the cis-acting sequences present near the 3' end of the pgRNA, was introduced at that location. Within this 72-nt region, we uncovered two new cis-acting sequences, which flank the acceptor site. We show that one of these sequences, named omega and located 3' of the acceptor site, base pairs with phi to contribute to the synthesis of minus-strand DNA. Thus, base pairing between three cis-acting elements (5' half of the upper stem of epsilon, phi, and omega) are necessary for the synthesis of HBV minus-strand DNA. We propose that this topology of pgRNA facilitates first-strand template switch and/or the initiation of synthesis of minus-strand DNA.

For hepadnaviruses, the RNA primer for plus-strand DNA synthesis is generated by the final RNase H cleavage of the pregenomic RNA at an 11 nt sequence called DR1 during the synthesis of minus-strand DNA. This RNA primer initiates synthesis at one of two distinct sites on the minus-strand DNA template, resulting in two different end products; duplex linear DNA or relaxed circular DNA. Duplex linear DNA is made when initiation of synthesis occurs at DR1. Relaxed circular DNA, the major product, is made when the RNA primer translocates to the sequence complementary to DR1, called DR2 before initiation of DNA synthesis. We studied the mechanism that determines the site of the final RNase H cleavage in hepatitis B virus (HBV). We showed that the sites of the final RNase H cleavage are always a fixed number of nucleotides from the 5' end of the pregenomic RNA. This finding is similar to what was found previously for duck hepatitis B virus (DHBV), and suggests that all hepadnaviruses use a similar mechanism. Also, we studied the role of complementarity between the RNA primer and the acceptor site at DR2 in HBV. By increasing the complementarity, we were able to increase the level of priming at DR2 over that seen in the wild-type virus. This finding suggests that the level of initiation of plus-strand DNA synthesis at DR2 is sub-maximal for wild-type HBV. Finally, we studied the role of the sequence at the 5' end of the RNA primer that is outside of the DR sequence. We found that substitutions or insertions in this region affected the level of priming at DR1 and DR2.

Hepadnaviruses utilize two template switches (primer translocation and circularization) during synthesis of plus-strand DNA to generate a relaxed-circular (RC) DNA genome. In duck hepatitis B virus (DHBV) three cis-acting sequences, 3E, M, and 5E, contribute to both template switches through base pairing, 3E with the 3' portion of M and 5E with the 5' portion of M. Human hepatitis B virus (HBV) also contains multiple cis-acting sequences that contribute to the accumulation of RC DNA, but the mechanisms through which these sequences contribute were previously unknown. Three of the HBV cis-acting sequences (h3E, hM, and h5E) occupy positions equivalent to those of the DHBV 3E, M, and 5E. We present evidence that h3E and hM contribute to the synthesis of RC DNA through base pairing during both primer translocation and circularization. Mutations that disrupt predicted base pairing inhibit both template switches while mutations that restore the predicted base pairing restore function. Therefore, the h3E-hM base pairing appears to be a conserved requirement for template switching during plus-strand DNA synthesis of HBV and DHBV. Also, we show that base pairing is not sufficient to explain the mechanism of h3E and hM, as mutating sequences adjacent to the base pairing regions inhibited both template switches. Finally, we did not identify predicted base pairing between h5E and the hM region, indicating a possible difference between HBV and DHBV. The significance of these similarities and differences between HBV and DHBV will be discussed.

There are two mutually exclusive pathways for plus-strand DNA synthesis in hepadnavirus reverse transcription. The predominant pathway gives rise to relaxed circular DNA, while the other pathway yields duplex linear DNA. At the completion of minus-strand DNA synthesis, the final RNase H cleavage generates the plus-strand primer at direct repeat 1 (DR1). A small fraction of viruses make duplex linear DNA after initiating plus-strand DNA synthesis from this site, a process called in situ priming. To make relaxed circular DNA, a template switch is necessary for the RNA primer generated at DR1 to initiate plus-strand DNA synthesis from the direct repeat 2 (DR2) located near the opposite end of the minus-strand DNA, a process called primer translocation. We are interested in understanding the mechanism that discriminates between these two processes. Previously, we showed that a small DNA hairpin forms at DR1 in the avihepadnaviruses and acts as an inhibitor of in situ priming. Here, using genetic approaches, we show that sequence identity between DR1 and DR2 is necessary, but not sufficient for primer translocation in the duck hepatitis B virus. The discrimination between in situ priming and primer translocation depends upon suppression of in situ priming, a process that is dependent upon both sequence identity between DR1 and DR2, and the presence of the hairpin at DR1. Finally, our analysis indicates the entire RNA primer can contribute to primer translocation and is translocated to DR2 before initiation of plus-strand DNA synthesis from that site.

Synthesis of minus-strand DNA of human hepatitis B virus (HBV) can be divided into three phases: initiation of DNA synthesis, the template switch, and elongation of minus-strand DNA. Although much is known about minus-strand DNA synthesis, the mechanism(s) by which this occurs has not been completely elucidated. Through a deletion analysis, we have identified a cis-acting element involved in minus-strand DNA synthesis that lies within a 27-nucleotide region between DR2 and the 3' copy of DR1. A subset of this region (termed Phi) has been hypothesized to base pair with the 5' half of epsilon (H. Tang and A. McLachlan, Virology, 303:199-210, 2002). To test the proposed model, we used a genetic approach in which multiple sets of variants that disrupted and then restored putative base pairing between the 5' half of epsilon and phi were analyzed. Primer extension analysis, using two primers simultaneously, was performed to measure encapsidated pregenomic RNA (pgRNA) and minus-strand DNA synthesized in cell culture. The efficiency of minus-strand DNA synthesis was defined as the amount of minus-strand DNA synthesized per encapsidation event. Our results indicate that base pairing between phi and the 5' half of epsilon contributes to efficient minus-strand DNA synthesis. Additional results are consistent with the idea that the primary sequence of phi and/or epsilon also contributes to function. How base pairing between phi and epsilon contributes to minus-strand DNA synthesis is not known, but a simple speculation is that phi base pairs with the 5' half of epsilon to juxtapose the donor and acceptor sites to facilitate the first-strand template switch.

Packaging of hepadnavirus pregenomic RNA (pgRNA) into capsids, or encapsidation, requires several viral components. The viral polymerase (P) and the capsid subunit (C) are necessary for pgRNA encapsidation. Previous studies of duck hepatitis B virus (DHBV) indicated that two cis-acting sequences on pgRNA are required for encapsidation: epsilon, which is near the 5' end of pgRNA, and region II, located near the middle of pgRNA. Later studies suggested that the intervening sequence between these two elements may also make a contribution. It has been demonstrated for DHBV that epsilon interacts with P to facilitate encapsidation, but it is not known how other cis-acting sequences contribute to encapsidation. We analyzed chimeras of DHBV and a related virus, heron hepatitis B virus (HHBV), to gain insight into the interactions between the various viral components during pgRNA encapsidation. We learned that having epsilon and P derived from the same virus was not sufficient for high levels of encapsidation, implying that other viral interactions contribute to encapsidation. Chimeric analysis showed that a large sequence containing region II may interact with P and/or C for efficient encapsidation. Further analysis demonstrated that possibly an RNA-RNA interaction between the intervening sequence and region II facilitates pgRNA encapsidation. Together, these results identify functional interactions among various viral components that contribute to pgRNA encapsidation.

The pregenomic RNA (pgRNA) of hepadnaviruses is packaged into capsids where it is reverse transcribed to yield mature DNA genomes. This report describes differences between the 3' region and other regions of the pgRNA isolated from capsids. Analysis of capsid pgRNA isolated by using an established method involving micrococcal nuclease treatment demonstrated reduced levels of the 3' region of the pgRNA compared to the 5' region. This underrepresentation of the 3' region was partly a result of microccocal nuclease digestion of the 3' region because isolation of capsid pgRNA by an alternative method that did not involve nuclease treatment led to a greater, but not complete, recovery of the 3' region. These results indicate that the 3' region of the capsid pgRNA is susceptible to micrococcal nuclease digestion during its isolation and that the 3' region can still be underrepresented when capsid pgRNA is isolated without nuclease digestion. Additional experiments show that the 3' ends of capsid pgRNA isolated by micrococcal nuclease treatment are heterogeneously dispersed from nucleotide 2577 to the poly(A) tail. These data provide evidence that the 3' region of the capsid pgRNA has biochemical properties different from those of its 5' region. Possibly, the 3' region of the pgRNA is not packaged into the interior of the capsid but rather is associated with a part of the capsid where it is susceptible to microccocal nuclease digestion.

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, 5'r, and 3'r 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.

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.

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.

Synthesis of the relaxed-circular (RC) DNA genome of hepadnaviruses requires two template switches during plus-strand DNA synthesis: primer translocation and circularization. Although primer translocation and circularization use different donor and acceptor sequences, and are distinct temporally, they share the common theme of switching from one end of the minus-strand template to the other end. Studies of duck hepatitis B virus have indicated that, in addition to the donor and acceptor sequences, three other cis-acting sequences, named 3E, M, and 5E, are required for the synthesis of RC DNA by contributing to primer translocation and circularization. The mechanism by which 3E, M, and 5E act was not known. We present evidence that these sequences function by base pairing with each other within the minus-strand template. 3E base-pairs with one portion of M (M3) and 5E base-pairs with an adjacent portion of M (M5). We found that disrupting base pairing between 3E and M3 and between 5E and M5 inhibited primer translocation and circularization. More importantly, restoring base pairing with mutant sequences restored the production of RC DNA. These results are consistent with the model that, within duck hepatitis B virus capsids, the ends of the minus-strand template are juxtaposed via base pairing to facilitate the two template switches during plus-strand DNA synthesis.

Pregenomic RNA (pgRNA) plays two major roles in the hepadnavirus life cycle. It is the mRNA for two proteins required for DNA replication, C and P, and it is the template for reverse transcription. pgRNA is a terminally redundant transcript whose synthesis does not involve RNA splicing. For duck hepatitis B virus (DHBV), a spliced RNA is derived from pgRNA by removal of a single intron. The mechanism for the simultaneous cytoplasmic accumulation of unspliced (pgRNA) and spliced RNA was not known. We found that mutations within two regions of the DHBV genome reduced the level of pgRNA while increasing the level of spliced RNA. One region is near the 5' end of pgRNA (region A), while the second is near the middle of pgRNA (region B). Inspection of the DHBV nucleotide sequence indicated that region A could base pair with region B. The 5' and 3' splice sites of the intron of the spliced RNA are within regions A and B, respectively. Substitutions that disrupted the predicted base pairing reduced the accumulation of pgRNA and increased the accumulation of spliced RNA. Restoration of base pairing, albeit mutant in sequence, resulted in restoration of pgRNA accumulation with a decrease in the level of spliced RNA. Our data are consistent with a model in which splicing of the pgRNA is suppressed by a secondary structure between regions A and B that occludes the splicing machinery from modifying pgRNA.

Previous analysis of duck hepatitis B virus (DHBV) indicated the presence of at least two cis-acting sequences required for efficient encapsidation of its pregenomic RNA (pgRNA), epsilon and region II. epsilon, an RNA stem-loop near the 5' end of the pgRNA, has been characterized in detail, while region II, located in the middle of the pgRNA, is not as well defined. Our initial aim was to identify the sequence important for the function of region II in DHBV. We scanned region II and the surrounding sequence by using a quantitative encapsidation assay. We found that the sequence between nucleotides (nt) 438 and 720 contributed to efficient pgRNA encapsidation, while the sequence between nt 538 and 610 made the largest contribution to encapsidation. Additionally, deletions between the two encapsidation sequences, epsilon and region II, had variable effects on encapsidation, while substitutions of heterologous sequence between epsilon and region II disrupted the ability of the pgRNA to be encapsidated efficiently. Overall, these data indicate that the intervening sequences between epsilon and region II play a role in encapsidation. We also analyzed heron hepatitis B virus (HHBV) for the presence of region II and found features similar to DHBV: a broad region necessary for efficient encapsidation that contained a critical region II sequence. Furthermore, we analyzed variants of DHBV that were substituted with HHBV sequence over region II and found that the chimeras were not fully functional for RNA encapsidation. These results indicate that sequences within region II may need to be compatible with other viral components in order to function in pgRNA encapsidation.

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.

We have identified and characterized a novel intracellular DNA replicative intermediate that is synthesized by heron hepatitis B virus (HHBV) and not by other avian hepadnaviruses. The new DNA form is synthesized in all host cells tested. The HHBV nucleic acid template, and not HHBV proteins, is responsible for the formation of the new form. The new form is comprised of a full-length minus-strand DNA and an incomplete plus-strand DNA whose 5' ends are mapped to DR2, predominantly. The 3' ends of its plus-strand are located between nucleotides 946 and 1046. Genetic analysis indicates that the sequences responsible for the formation of the new form lie between nucleotides 910 and 1364. The endogenous polymerase activity of capsids isolated from cells converted the new form into RC DNA. Intracellular capsids containing the new form are secreted inefficiently as virions, in comparison to RC- and DL DNA-containing capsids. Our analysis suggests that the new form is an incomplete RC DNA molecule that is due to a specific block or pause in the synthesis of plus-strand DNA. Our analysis also suggests that capsids become competent for efficient secretion sometime after the synthesis of 1500 nucleotides of plus-strand DNA., ((c) 2002 Elsevier Science (USA).)

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.

There are two mutually exclusive pathways for plus-strand DNA synthesis in hepadnavirus reverse transcription. The predominant pathway gives rise to relaxed circular DNA, while the other pathway yields duplex linear DNA. Both pathways use the same RNA primer, which is capped and 18 or 19 nucleotides in length. At the completion of minus-strand DNA synthesis, the final RNase H cleavage generates the plus-strand primer. To make relaxed circular DNA, primer translocation must occur, resulting in the transfer of the primer generated at DR1 to the acceptor site (DR2) near the opposite end of the minus-strand DNA. A small fraction of viruses instead make duplex linear DNA after initiating plus-strand DNA synthesis from DR1, a process called in situ priming. We are interested in understanding the mechanism of discrimination between these two pathways. Some variants of duck hepatitis B virus exhibit high levels of in situ priming due to cis-acting mutations. The mechanism by which these mutations act has been obscure. Sequence inspection predicted formation of a small DNA hairpin in the region overlapping these mutations. We have shown that substitutions disrupting base pairing potential in this hairpin led to increased levels of in situ priming. The introduction of compensatory changes to restore base pairing potential led to reduced levels of in situ priming. Thus, formation of the small DNA hairpin overlapping the 5' end of DR1 in the minus strand contributes to the regulation of primer translocation, at least, through inhibition of in situ priming by making the 3' end of the minus-strand DNA a poor template for initiation.

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.

Unique to hepadnavirus reverse transcription is the process of primer translocation, in which the RNA primer for the initiation of plus-strand DNA synthesis is generated at one site on its template, DR1, and is moved to a new site, DR2. For duck hepatitis B virus (DHBV), DR2 is located within 50 nucleotides of the 5' end of the minus-strand DNA template. When the synthesis of plus-strand DNA proceeds to the 5' terminus of the minus strand, the 3' end of the minus strand becomes the template for DNA synthesis. This switch in templates circularizes the nascent genome and is required for the genesis of the relaxed circular form of the DNA and the mature capsid. Maturation of the capsid is a prerequisite for virus egress. We have analyzed a series of DHBV variants in which plus-strand DNA synthesis was initiated from a new position relative to the 5' end of the template. For these variants, the subsequent circularization was inhibited. We found that when the number of nucleotides between the site of initiation of plus-strand DNA synthesis and the 5' end of its template was restored to 54 nucleotides, circularization was substantially restored. These results mean that the process of circularization is influenced by the earlier steps in DNA replication. This sensitivity is consistent with the notion that this region of the nascent genome is in a dynamic structure that is crucial for successful DNA replication.

Duck hepatitis B virus (DHBV) is a DNA virus that replicates via reverse transcription of a pregenomic RNA (pgRNA). Synthesis of the first strand of DNA (minus-strand DNA) for DHBV can be divided into two steps: (i) synthesis of the first four nucleotides of minus-strand DNA, which is primed by the viral polymerase (P) protein and copied from the sequence 5'-UUAC-3' within the phylogenetically conserved bulge in the encapsidation signal (epsilon) near the 5' end of pgRNA; and (ii) a template switch of the four-nucleotide minus-strand DNA from epsilon to an acceptor site near the 3' end of pgRNA and synthesis of a complete minus-strand DNA. To understand why only four nucleotides of minus-strand DNA were synthesized before the template switch, we introduced small insertions immediately 5' to the UUAC sequence in epsilon and determined whether these epsilon variants were competent for protein priming and whether minus strands longer than four nucleotides were synthesized. Then we determined, in cell culture, whether the longer minus-strand DNAs were competent to undergo a template switch. Also, we analyzed the structure of the epsilon variants, in solution. We found that the epsilon variants were functional for protein priming and RNA encapsidation and that the insertions were copied into minus-strand DNA. However, two mutant viruses that contained two different three-nucleotide insertions failed to synthesize minus-strand DNA efficiently from the acceptor site, even though seven nucleotides of the donor and acceptor sites were identical. These results suggest that the length and/or sequence of the minus-strand DNA copied from epsilon can be important for an efficient template switch. The RNA structural analysis of the epsilon variants indicated alteration in the position and size of the bulge. Overall, these results are consistent with the notion that the template within epsilon is limited to four nucleotides because the remaining two nucleotides located within the bulge are inaccessible for polymerization.

A characteristic of all hepadnaviruses is the relaxed-circular conformation of the DNA genome within an infectious virion. Synthesis of the relaxed-circular genome by reverse transcription requires three template switches. These template switches, as for the template switches or strand transfers of other reverse-transcribing genetic elements, require repeated sequences (the donor and acceptor sites) between which a complementary strand of nucleic acid is transferred. The mechanism for each of the template switches in hepadnaviruses is poorly understood. To determine whether sequences other than the donor and acceptor sites are involved in the template switches of duck hepatitis B virus (DHBV), a series of molecular clones which express viral genomes bearing deletion mutations were analyzed. We found that three regions of the DHBV genome, which are distinct from the donor and acceptor sites, are required for the synthesis of relaxed-circular DNA. One region, located near the 3' end of the minus-strand template, is required for the template switch that circularizes the genome. The other two regions, located in the middle of the genome and near DR2, appear to be required for plus-strand primer translocation. We speculate that these cis-acting sequences may play a role in the organization of the minus-strand DNA template within the capsid particle so that it supports efficient template switching during plus-strand DNA synthesis.

The retroviral proteases (PRs) have a structural feature called the flap, which consists of a short anti-parallel beta-sheet with a turn. The flap extends over the substrate binding cleft and must be flexible to allow entry and exit of the polypeptide substrates and products. We analyzed the sequence requirements of the amino acids within the flap region (positions 46-56) of the HIV-1 PR. The phenotypes of 131 substitution mutants were determined using a bacterial expression system. Four of the mutant PRs with mutations in different regions of the flap were selected for kinetic analysis. Our phenotypic analysis, considered in the context of published structures of the HIV-1 PR with a bound substrate analogs, shows that: (i) Met-46 and Phe-53 participate in hydrophobic interactions on the solvent-exposed face of the flap; (ii) Ile-47, Ile-54, and Val-56 participate in hydrophobic interactions on the inner face of the flap; (iii) Ile-50 has hydrophobic interactions at the distance of both the delta and gamma carbons; (iv) the three glycine residues in the beta-turn of the flap are virtually intolerant of substitutions. Among these mutant PRs, we have identified changes in both kcat and Km. These results establish the nature of the side chain requirements at each position in the flap and document a role for the flap in both substrate binding and catalysis.

The template for hepadnavirus plus-strand DNA synthesis is a terminally redundant minus-strand DNA. An intramolecular template switch during plus-strand DNA synthesis, which permits plus-strand DNA elongation, has been proposed to be facilitated by this terminal redundancy, which is 7 to 9 nucleotides long. The aim of this study was to determine whether the presence of identical copies of the redundancy on the minus-strand DNA template was necessary and/or sufficient for the template switch and at what position(s) within the redundancy the switch occurs for duck hepatitis B virus. When dinucleotide insertions were placed within the copy of the redundancy at the 3' end of the minus-strand DNA template, novel sequences were copied into plus-strand DNA. The generation of these novel sequences could be explained by complete copying of the redundancy at the 5' end of the minus-strand DNA template followed by a template switch and then extension from a mismatched 3' terminus. In a second set of experiments, it was found that when one copy of the redundancy had either three or five nucleotides replaced the template switch was inhibited. When the identical, albeit mutant, sequences were restored in both copies of the redundancy, template switching was not necessarily restored. Our results indicate that the terminal redundancy on the minus-strand DNA template is necessary but not sufficient for template switching.

The initial aim of this study was to examine the role of complementarity between the plus-strand primer and the minus-strand DNA template for translocation of the plus-strand primer in hepadnaviral replication. We show that when a 5-nucleotide substitution was placed in either DR1 or DR2, translocation of the primer at a detectable level did not occur. Placing the mutation in both DR1 and DR2 did not restore primer translocation, which indicates that complementarity is not the sole determinant for primer translocation. These mutants, in which primer translocation has been inhibited, have been additionally informative. The mutation in DR1 led to efficient synthesis of plus-strand DNA, albeit primed in situ. In contrast, the mutation in DR2 resulted in a reduction in the amount of plus-strand DNA synthesized per unit of minus-strand DNA. These findings were interpreted as indicating that a mutation at DR2, the primer acceptor site, can inhibit both primer translocation and in situ priming. Lastly, we show that mutations within DR2 can result in a reduction in the synthesis of minus-strand DNA and that this reduction is occurring at an early phase of the process. We speculate that this reduction in the amount of minus-strand DNA synthesized could be due to an inhibition of the template switch during minus-strand DNA synthesis.

Heron hepatitis B virus (HHBV) is an avian hepadnavirus that is closely related to duck hepatitis B virus (DHBV). To learn more about the mechanism of hepadnavirus replication, we characterized a clone of HHBV that contains a substitution of DHBV sequence from nucleotide coordinates 403 to 1364. This clone, named HDE1, expresses a chimeric pregenomic RNA, a chimeric polymerase (P) protein, and a core (C) protein with a one-amino-acid substitution at its carboxy terminus. We have shown that HDE1 is defective for minus-strand DNA synthesis, resulting in an overall reduction of viral DNA. HDE1 was also defective for plus-strand DNA synthesis, resulting in aberrant ratios of replication intermediates. Genetic complementation assays indicated that HDE1 replication proteins, C and P, are functional for replication and wild-type HHBV proteins do not rescue either defect. These findings indicate that the HDE1 substitution mutation acts primarily in cis. By restoring nucleotides 403 to 902 to the HHBV sequence, we showed that cis-acting sequences for plus-strand DNA synthesis are located in the 5' half of the HDE1 chimeric region. These data indicate the presence of one or more formerly unrecognized cis-acting sequences for DNA synthesis within the chimeric region (nucleotides 403 to 1364). These cis-acting sequences in the middle of the genome might interact directly or indirectly with known cis elements that are located near the ends of the genome. Our findings suggest that a specific higher-order template structure is involved in the mechanism of hepadnavirus DNA replication.

The current model for replication of duck hepatitis B virus has reverse transcription initiating and copying a UUAC motif within the encapsidation signal, epsilon, near the 5' end of the RNA template. This results in synthesis of four nucleotides of DNA. This short minus-strand DNA product is then transferred to a complementary position, at DR1, near the 3' end of the RNA template. Elongation of minus-strand DNA then ensues. We have examined the transfer of minus-strand DNA during replication of duck hepatitis B virus in cell culture. The initial aim of this work was to examine the effect of mutations at DR1 on the transfer process. We found that when mutations were introduced into the UUAC motif overlapping DR1, the 5' end of minus-DNA no longer mapped to position 2537 but was shifted two or four nucleotides. Mismatches were predicted to exist at the new sites of elongation. Elongation from nucleotide 2537 could be restored in these mutants by making compensatory changes in the UUAC motif within epsilon. This finding led us to examine limitations in the shifting of the site of transfer. When the UUAC motif in epsilon was changed to six different tetranucleotide motifs surrounding position 2537, transfer of minus-strand DNA shifted predictably, albeit inefficiently. Also, when multiple UUAC motifs were introduced near DR1, the UUAC motif at nucleotide 2537 was used preferentially. Overall, our findings confirm the current minus-strand DNA transfer model and demonstrate a marked preference for the site of the transfer.

Substitution of glycine with glutamic acid at position 48 of the human immunodeficiency virus protease resulted in an enzyme with reduced activity on one of the protease processing sites in the viral Pol polyprotein precursor. Cleavage at this site was restored by a second-site substitution in the substrate replacing an aspartic acid with either glycine or asparagine. These results suggest that the glutamic acid side chain in the mutant protease has an unfavorable charge-charge interaction with this position in the substrate. Cleavage of a processing site in the viral Gag polyprotein precursor with the mutant enzyme was enhanced, and this enhancement was dependent on the presence of an arginine residue in the substrate, again suggesting a charge-charge interaction. The potential for such interactions was confirmed using molecular modeling. The effect of the position 48 substitution was attributed to a 10-fold increase in Km for the processing site in Pol. These results indicate that the addition of a side chain at position 48 can alter the specificity of the HIV-1 protease to substrate in a sequence specific manner and that compensatory changes can be made in the substrate.

Human immunodeficiency virus type 1 encodes a protease whose activity is required for the production of infectious virus. An Escherichia coli expression and processing assay system was used to screen 285 protease mutants for temperature-sensitive activity. Fourteen protease mutants had a temperature-sensitive phenotype, and approximately half resulted from conservative amino acid substitutions. Of the 14 substitutions that conferred a temperature-sensitive phenotype, 11 substitutions occurred at 6 positions that represent 3 pairs of residues in the protease that contact each other in the three-dimensional structure. These mutants assist in pinpointing regions of the protease that are important for enzyme activity and stability.

Reverse transcription of RNA into duplex DNA requires accurate initiation of both minus and plus strand DNA synthesis; this in turn requires the generation of specific primer molecules. We have examined plus strand primer generation in the hepatitis B viruses, small DNA viruses that replicate via reverse transcription. The plus strand primer in these viruses is a short capped RNA derived from the 5' end of the RNA template by cleavage at a specific set of sites. To elucidate the cleavage mechanism we constructed a series of viral mutants bearing alterations in and around the cleavage sites. Our results reveal that the cleavage reaction is sequence-independent and indicate that the cleavage sites are positioned by measurement of the distance from the 5' end of the RNA. Comparison of these findings with what is known about RNase H-mediated primer generation in retroviruses and other retroid elements suggests that, despite many divergent features, some common molecular features are preserved.

DNA sequence analysis of the same chromosomal region from two haplotypes of Mus musculus and from the related species M. caroli and M. pahari reveals the presence of long interspersed sequence one (LINES-1, or L1) elements residing at the same nucleotide position in the two most distantly related of the species (M. musculus and M. pahari). The DNA sequence of each of these L1 elements is more similar to that of other L1 elements from its own species than to the other. Thus, the L1 sequence at each of these sites is recent with respect to the divergence of the species. This could be a result of recent gene conversion of L1 elements inherited from a common ancestor or of two recent independent L1 insertion events at the same nucleotide position in the two species. Such specificity of insertion would be quite different from the apparent randomness of other characterized L1 insertion events, such as those in the beta-globin locus. If the recent L1 sequences arose at this site by gene conversion of an ancestral L1 element, then the absence of an L1 element at this location in the M. caroli chromosome examined could arise either from its precise deletion from M. caroli or from the segregation into M. caroli of a polymorphic chromosome present in the ancestral population which was missing this L1 element.

Retroviruses encode a protease which cleaves the viral Gag and Gag/Pol protein precursors into mature products. To understand the target sequence specificity of the viral protease, the amino acid sequences from 46 known processing sites from 10 diverse retroviruses were compared. Sequence preference was evident in positions P4 through P3' when compared to flanking sequences. Approximately 80% of all cleavage site sequences could be grouped into two classes based on the sequence composition flanking the scissile bond. The sequences at the amino-terminal cleavage site of the major capsid protein of Gag is always a member of one of the two classes while the carboxyl-terminal cleavage site is of the other class, suggesting a biological role for the two classes. Known processing site sequences proved useful in a motif searching strategy to identify processing sites in retroviral protein sequences, particularly in Gag. In all known cleavage sites, the P1 amino acid is hydrophobic and unbranched at the beta-carbon. The sequence requirements of the P1 position were tested by site-directed mutagenesis of the P1 Phe codon in an HIV-1 Pol cleavage site. Mutations were tested for protease-mediated cleavage of the Pol precursor expressed in Escherichia coli.

Hepadnavirus reverse transcription requires that pregenomic RNA first be selectively packaged into a cytoplasmic core particle. This process presumably requires the presence of specific recognition sequences on the pregenomic RNA. To define the cis-acting sequences required for pregenome encapsidation in the duck hepatitis B virus (DHBV), we assayed the packaging efficiency of a series of pregenomic RNA deletion mutants and hybrid DHBV/lacZ fusion transcripts. The 5' boundary of the packaging signal lies within the precore region, starting approximately 35 nucleotides from the cap site of pregenomic RNA; thus, the DR1 sequence required for proper viral DNA synthesis is not included in this signal. To define the 3' boundary of the encapsidation signal, fusion transcripts bearing foreign (lacZ) sequences fused to DHBV at different sites 3' to the pregenomic RNA start site were examined. A surprisingly large region of the DHBV genome proved to be required for packaging of such chimeras, which are efficiently encapsidated only when they contain the first 1,200 to 1,400 nucleotides of DHBV pregenomic RNA. However, mutant genomes bearing insertions within this region are packaged efficiently, making it likely that the actual recognition elements for encapsidation are smaller discontinuous sequences located within this region.

Hepadnaviruses replicate their circular DNA genomes via reverse transcription of an RNA intermediate. The initial product of reverse transcription, minus-strand DNA, contains two copies of a short direct repeat (DR) sequence, termed DR1 and DR2. Plus-strand DNA synthesis initiates at DR2 on minus-strand DNA, using as a primer a short, DR1-containing oligoribonucleotide derived by cleavage and translocation from the 5' end of pregenomic RNA. To clarify the sequence requirements for plus-strand primer cleavage and translocation, we have constructed mutants of the duck hepatitis B virus bearing base changes in or around the DR1 sequence in the primer. A point mutation at the terminal nucleotide of DR1 has a striking phenotype: normal levels of duplex viral DNA are produced, but nearly all of the DNA is linear rather than circular. Mapping of the 5' end of plus-strand DNA reveals that primer cleavage occurs with normal efficiency and accuracy, but the primer is not translocated to DR2; rather, it is extended in situ to generate duplex linear DNA. Other mutations just 3' to DR1 similarly affect primer translocation, although with differing efficiencies. Linear DNA found in wild-type virus preparations has the same fine structure as the mutant linears described above. These results indicate that (i) plus-strand primer cleavage and translocation are distinct steps that can be dissociated by mutation, (ii) lesions in sequences not included in the primer can severely inhibit primer translocation, and (iii) elongation of such untranslocated primers is responsible for the variable quantities of linear DNA that are found in all hepadnaviral stocks.

Herpesvirus papio (HVP) is a lymphotropic virus of baboons which is related to Epstein-Barr virus (EBV) and produces latent infection. The nucleotide sequence of the 5,775-base-pair (bp) EcoRI K fragment of HVP, which has previously been shown to confer the ability to replicate autonomously, has been determined. Within this DNA fragment is a region which bears structural and sequence similarity to the ori-P region of EBV. The HVP ori-P region has a 10- by 26-bp tandem array which is related to the 20- by 30-bp tandem array from the EBV ori-P region. In HVP there is an intervening region of 764 bp followed by five partial copies of the 26-bp monomer. Both the EBV and HVP 3' regions have the potential to form dyad structures which, however, differ in arrangement. We also demonstrate that a transcriptional enhancer which requires transactivation by a virus-encoded factor is present in the HVP ori-P.