Live Cell Imaging Reveals HBV Capsid Translocation from the Nucleus To the Cytoplasm Enabled by Cell Division.

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.

Modulation of hepatitis B virus pregenomic RNA stability and splicing by histone deacetylase 5 enhances viral biosynthesis.

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.

Encapsidated hepatitis B virus reverse transcriptase is poised on an ordered RNA lattice.

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.

Snow goose hepatitis B virus (SGHBV) envelope and capsid proteins independently contribute to the ability of SGHBV to package capsids containing single-stranded DNA in virions.

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.

Roles of the envelope proteins in the amplification of covalently closed circular DNA and completion of synthesis of the plus-strand DNA in hepatitis B virus.

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.

Serine phosphoacceptor sites within the core protein of hepatitis B virus contribute to genome replication pleiotropically.

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.

cis-Acting sequences that contribute to synthesis of minus-strand DNA are not conserved between hepadnaviruses.

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.

Development of cell cultures that express hepatitis B virus to high levels and accumulate cccDNA.

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.

Full-length hepatitis B virus core protein packages viral and heterologous RNA with similarly high levels of cooperativity.

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.

The topology of hepatitis B virus pregenomic RNA promotes its replication.

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.

Base pairing between cis-acting sequences contributes to template switching during plus-strand DNA synthesis in human hepatitis B virus.

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.

Base pairing between the 5' half of epsilon and a cis-acting sequence, phi, makes a contribution to the synthesis of minus-strand DNA for human hepatitis B virus.

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.

Chimeras of duck and heron hepatitis B viruses provide evidence for functional interactions between viral components of pregenomic RNA encapsidation.

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.

Underrepresentation of the 3' region of the capsid pregenomic RNA of duck hepatitis B virus.

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.

cis-Acting sequences that contribute to the synthesis of relaxed-circular DNA of human hepatitis B virus.

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.

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.

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.

Template switches during plus-strand DNA synthesis of duck hepatitis B virus are influenced by the base composition of the minus-strand terminal redundancy.

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.

Base pairing among three cis-acting sequences contributes to template switching during hepadnavirus reverse transcription.

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.

A secondary structure that contains the 5' and 3' splice sites suppresses splicing of duck hepatitis B virus pregenomic RNA.

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.

Characterization of the cis-acting contributions to avian hepadnavirus RNA encapsidation.

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.