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

Thomas B Lentz; Daniel D Loeb

doi:10.1128/JVI.05373-11

. 2011 Nov;85(22):11916–11927. doi: 10.1128/JVI.05373-11

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 ^{^▿}

Thomas B Lentz ^1,², Daniel D Loeb ^1,^*

PMCID: PMC3209309 PMID: 21900164

Abstract

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.

INTRODUCTION

Hepatitis B virus (HBV) is a member of the family Hepadnaviridae. Features of this family include a small, double-stranded DNA genome and enveloped virions (14). The envelope of HBV consists of a host cell-derived phospholipid bilayer that contains the virally encoded large (L), middle (M), and small (S) envelope proteins (21). This envelope surrounds the nucleocapsid, which is composed of the viral core (C) protein and contains the DNA genome (11). The double-stranded DNA genome within the virion is in either a relaxed circular (RC) or a duplex linear (DL) conformation, of which RC is the predominant form (12, 44, 48). RC DNA contains a full-length minus strand, which has the viral polymerase (P) protein covalently attached to its 5′ end (17). The plus strand has a specific 5′ end but is extended to various lengths, which can leave a single-stranded (SS) region (12, 45, 48). The reason the plus strand is not completely synthesized prior to the release of the virion is not understood. It is not known if the process of the nucleocapsid acquiring an envelope is related to the incomplete state of the plus strand found in virions.

The RC DNA molecule from the virion is converted into a covalently closed circular DNA (cccDNA) molecule in the nucleus of a cell (38, 40, 51). The mechanism of conversion of RC DNA into cccDNA is not well understood. Minimally, this involves removal of the 5′-terminal moieties from the minus strand (P protein) and the plus strand (RNA primer), completion of synthesis of the plus strand, and ligation of 5′ and 3′ ends of each of the strands (14). cccDNA is the template for transcription of the viral RNAs, which includes the pregenomic RNA (pgRNA). The pgRNA associates with the P protein in the cytoplasm and is encapsidated into a nascent nucleocapsid (22). Reverse transcription of the pgRNA within the nucleocapsid results in the accumulation of nucleocapsids containing RC DNA (47). RC DNA-containing nucleocapsids participate in two known processes. They can be enveloped at an endoplasmic reticulum (ER)/Golgi organelle and secreted as virions (7, 52). Alternatively, RC DNA in these capsids can be converted into cccDNA in a process known as intracellular amplification (56).

Envelopment and secretion require interaction of the nucleocapsid with the L and S proteins at the membrane of the ER/Golgi. The envelope proteins are cotranslationally inserted into the membrane of the ER and assume a specific transmembrane orientation (5, 13). Signal sequences (termed signals I and II), common to all three proteins, direct this transmembrane orientation such that the amino and carboxyl ends of the proteins are luminally disposed and only an intervening loop remains cytoplasmically disposed. The L protein shares this orientation but exhibits another orientation that is not observed with the M and S proteins (8, 26, 42, 43). In this alternative orientation, signal I is not inserted into the membrane and the amino terminus projects into the cytosol. Regions of the L and S proteins required for the secretion of virus, and thus predicted to interact with the nucleocapsid, have been mapped to residues 83 to 114 (subtype ayw) of L, present in the amino terminal sequence, and 33 to 59 of S, present in the cytosolic loop (6, 9, 33–35, 37). The dual topology of L places these residues on the cytosolic face of the ER/Golgi and is consistent with the L protein performing functions on both the cytosolic (envelopment) and luminal face (infectivity) of the viral membrane. Ablation of expression of the M protein results in decreased levels of virus production, suggesting that this protein may contribute but is not necessary for envelopment (16, 41, 52). The S protein can also self-assemble into nucleocapsid-free subviral particles, which are secreted from the cell (21). L and M proteins are not secreted when expressed alone but can be incorporated into particles composed predominantly of S protein. The role of subviral particles in HBV biology is not well understood but their formation appears to be mechanistically distinct from the virion formation pathway (16).

Studies performed with duck hepatitis B virus (DHBV) indicate that ablation of expression of the L protein (DHBV expresses only L and S envelope proteins) leads to hyperamplification (∼28-fold at day 10 postinfection) of cccDNA, and the expression of only L results in normal amplification (49, 50). Studies to determine if expression of an envelope protein of HBV provides a similar function have produced conflicting conclusions. Gao and Hu (15) and Guo et al. (18) have demonstrated that the level of cccDNA is increased in the absence of envelope protein expression, though in both cases the increase was <1.5-fold (15, 18). In contrast, Ling and Harrison (36) and Sprinzl et al. (46) reported finding no amplification of cccDNA when the expression of all three envelope proteins or only L protein was ablated. The reason for these conflicting conclusions is not apparent. Thus, the role of the envelope proteins in regulating cccDNA synthesis in HBV is unresolved. The maintenance of cccDNA in the nuclei of infected cells as a result of intracellular amplification is thought to contribute to the persistence of viral infections (57). Understanding the mechanism of the regulation of cccDNA synthesis will improve our understanding of chronic HBV infection.

We investigated the role of the viral envelope proteins in regulating cccDNA synthesis in HBV. A cell line, derived from Huh7 cells, that accumulates cccDNA to readily detectable levels upon transfection with an HBV expression plasmid was used (32). We found that simultaneous ablation of the expression of the three envelope proteins (L⁻M⁻S⁻) led to an increase in the level of cccDNA of >6-fold over the level of the wild type (WT). Comparison of the relative levels of full-length RC DNA (fl-RC DNA) between the L⁻M⁻S⁻ mutant and WT HBV revealed an increase of ∼4-fold in the absence of the envelope proteins. Restoration of expression of the envelope proteins by genetic complementation resulted in a decrease in the level of cccDNA and RC DNA. To determine the contribution of the individual envelope proteins, the L⁻M⁻S⁻ mutant was complemented with the different combinations of envelope proteins (LMS, LS, LM, MS, L, M, S). Results from this analysis indicated that L protein is required but not sufficient for the regulation of cccDNA synthesis. Completion of the plus-strand DNA was not limited by expression of any of the different subsets of envelope proteins, indicating that all three are required for this effect. The level of fl-RC DNA correlated inversely with the level of secreted virions. These results, and the previously reported observations that the endogenous polymerase can extend the plus strand within the nucleocapsid once the viral envelope is removed, suggest that the lack of completion of plus-strand DNA synthesis is related to envelopment of the nucleocapsid and may explain the long-standing observation that virions contain genomic molecules with an incomplete plus strand (12, 22, 23, 48).

MATERIALS AND METHODS

Molecular clones and plasmids.

All molecular clones of HBV were derived from the sequence found at GenBank accession number V01460 (subtype ayw, genotype D). The C of the unique EcoRI site (GAATTC) was designated nucleotide position 1.

Plasmids TL25 (WT) and TL7 (L⁻M⁻S⁻ mutant) contain ∼1.1 copies of the HBV genome, nucleotide (nt) coordinates 1806 to 3182/1 to 1986, and express the pgRNA from the human cytomegalovirus major immediate early (CMV) promoter (32). Mutants of the genomic expression plasmid TL25, which express different combinations of the envelope proteins (TL24, TL26, TL35, TL36, TL38, TL39), were generated by site-directed mutagenesis of the start codons of the genes for the individual envelope proteins. Donor plasmids TL34 (LMS) and TL27 (L⁻M⁻S⁻) were created by removal of the CMV promoter and a portion of the HBV sequence from plasmids TL25 and TL7, respectively. These plasmids contain HBV sequences with coordinates 2627 to 3182/1 to 1986 and were designed to express the mRNAs for the envelope proteins from their endogenous promoters. Donor plasmids TL28 (S), TL29 (M), TL30 (L), TL31 (MS), TL32 (LS), and TL33 (LM), which express the different permutations of the envelope proteins, were created by site-directed mutagenesis of the start codons of the genes for the individual envelope proteins. Nucleotides 2847 (L knockout), 3171 (M knockout), and 154 (S knockout) in the HBV sequence were changed from thymidine to cytidine, resulting in a methionine-to-threonine change in the protein sequence. Plasmids in which the CMV promoter drives expression of the mRNA for the L protein (CMV donor) were created by juxtaposing the transcription start site of the CMV promoter to nucleotide 2805 of the HBV sequence, which is the 5′ nucleotide of the L mRNA (29). All plasmids are listed in Table 1.

Table 1.

Description of HBV and envelope protein expression plasmids

Plasmid name	pgRNA promoter^a	Envelope promoter^b	Envelope protein status^c
TL7	CMV IE	HBV	L⁻M⁻S⁻
TL25	CMV IE	HBV	L⁺M⁺S⁺
TL24	CMV IE	HBV	L⁻M⁺S⁺
TL26	CMV IE	HBV	L⁺M⁻S⁺
TL38	CMV IE	HBV	L⁺M⁺S⁻
TL36	CMV IE	HBV	L⁺M⁻S⁻
TL35	CMV IE	HBV	L⁻M⁺S⁻
TL39	CMV IE	HBV	L⁻M⁻S⁺
TL27	−	HBV	L⁻M⁻S⁻
TL28	−	HBV	L⁻M⁻S⁺
TL29	−	HBV	L⁻M⁺S⁻
TL30	−	HBV	L⁺M⁻S⁻
TL31	−	HBV	L⁻M⁺S⁺
TL32	−	HBV	L⁺M⁻S⁺
TL33	−	HBV	L⁺M⁺S⁻
TL34	−	HBV	L⁺M⁺S⁺
TL42	−	CMV IE	L⁺M⁺S⁻
TL51	−	CMV IE	L⁻M⁻S⁻

Open in a new tab

Promoter for expression of the pgRNA. This promoter is absent (−) from envelope protein donor plasmids.

Promoter for expression of the viral envelope proteins. HBV, endogenous promoters. IE, immediate early.

Status of coding for the L, M, and S envelope proteins within each plasmid. + indicates expressed; − indicates that expression is ablated.

All plasmids contain the Epstein-Barr virus (EBV) origin of plasmid replication (oriP) and the Tet operator (TetO) sequence. The oriP sequence is bound by EBV nuclear antigen 1 (EBNA-1) protein, which is stably expressed in Huh7-H1 cells (discussed below). Previous characterization demonstrated that expression of HBV from these plasmids (TL25 and TL7) is increased in Huh7-H1 cells compared with parental Huh7 cells (32). The TetO sequence is positioned adjacent to the CMV (TL7, TL25, TL42, and TL51) or the endogenous envelope (TL27 to TL34) promoter in each of these constructs and can be used to regulate gene expression, though it was not used for that purpose in this study.

Cell cultures and transfections.

The cell line Huh7-H1 has been described previously (32). These cells stably express EBNA-1 and a chimeric protein composed of the Tet repressor fused with the Kox1 transcriptional repression domain (TetR-KRAB). They are maintained in G418 selection medium. Cultures were transfected as described previously with the exceptions that Tet was added to relieve the repression of gene expression by TetR-KRAB and 5% fetal bovine serum (FBS) was used in all experiments. Briefly, cells were cultured in a 60-mm dish to ∼70% confluence and DNA was added to culture medium (without G418) as a calcium phosphate precipitate (Table 2). The presence of G418 in the culture medium during transfection resulted in cell death and so it was excluded. In experiments in which plasmids TL25 and TL7 were transfected singly (see Fig. 1 and 3), 9 μg of the respective plasmid and 1 μg of a plasmid that expresses green fluorescent protein (GFP) was used. For experiments in which the plasmid TL7 was cotransfected with either plasmid TL34 or TL27 (see Fig. 2), 9.5 μg of plasmid TL7, 1 μg of the GFP plasmid, and 9.5 μg of plasmid TL34 or TL27 was used. For experiments in which TL7 was cotransfected with varying amounts of donor plasmids expressing the different combinations of envelope proteins (see Fig. 5), 8.5 μg of plasmid TL7, 0.5 μg of the GFP plasmid, and 16 μg of donor plasmid was used. Though the amount of each donor plasmid was varied, the total mass of donor plasmid was kept constant by adding plasmid TL27 for a total of 16 μg. In experiments in which TL7 was cotransfected with the CMV donor plasmids, 10 μg of plasmid TL7, 10 μg of donor plasmid, and 2 μg of CMV donor plasmid were used. The total mass of donor plasmid and CMV donor plasmid used was kept constant using TL27 (donor) or TL51 (CMV donor). Finally, for experiments in which the donor or CMV donor plasmids were transfected alone to detect expression of the M protein (see Fig. 6B and 7A), 10 μg of the respective plasmid was used. Culture medium was replaced ∼16 to 20 h after transfection to include G418 at 600 μg/ml and Tet at 100 ng/ml and changed as necessary over 4 days when cells were collected.

Table 2.

Description of transfection conditions

Expt shown in:	Mass (μg) of indicated plasmid
Expt shown in:	TL7 (L⁻M⁻S⁻) or TL25 (LMS)	Donor plasmid	CMV donor plasmid	GFP plasmid
Fig. 1 and 3	9			1
Fig. 2	9.5	9.5		0.5
Fig. 4 and 5A	8.5	16^a		0.5
Fig. 5B and 6A	10
Fig. 6B	10	10	2^b

Open in a new tab

The mass of the envelope protein donor plasmid was brought to a total of 16 μg by the addition of the donor TL27 (L⁻M⁻S⁻) as necessary.

The mass of the CMV donor plasmid was brought to a total of 2 μg by the addition of the CMV donor TL51 (L⁻M⁻S⁻) as necessary.

Fig. 1. — Effect of ablation of expression of the envelope proteins on the level of fl-RC DNA and cccDNA. Huh7-H1 cells were transfected with plasmids that express either WT or L⁻M⁻S⁻ mutant HBV. (A) Immunoblotting for viral envelope proteins and GFP in total cell lysates using the anti-HBs (Fitzgerald) and anti-GFP (Santa Cruz) primary antibodies. Lysate from nontransfected cells (naïve) was included. (B) Southern blot of intracellular vDNA (intracell), extracellular vDNA (extracell), and DNA associated with immunoprecipitated (IP) virions (anti-HBs) and free capsids (anti-HBc). Intermediates of genome replication containing a full-length minus strand, fl (−) DNA (bracket), as well as distinct species, fl-RC, DL, and SS (arrows), are indicated. A linear monomer of the HBV genome (linear HBV) was included to identify the position of DL DNA. The proportion of fl-RC (ng/plate) to fl (−) DNA (ng/plate) in intracellular and extracellular vDNA was determined for cultures expressing the WT and L⁻M⁻S⁻ mutant and is represented as fold of the proportion in the WT in the histogram. (C) Southern blot of cccDNA. Both the transfected plasmid and cccDNA were detected (arrows). Markers for plasmid DNA and linear monomer HBV DNA (linear HBV) are indicated. Circular monomers of HBV DNA (ccc marker) were produced by ligation of linear monomer HBV DNA. Intracellular vDNA (vDNA) was run to demonstrate the position of cccDNA relative to other intermediates of viral genome replication. The proportion of cccDNA (ng/plate) to fl (−) DNA (ng/plate) was determined for cultures expressing the WT and L⁻M⁻S⁻ mutant and is represented as fold of the proportion in the WT in the histogram. vDNA and cccDNA were detected with a ribonucleotide probe specific for coordinates 3096 to 3182/1 to 264 of the minus strand of HBV. *, P < 0.05.

Fig. 3. — *In vitro* extension of the plus-strand DNA by EPR. Nucleocapsids were immunoprecipitated from cell lysates expressing either WT or L⁻M⁻S⁻ mutant HBV. Separate fractions of nucleocapsids were then either left untreated or incubated with EPR buffer containing dNTPs (EPR) or lacking dNTPs (mock). Then, DNA was isolated from the different fractions and analyzed by Southern blotting. Intracellular vDNA (Intracell) was included for comparison. Each lane represents an equal fraction of a 60-mm plate. Intermediates of viral genome replication, fl-RC, DL, and SS, are indicated (arrows), as is linear monomer of HBV DNA (linear HBV). The histogram shows the levels of fl-RC (ng/plate).

Fig. 2. — Effect of restoration of envelope protein expression on the level of fl-RC DNA and cccDNA. Huh7-H1 cells were cotransfected with the plasmid for expression of the L⁻M⁻S⁻ mutant and a donor plasmid either capable of expressing the viral envelope proteins (LMS) or deficient for envelope protein expression (L⁻M⁻S⁻). (A) Immunoblotting for viral envelope proteins and GFP in total cell lysates by using anti-HBs (Fitzgerald) and anti-GFP (Santa Cruz) primary antibodies. (B) Native particle blotting of virions and free capsids in culture medium. Culture medium was concentrated ∼20-fold, electrophoresed through a nondenaturing agarose gel, and blotted to membrane. DNA associated with virions and free capsids was detected by hybridization with a ribonucleotide probe specific for coordinates 3096 to 3182/1 to 264 of the minus strand of HBV. Southern blotting of vDNA (C) and cccDNA (D) was performed. Intermediates of genome replication containing a full-length minus strand, fl (−) DNA (bracket), as well as distinct species, fl-RC, DL, and SS (arrows), are indicated. A linear monomer of the HBV genome (linear HBV) was included to identify the position of DL DNA. The proportion of fl-RC DNA (ng/plate) (C) or cccDNA (ng/plate) (D) to fl (−) DNA (ng/plate) was determined and is represented in the histograms as the percentage of the proportion in the WT. *, P < 0.05.

Fig. 5. — Effect of complementing the L⁻M⁻S⁻ mutant with the different combinations of envelope proteins on fl-RC DNA and cccDNA. Cells were cotransfected with the L⁻M⁻S⁻ mutant expression plasmid and one of a series of donor plasmids that express the different combinations of envelope proteins. In each case, the amount of the donor plasmid was varied in order to produce different levels of protein. A donor plasmid deficient for envelope protein expression (L⁻M⁻S⁻) was used for comparison. (A) The levels of L and S proteins, determined by immunoblotting of total cell lysates, are shown in the histograms, represented as percentages of the level observed when the greatest amount of LMS donor plasmid was used. (B) Native particle blot of virions and free capsids in concentrated culture medium. The proportion of fl-RC DNA (ng/plate) (C) and cccDNA (ng/plate) (D) to fl (−) DNA (ng/plate) was determined from Southern blotting of intracellular vDNA and cccDNA samples as in Fig. 1. Histograms show these proportions as percentages of the proportion when the L⁻M⁻S⁻ donor plasmid was used.

Fig. 6. — Relationship between the level of cccDNA and the level of L protein when different combinations of envelope proteins are expressed. (A) The relative level of cccDNA (y axis) is taken from Fig. 4A and equals the level of cccDNA (ng/plate) normalized to fl (−) DNA (ng/plate) for each amount of donor plasmid as a proportion of the level when the L⁻M⁻S⁻ donor was used. The relative level of L protein (x axis) is taken from Fig. 4D and equals the level of L protein for each amount of donor plasmid used as a proportion of the level of L when the greatest amount of the LMS donor was used. The points on each line represent different amounts of the L protein. (B) Immunoblot for expression of the M protein. Protein soluble in the detergent Triton X-114 was isolated from cells transfected with the indicated plasmid and envelope proteins were detected with either a polyclonal anti-HBs antibody (Fitzgerald) or a monoclonal anti-PreS2 antibody (Virogen).

Fig. 7. — Relationship between cccDNA and L protein levels when expression of L^m109 is driven by the CMV promoter. Huh7-H1 cells were cotransfected with the L⁻M⁻S⁻ mutant expression plasmid and various amounts of the LMS or CMV-LM donor plasmid. The level of cccDNA was measured by Southern blotting and normalized to the level of fl (−) DNA, measured by Southern blotting of vDNA. The level of L protein was measured in total cell lysates by using the polyclonal anti-HBs antibody (Fitzgerald). (A) Immunoblot of envelope proteins expressed from either the LMS or CMV-LM donor plasmids. Protein soluble in the detergent Triton X-114 was isolated and detected with either a polyclonal anti-HBs antibody (Fitzgerald) or a monoclonal anti-PreS2 antibody (Virogen). (B) The graph shows the level of cccDNA (y axis) as a proportion of the level when the L⁻M⁻S⁻ donor was used, plotted against the level of L protein (x axis) as a proportion of the level of L when the greatest amount of the LMS donor was used. The points on each line represent the different amounts of each donor plasmid transfected.

Isolation of vDNA and Southern blotting.

Isolation of intracellular encapsidated viral DNA (vDNA) and cccDNA and Southern blotting were performed as described previously (32). Briefly, cells were lysed in 10 mM Tris, 1 mM EDTA, 0.2% NP-40, pH 8.1. For vDNA, nuclei were pelleted and the supernatant was treated with micrococcal nuclease for 1.5 h at 37°C. The sample was then adjusted to 10 mM EDTA, 0.4% SDS, 100 mM NaCl, 0.4 mg/ml pronase and incubated for 2 h at 37°C before extraction with phenol and precipitation in ethanol. DNA was resolved on a 1.25% agarose gel buffered with TBE (90 mM Tris-borate, 2.5 mM EDTA, pH 8.5) and transferred to a Hybond-N membrane. For cccDNA, alkaline lysis with an equal volume of 6% SDS, 0.15 M NaOH and incubated at 37°C for 15 min was used to prepare a total cell lysate. The lysate was brought to 0.6 M potassium acetate (KOAc), pH 5.0, and the precipitate was removed by centrifugation. Extraction with phenol removed single-stranded DNA in the organic phase and DNA in the aqueous phase was precipitated with ethanol. Samples were treated with RNase A prior to electrophoresis through a 1.25% agarose gel containing 0.5 μg/ml ethidium bromide (EtBr) and buffered with TBE. The gel was subsequently soaked in 0.025 N HCl-1.5 M NaCl and transferred to a Hybond-N membrane. A 351-nt ribonucleotide probe specific for coordinates 3096 to 3182/1 to 264 of the HBV minus strand was used to detect viral DNA. This probe does not detect DNA produced by reverse transcription of most spliced pgRNA molecules, which may be present in these cultures (1). The masses of vDNA and cccDNA were determined by comparison to known masses of a linear double-stranded fragment of the HBV genome and a plasmid containing the HBV sequence, respectively.

Immunoblotting.

Protein levels were determined by immunoblotting as described previously (32). Viral envelope proteins and GFP were detected with primary antibodies rabbit anti-HBsAg (catalog number [Cat. #] 20-HR20; Fitzgerald) at a dilution of 1:500 and mouse anti-GFP (Cat. # sc-9996; Santa Cruz) at a dilution of 1:1,000, respectively. Secondary antibodies goat anti-rabbit IRDye 680 (Cat. # 926-32221; Li-Cor) and goat anti-mouse IRDye 800CW (Cat. # 926-32210; Li-Cor) were used at a dilution of 1:10,000. To reduce the level of nonspecific binding of the anti-HBsAg antibody, preparations of this antibody were absorbed against immunoblots produced with naïve cell lysate.

To detect M protein, viral envelope proteins were purified based on their solubility in Triton X-114. Cells were lysed on the plate in 10 mM Tris, 1 mM EDTA, 0.2% NP-40, pH 8.1. A fraction of the cell lysate was centrifuged to pellet nuclei. Then, 50 μl of the supernatant was mixed with 25 μl of a 10% Triton X-114 solution and incubated at 37°C for 5 min. Subsequently, 1 ml of 10 mM Tris, 1 mM EDTA, pH 8, was added to each sample and incubated at 37°C for 10 min. Aqueous (top) and detergent (bottom) phases of the solution were separated by centrifugation. Protein was precipitated from the detergent phase with 500 μl of acetone and suspended in Laemmli buffer (2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.002% bromophenol blue, 0.0625 M Tris-HCl, pH 7). These samples were analyzed in the same manner as protein from total cell lysates, with the following exception. The anti-HBsAg antibody was used without prior absorption against naïve cell lysates and the primary antibody mouse anti-PreS (Cat. # 031-A; Virogen), specific for the PreS2 region of the envelope proteins, was used at a dilution of 1:500 with the goat anti-mouse secondary.

Immunoblots were visualized and quantified using a Li-Cor Odyssey Imager and software. The linearity of detection was established over a ≥10-fold range in each analysis using a serial dilution of one sample.

Native particle gel blotting.

Virions and nonenveloped (free) nucleocapsids in the culture medium were measured as described previously (32), with the following changes. Culture medium was clarified by centrifugation and 400 μl of the supernatant was concentrated ∼20-fold with either a 100-kDa Microcon filter device (Millipore) or a 100-kDa Nanosep filter device (Pall).

IP and endogenous polymerase reaction (EPR).

Immunoprecipitation (IP) of virions or nucleocapsids was performed using either rabbit anti-HBsAg (Cat. # 20-HR20; Fitzgerald) or rabbit anti-HBcAg (Cat. # B0586; Dako) antibody, respectively, bound to Sepharose CL-4B beads (Boehringer Mannheim). Antibody-bound beads were incubated with clarified culture medium for IP of virions or cell lysates treated with micrococcal nuclease for IP of intracellular nucleocapsids. Beads were collected by centrifugation. Extracellular free capsids were immunoprecipitated from clarified culture medium after the depletion of virions by IP.

For the EPR, beads bound with intracellular nucleocapsids were mixed with 50 μl of EPR buffer (50 mM Tris, 75 mM NH₄Cl, 1 mM EDTA, 20 mM MgCl₂, 0.1% beta-mercaptoethanol, 0.5% NP-40, 0.4 mM deoxynucleoside triphosphates [dNTPs], pH 8), mock buffer (same composition as EPR buffer but lacking dNTPs), or phosphate-buffered saline (PBS) (untreated) and incubated for ∼22 h at 37°C. Beads were suspended in 100 μl of PBS and 100 μl of 1% SDS, 10 mM Tris, 10 mM EDTA, 0.6 mg/ml pronase, pH 8, was added to isolate DNA from each EPR reaction. The suspension was incubated at 37°C for 45 min. The sample was then extracted with phenol, 20 μg of glycogen was added, and DNA was precipitated with ethanol and analyzed by Southern blotting.

To isolate total extracellular encapsidated DNA (see Fig. 1B, lanes 2, 4, and 7), clarified culture medium or supernatant from IP samples was brought to 0.2% NP-40, 75 mM Tris, 2 mM CaCl₂ and treated with 44 units of micrococcal nuclease at 37°C for 30 min (Worthington). The sample was then brought to 10 mM EDTA, 0.4% SDS, 100 mM NaCl, and 0.4 mg/ml pronase and incubated at 37°C for 2 h. After extraction once with 1:1 phenol-chloroform and once with chloroform, the sample was brought to 0.2 M NaCl and 20 μg glycogen was added. DNA was precipitated with ethanol and analyzed by Southern blotting.

Statistical analysis.

Statistical comparisons were made using the Wilcoxon signed-rank test between paired data samples. Paired data samples were defined as samples that were transfected, isolated, and analyzed at the same time.

RESULTS

Rationale.

The L protein is sufficient to regulate the amplification of cccDNA for DHBV (49, 50). Whether one or more of the envelope proteins regulates the amplification of cccDNA in HBV was unresolved because studies addressing this question have produced conflicting conclusions (15, 18, 36, 46). We decided to determine whether the level of cccDNA increased upon ablation of expression of the three envelope proteins and if the restoration of expression restored cccDNA to lower levels. If ablation of envelope protein expression did increase levels of cccDNA, we wanted to learn which of the envelope proteins were responsible. We used the Huh7-derived cell line Huh7-H1. Unlike primary hepatocytes, which are nondividing cells, Huh7-H1 cells are a proliferating cell line and more closely reflect cells of the liver during active viral replication, when there is significant cell turnover (39). These cultures provide insight into the regulation of synthesis of cccDNA during amplification and maintenance of the virus but may not reflect the formation of the initial pool of cccDNA (see Discussion). This cell line stably expresses the EBV EBNA-1 protein and, when transfected with HBV expression plasmids that contain EBV oriP, accumulates cccDNA to levels 6-fold higher than parental Huh7 cell levels (32). This cell culture system allowed the reliable detection of cccDNA in cotransfection experiments, necessary for manipulating expression of the viral envelope proteins, when Huh7 and HepG2 cells did not. The level of cccDNA was normalized to the level of viral genome replication in our experiments to account for differences in viral genome replication that might affect the synthesis of cccDNA. Initially, cccDNA was normalized to the level of fl-RC DNA, which is the immediate precursor to cccDNA. It was later determined that the level of fl-RC DNA changed in response to expression of the envelope proteins (Fig. 1 and 2). This effect of the envelope proteins was limited to fl-RC DNA and did not influence the level of other intermediates of viral DNA replication (Fig. 3 ). Thus, full-length, minus-strand [fl (−)] DNA was used for the normalization of cccDNA to account for differences in viral genome replication. Finally, the level of envelope protein was varied to determine if the level of cccDNA correlated inversely with the level of envelope protein expression.

Coexpression of the three viral envelope proteins limits amplification of cccDNA and accumulation of fl-RC DNA.

We compared the replication of WT virus (TL25) and a mutant deficient in expression of the three envelope proteins (TL7; L⁻M⁻S⁻) to determine whether coexpression of the three envelope proteins influenced the synthesis of cccDNA in HBV. Expression of the viral envelope proteins was examined by immunoblotting (Fig. 1A). Both the S protein, which appears in two forms corresponding in size with the nonglycosylated (24-kDa) and singly glycosylated (27-kDa) forms, and the L protein were detected in the cells expressing WT virus. Envelope proteins were not detected with the L⁻M⁻S⁻ mutant. The M protein was not reproducibly detected in this analysis. The sequence of the S protein is contained within the M protein, and thus the antibody is predicted to detect M at least as efficiently as S. The inability to detect M protein may be due to low levels of expression of this protein. In later experiments (see Fig. 6 and 7), an alternative isolation protocol was used that allowed reproducible detection of M. Secreted virions were detected in the culture medium of cells expressing WT virus, but not the L⁻M⁻S⁻ mutant, as determined by native particle gel blotting (data not shown). The levels of free capsids in the medium were similar in WT and L⁻M⁻S⁻ cultures.

Next we analyzed vDNA to determine whether ablation of expression of the envelope proteins affected viral genome replication. Southern blotting indicated that the levels of intermediates of viral genome replication containing fl (−) DNA, which includes SS molecules as well as molecules that contain a complete plus strand, were similar in cultures expressing WT (47 ± 17 ng/plate) and L⁻M⁻S⁻ (44 ± 11 ng/plate) viruses (Fig. 1B, lanes 1 and 3). However, the level of fl-RC molecules (relaxed circular conformation containing a complete minus strand and a complete plus strand) was higher for the L⁻M⁻S⁻ mutant (1.17 ± 0.31 ng/plate) than for the WT virus (0.40 ± 0.11 ng/plate). The difference was >4-fold upon normalization to fl (−) DNA.

The envelope proteins facilitate the envelopment and secretion of nucleocapsids from the cell as virions (7, 52). Therefore, a reason for the reduced level of intracellular fl-RC DNA in the cultures expressing WT virus could be that nucleocapsids containing fl-RC DNA were secreted from the cell. Total extracellular encapsidated DNA, including virions and free capsids, from both WT and the L⁻M⁻S⁻ mutant was analyzed by Southern blotting to determine if this was the case (Fig. 1B, lanes 2 and 4). The level of extracellular fl-RC DNA in WT culture medium (0.22 ± 0.11 ng/plate) was less than that observed for the L⁻M⁻S⁻ mutant (0.32 ± 0.17 ng/plate). The difference in fl-RC DNA was >3.5-fold after normalization to fl (−) DNA. Thus, the total amount of fl-RC DNA produced by the L⁻M⁻S⁻ mutant was greater than the amount produced by the WT virus (1.17 + 0.32 ng/plate in the L⁻M⁻S⁻ versus 0.40 + 0.22 ng/plate in the WT). Therefore, secretion of nucleocapsids containing fl-RC DNA could not account for the lower levels of intracellular fl-RC DNA in the cultures expressing WT virus. Previous studies have also observed accumulation of RC DNA within the cell when expression of the envelope proteins is ablated (15, 18, 19, 24). In particular, Kock et al. (24) found that the accumulation of RC DNA was more pronounced in the nucleus than in the cytoplasm of the cell. These findings are consistent with a role of the envelope proteins in regulating the synthesis of cccDNA via preventing trafficking of the nucleocapsid to the nucleus.

DNA from virions and free capsids in the culture medium was isolated individually by selective immunoprecipitation and analyzed by Southern blotting (Fig. 1B, lanes 5 to 7). Virions predominantly contained DNA with an electrophoretic mobility between that of SS DNA and fl-RC DNA, indicating these molecules contain a complete minus strand and an incomplete plus strand (Fig. 1B, lane 5). DNA isolated from virions in patient sera has been shown to contain RC DNA with an incomplete plus strand (12, 48). Free capsids from WT cultures contained molecules with mobility similar to that of SS DNA (Fig. 1B, lane 6). Therefore, nucleocapsids containing double-stranded DNA appear to be secreted preferentially over those containing single-stranded DNA, and few of these molecules have a full-length plus strand. These data suggest that the expression of envelope proteins limits completion of the elongation of plus-strand DNA.

cccDNA was isolated from cultures expressing the WT or the L⁻M⁻S⁻ mutant and measured by Southern blotting to determine if expression of the envelope proteins affected the level of cccDNA (Fig. 1C). Similar to what was observed for fl-RC, the level of cccDNA was greater in the L⁻M⁻S⁻ mutant (66 ± 22 pg/plate) than in the WT virus (16 ± 9 pg/plate). The level of cccDNA, after normalization to fl (−) DNA, in the L⁻M⁻S⁻ mutant was >6-fold higher than the level in the WT virus.

If the increase in cccDNA and fl-RC DNA was due to the absence of the envelope proteins, then restoration of the expression of the envelope proteins for the L⁻M⁻S⁻ mutant should decrease the level of these molecules. Cells were cotransfected with an expression plasmid for the L⁻M⁻S⁻ mutant (TL7) and an expression plasmid for LMS proteins (TL34). An L⁻M⁻S⁻ donor plasmid (TL27), deficient in expression of the envelope proteins, was cotransfected with TL7 as a comparison. Cotransfection of the L⁻ M⁻S⁻ mutant with the LMS donor restored expression of the envelope proteins (Fig. 2A). Additionally, virion secretion was restored, demonstrating that functional levels of envelope proteins were expressed (Fig. 2B). Analysis of vDNA and cccDNA revealed decreases in the levels of fl-RC DNA and cccDNA (Fig. 2C and D). The levels of fl-RC DNA and cccDNA, after normalization to fl (−) DNA, in cultures cotransfected with the L⁻M⁻S⁻ mutant and the LMS donor were ∼40% (Fig. 2C) and ∼50% (Fig. 2D), respectively, of the level when the L⁻M⁻S⁻ donor was used.

These results provide evidence that expression of the envelope proteins limits the synthesis of cccDNA in HBV. Increased accumulation of cccDNA in response to the ablation of envelope protein expression is consistent with the findings of Gao and Hu (15) and Guo et al. (18), though here we report a greater difference (∼1.5-fold versus ∼6-fold in our studies). The reason for the greater difference observed in our system is not known, but it may be due to differences in the method of expressing the virus. The cell cultures used in this report express HBV to higher levels than do Huh7 cells (used by Guo et al.) or HepG2 cells (used by Gao and Hu). Differences in the levels of viral DNA replication and protein expression would affect the synthesis of cccDNA and may account for this difference. Additionally, the difference reported by Guo et al. was observed in a cell line expressing envelope-deficient HBV in which expression of the envelope proteins was restored via transfection with an expression plasmid, which would enter only a subset of cells. Thus, the change in the level of cccDNA they report is likely an underestimate of the real effect. The finding that the level of fl-RC DNA is increased in the L⁻M⁻S⁻ mutant, as compared with WT virus, indicates that the envelope proteins influence the synthesis of fl-RC DNA. The effect on fl-RC DNA seen in these studies has not been previously reported to our knowledge and was further investigated.

Expression of the viral envelope proteins limits completion of the plus-strand DNA.

The observation that the level of fl-RC DNA was reduced in WT in comparison to L⁻M⁻S⁻ mutant, despite these viruses having similar levels of fl (−) DNA, suggested that the expression of the envelope proteins was affecting the synthesis of plus-strand DNA. After the completion of synthesis of minus-strand DNA, synthesis of fl-RC DNA requires translocation of the RNA primer from the 3′ end of the minus strand (DR1) to a complementary site near the 5′ end of the minus strand (DR2) and initiation of plus-strand DNA synthesis from this position. The level of 5′ ends of plus-strand DNA at DR2 was measured for the WT and L⁻M⁻S⁻ mutant to determine if the envelope proteins were affecting early steps of the plus-strand DNA synthesis. Primer extension analysis of vDNA indicated that the numbers of 5′ ends at DR2 were similar (<50% difference), after normalization to the level of minus-strand DNA, in the WT and L⁻M⁻S⁻ viruses (data not shown). This result indicated that expression of the envelope proteins was limiting the completion of the plus strand, leading to the accumulation of nucleocapsids containing genomes with a full-length minus strand but incomplete plus strand for WT virus. If true, then extension of the plus-strand DNA in vitro should result in the accumulation of similar levels of fl-RC DNA for the L⁻M⁻S⁻ mutant and WT viruses. We performed an endogenous polymerase reaction (EPR) on intracellular capsids isolated from cultures expressing either the WT or L⁻M⁻S⁻ mutant and compared the resulting levels of fl-RC DNA (25, 27). A mock treatment in which dNTPs, present in EPR-treated samples, were omitted from the reaction was included for comparison. DNA was isolated after the reaction and analyzed by Southern blotting (Fig. 3). Relative to the untreated sample, the level of fl-RC DNA was increased ∼5-fold in the EPR-treated sample for the WT, accumulating to a level similar to that observed in the EPR-treated L⁻M⁻S⁻ sample. The mass of fl-RC DNA in untreated and mock-treated nucleocapsids for the L⁻M⁻S⁻ mutant (0.84 ± 0.26 ng/plate and 0.65 ± 0.17 ng/plate, respectively) was ∼3-fold higher than the mass in the WT virus (0.30 ± 0.15 ng/plate and 0.21 ± 0.09 ng/plate, respectively), similar to the difference seen in intracellular vDNA. In contrast, the difference in EPR-treated samples was <1.1-fold (1.34 ± 0.38 ng/plate in the L⁻M⁻S⁻ and 1.39 ± 0.59 ng/plate in the WT). These data indicate that the numbers of nucleocapsids capable of synthesizing fl-RC DNA are similar with the L⁻M⁻S⁻ mutant and WT viruses and support the interpretation that the majority of WT nucleocapsids contain plus-strand DNA that is partially elongated. It is possible that DNA synthesis is arrested after a nucleocapsid is enveloped.

Analysis of envelope proteins involved in regulation of amplification of cccDNA and limiting completion of plus-strand DNA.

We wanted to identify which envelope proteins were responsible for regulating the amplification of cccDNA and limiting the completion of plus-strand DNA. We constructed a series of plasmids, derived from the expression plasmid for WT HBV (TL25), in which expression of different combinations of the envelope proteins were ablated. These plasmids serve as the template for the expression of the pgRNA as well as the template for the expression of the envelope proteins. Upon transfection into cells, these plasmids led to accumulation of similar levels of vDNA (data not shown). Unfortunately, there were unanticipated and undesirable consequences on expression of the envelope proteins. We found that the levels of the expressed envelope proteins changed when expression of one or more of the proteins was knocked out (Fig. 4A and B). For example, when M protein was knocked out, the levels of L and S proteins increased compared to those for the WT. This was undesirable because two parameters were changing simultaneously, the combination of proteins being expressed and the levels of these proteins, making it impossible to attribute changes in phenotype to removal of the targeted protein. We wanted to remove one (or two) envelope protein(s) while maintaining normal levels of expression of the remaining proteins. We devised an alternative strategy to accomplish this goal. The L⁻M⁻S⁻ expression plasmid (TL7) was cotransfected with donor plasmids that were designed to express the different combinations of envelope proteins from their endogenous promoters (Table 1). In addition, the amount of each donor plasmid was varied to find a condition in which normal levels (levels similar to those produced by the LMS donor) of the remaining envelope proteins were synthesized. Cotransfection of the L⁻M⁻S⁻ mutant (TL7) with the L⁻M⁻S⁻ donor (TL27) served as the reference for virus replication in the absence of envelope proteins. The intracellular levels of L and S protein produced by each donor plasmid are shown relative to the level produced by the LMS donor, as determined by immunoblotting (Fig. 5A). M protein was detected in some, but not all, experiments, and thus its level was not measured. The level of secreted virions was determined by native particle gel blotting (Fig. 5B). Only the LMS donor plasmid restored virion secretion to the L⁻M⁻S⁻ virus. The level of fl-RC DNA was measured by Southern blotting (Fig. 5C). A reduction in the level of fl-RC DNA was observed only with the LMS donor plasmid and not with the other envelope protein expression plasmids. The level of fl-RC DNA correlated inversely with the level of expression of envelope proteins and the level of virion secretion. Measurements of cccDNA levels showed that only three donor plasmids, L, LM, and LS, led to a reduction in cccDNA levels (Fig. 5D). The other donor plasmids, S, M, and MS, did not change the level of cccDNA from the L⁻M⁻S⁻ virus. This result indicates that L protein contributes to the regulation of synthesis of cccDNA in HBV. Additionally, these data indicate that the processes of limiting the completion of plus-strand DNA synthesis and regulation of synthesis of cccDNA require different combinations of envelope proteins.

Fig. 4. — Envelope protein levels in mutants of the genomic expression plasmid TL25. Cells were transfected with mutant genomic plasmids that express the pgRNA and different combinations of the envelope proteins. For each plasmid the indicated proteins were knocked out by point mutation of their respective start codons. (A) Immunoblot of envelope proteins expressed from the genomic mutant plasmids. Proteins were detected in total cell lysates by using anti-HBs (Fitzgerald) and anti-GFP (Santa Cruz) primary antibodies. Bands attributable to nonspecific binding of the antibody are indicated with an asterisk. (B) Histogram showing the levels of L and S proteins in each genomic mutant relative to the levels in the WT (TL25) expression plasmid.

We wanted to know if expression of LM, LS, or L regulated the amplification of cccDNA and when LMS were expressed. The level of cccDNA synthesized in each condition was plotted versus the relative level of L protein to compare cccDNA levels with these four conditions under circumstances of similar levels of expression of L protein (Fig. 6A). These plots would be similar for all permutations, and similar to that of the LMS donor, if L protein was sufficient to regulate cccDNA synthesis. Alternatively, if the M or S protein, in combination with L, contributed to regulation, then only the donor plasmid that expresses the required proteins will recapitulate the behavior of the LMS donor. We found that expression of L or LS did not reduce cccDNA levels as a function of the level of L protein as well as the expression of LMS. Also, the LM donor (TL33) produced a relationship similar to the LMS donor (Fig. 6). These data suggest that L protein is necessary but not sufficient for the regulation of cccDNA synthesis in HBV. However, we were hesitant to conclude that the combination of L and M are sufficient for normal regulation because we were unable to detect and measure M protein.

The inability to detect M prevented us from drawing conclusions about the involvement of this protein. To determine whether the donor plasmids were capable of expressing M protein, we transfected cells with 10 μg of the envelope donor plasmids and used an isolation procedure that enriches for membrane proteins based on solubility in Triton X-114 (3). Envelope protein was analyzed by immunoblotting and detected with either the polyclonal anti-HBs antibody or a monoclonal anti-PreS antibody (Fig. 6B). L protein corresponding in size to the nonglycosylated (39-kDa) and singly glycosylated (42-kDa) forms were detected in all samples. M protein corresponding in size with nonglycosylated (30-kDa), singly glycosylated (33-kDa), and doubly glycosylated (36-kDa) forms were detected only in the cells transfected with the LMS and LM donor plasmids. The level of M protein was lower when expressed from the LM donor than it was when expressed from the LMS donor. Normalizing to the level of L protein, we found that the LMS donor expressed M at 300% the level of L and that the LM donor expressed M protein at ∼3 to 4% the level of L. Thus, the donor plasmids are capable of producing M protein, albeit to different levels relative to L protein expression.

L protein is necessary but not sufficient for regulation of cccDNA synthesis.

The analysis depicted in Fig. 6 indicated that the LM donor was as efficient as the LMS donor and better than the LS and L donors in regulating cccDNA synthesis. This suggests that L is necessary but not sufficient for this function. An alternative possibility is that L protein is sufficient for the regulation of cccDNA synthesis but that the methionine at residue 109 of the L protein (mutated to a threonine in the LS and L donors) is required. We constructed a second plasmid for the expression of L and M. This plasmid, TL42 (Table 1), expresses L mRNA from the CMV promoter. When transfected with plasmid TL42 (CMV-LM) alone and analyzed by Triton X-114 isolation and immunoblotting, cells produced M protein at 33% the level of L protein (Fig. 7A). Complementation of the L⁻M⁻S⁻ mutant with the CMV-LM donor plasmid did lower cccDNA levels, but not to the level seen with the LMS and LM donor plasmids (Fig. 7B). This result indicates that neither L protein with methionine at residue 109 nor the mere expression of L and M is sufficient for regulation of cccDNA synthesis. When expressed from their endogenous promoters, L and M appear to be sufficient to regulate the synthesis of cccDNA, but when an exogenous promoter (CMV) is used for the expression of L, the regulation of cccDNA is compromised. Thus, expression of the two proteins is not sufficient for regulation and other factors must also play a role. How the promoter is influencing the regulatory mechanism cannot be determined from these studies. The result in Fig. 7 suggests that the effect of the promoter is independent of the level of the envelope proteins, which was varied over a range. One possible explanation is that the promoter influences the localization of the envelope proteins within the cell. If L and M must assemble at a common intracellular membrane in order to regulate the synthesis of cccDNA, then alteration in the localization of L or M due to expression from different promoters could disrupt this mechanism. Further investigation is required to determine if this is the case.

DISCUSSION

Prior to the present report, several observations were consistent with the idea that intracellular amplification of cccDNA in HBV is regulated. First, DHBV uses the L protein to regulate the amplification of cccDNA (30, 31, 49, 50). Second, measurement of the level of cccDNA in HBV-infected patients, as well as experimental systems for the expression of HBV, indicates this molecule is present at ∼1 to 10 copies per cell (2, 4, 10, 15, 18, 20, 28, 53–55), suggesting that its synthesis is regulated. Third, some studies observed changes in the level of cccDNA in response to ablating or restoring expression of the envelope proteins (15, 18). However, due to moderate changes seen in the levels of cccDNA in these studies (<1.5-fold) and the appearance of conflicting reports (36, 46), the role of the HBV envelope proteins in regulating the synthesis of cccDNA in HBV was not unequivocally established. Our findings demonstrate that the expression of the envelope proteins regulates the amplification of cccDNA in HBV. We found that the level of cccDNA increased when expression of the envelope proteins was ablated (Fig. 1) and subsequently decreased when expression of the envelope proteins was restored (Fig. 2). Additionally, the level of cccDNA correlated inversely with the level of envelope protein (Fig. 5).

In this study, cccDNA synthesis is studied in the context of a proliferating cell line, Huh7-H1. These cultures are different from nondividing hepatocytes of the liver, which the virus would encounter initially upon infection. Instead, these cells more closely resemble hepatocytes undergoing division, as is the case during liver turnover in response to active replication of the virus (39). It is unknown if the mechanisms of regulating cccDNA synthesis are different in nondividing cells, in which the nuclear membrane remains intact, and in dividing cells, in which the nuclear membrane breaks down. Thus, the mechanism of regulation of cccDNA synthesis reported here may be active during later stages of viral replication when considerable cell turnover is observed in the liver. These results may not reflect the mechanism of regulation in place during formation of the initial pool of cccDNA, when there is little cell division.

The cell culture system used in this study employs elements of Epstein-Barr virus to engender high levels of HBV expression and accumulation of cccDNA. Previous characterization of this system indicates that the profile of DNA replication is not significantly altered in this cell line compared with parental Huh7 cells. All aspects of HBV expression studied were increased: core protein, viral DNA, and virus production. There was no indication that trafficking of the capsid was altered. EBNA-1 does not interact with cccDNA as determined by EMSA with overlapping fragments representing the entire HBV genome (data not shown). Thus, we do not believe that the use of this system affects the conclusions of this report.

We found that the L protein contributes to the regulation of amplification of cccDNA. When the M and S proteins were expressed alone or together, cccDNA was synthesized to levels similar to that observed in the absence of envelope proteins. Expression of the L protein alone or in combination with S protein did produce a decrease in the level of cccDNA, but not as great as when all three envelope proteins were present. Only the LM donor, which was designed to express L and M mRNAs from their endogenous promoters, produced levels of cccDNA similar to that observed with the LMS donor (Fig. 5). This result suggests that either the methionine at amino acid position 109 of L protein or M protein make a contribution to the regulation of cccDNA synthesis. However, complementation of the L⁻M⁻S⁻ mutant with a different LM donor plasmid, CMV-LM, did not produce WT levels of cccDNA. If an L protein with methionine at position 109 is sufficient for the regulation of cccDNA synthesis, then the CMV-LM donor would have produced WT levels of cccDNA. Instead, the CMV-LM donor did not produce WT levels of cccDNA, indicating that L^m109 is not sufficient to regulate cccDNA. Therefore, one or more additional parameters or conditions must be met for the regulation of cccDNA synthesis. This new finding suggests that the mechanism of regulation in HBV is different than that in DHBV. The L protein was found to be sufficient for the regulation of cccDNA in DHBV, and the proposed mechanism for regulation is direct interaction between the nucleocapsid and the L protein. Our results suggest that factors in addition to L are required for this interaction in HBV. Unfortunately, we cannot draw compelling conclusions about the involvement of M protein in cccDNA synthesis. Measurement of M protein in cells transfected with the LMS, LM, and CMV-LM donor alone suggested that all three plasmids express M protein but at different levels relative to L protein. The fact that the CMV-LM donor expresses both L and M proteins but does not regulate cccDNA synthesis as well as the LMS donor plasmid suggests that the expression of L and M proteins is not sufficient. Further investigation is required to determine the additional factors or conditions that contribute to the regulation of cccDNA in HBV.

The mechanism HBV uses to regulate the synthesis of cccDNA is likely more complex than interaction between the nucleocapsid and monomeric L and/or M proteins. We postulate that the envelope proteins assemble and oligomerize within a membrane and present a specific conformational surface on the cytosolic face of the ER/Golgi apparatus and that the mature nucleocapsid interacts with this functional surface. Assembly of this functional surface requires proper localization and/or relative abundance of L and M. Changing the promoter for expression of the L mRNA or the relative ratio of the proteins could alter the configuration of this surface, by altering either the localization of the proteins or their arrangement in the membrane. Such perturbations might not change the level of the envelope proteins but could disrupt interaction with nucleocapsid and hence the regulation of cccDNA synthesis.

It has been reported that M is dispensable for the formation of virions (7). However, the level of secreted virus is reduced as compared with that for the WT when the expression of M protein is ablated (16, 41, 52). We found that the L and S proteins were not sufficient to support normal levels of secretion of virus (Fig. 5). Complementation of the L⁻M⁻S⁻ mutant with the LS donor plasmid produced levels of L and S protein greater than that observed when the LMS donor plasmid was used (Fig. 5). However, secreted virus was below the limit of detection in cultures expressing only the L and S proteins. It is unclear why the L and S proteins did not support secretion of virus in this context, but this suggests that either M protein or the methionine at amino acid position 109 of L contributes to this process.

We further found that expression of the envelope proteins limited completion of the plus-strand DNA. We observed an increase in the level of fl-RC DNA (>4-fold) (Fig. 1) when the expression of the envelope proteins was ablated. Conversely, restoration of the expression of the envelope proteins led to a decrease in the level of fl-RC DNA (Fig. 2). The magnitude of the decrease correlated with the level of envelope protein expression. These data demonstrate that this effect is due to expression of the envelope proteins and provides a new insight into the mechanism of plus-strand DNA synthesis. Inhibition of plus-strand DNA synthesis by the viral envelope proteins may explain the long-standing observation that the plus-strand DNA in virions from patient serum is not completely extended (12, 48). Though the mechanism through which the envelope proteins limit completion of the plus strand is not completely understood, our analysis (Fig. 5) has determined that all three envelope proteins are required for this effect. It has been reported that the endogenous polymerase reaction requires removal of the envelope (23). A possible mechanism is that further DNA synthesis is prevented within the nucleocapsid upon acquiring an envelope due to limited access to nucleotides or some other necessary substrate.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grants P01 CA022443, R01 AI060018, and T32 CA009135.

Footnotes

^▿

Published ahead of print on 7 September 2011.

REFERENCES

1. Abraham T. M., Lewellyn E. B., Haines K. M., Loeb D. D. 2008. Characterization of the contribution of spliced RNAs of hepatitis B virus to DNA synthesis in transfected cultures of Huh7 and HepG2 cells. Virology 379: 30–37 [DOI] [PMC free article] [PubMed] [Google Scholar]
2. Bock C. T., Schranz P., Schroder C. H., Zentgraf H. 1994. Hepatitis B virus genome is organized into nucleosomes in the nucleus of the infected cell. Virus Genes 8: 215–229 [DOI] [PubMed] [Google Scholar]
3. Bordier C. 1981. Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256: 1604–1607 [PubMed] [Google Scholar]
4. Bourne E. J., et al. 2007. Quantitative analysis of HBV cccDNA from clinical specimens: correlation with clinical and virological response during antiviral therapy. J. Viral Hepat. 14: 55–63 [DOI] [PubMed] [Google Scholar]
5. Bruss V. 2004. Envelopment of the hepatitis B virus nucleocapsid. Virus Res. 106: 199–209 [DOI] [PubMed] [Google Scholar]
6. Bruss V. 1997. A short linear sequence in the pre-S domain of the large hepatitis B virus envelope protein required for virion formation. J. Virol. 71: 9350–9357 [DOI] [PMC free article] [PubMed] [Google Scholar]
7. Bruss V., Ganem D. 1991. The role of envelope proteins in hepatitis B virus assembly. Proc. Natl. Acad. Sci. U. S. A. 88: 1059–1063 [DOI] [PMC free article] [PubMed] [Google Scholar]
8. Bruss V., Lu X., Thomssen R., Gerlich W. H. 1994. Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein. EMBO J. 13: 2273–2279 [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Bruss V., Thomssen R. 1994. Mapping a region of the large envelope protein required for hepatitis B virion maturation. J. Virol. 68: 1643–1650 [DOI] [PMC free article] [PubMed] [Google Scholar]
10. Chou Y. C., et al. 2005. Evaluation of transcriptional efficiency of hepatitis B virus covalently closed circular DNA by reverse transcription-PCR combined with the restriction enzyme digestion method. J. Virol. 79: 1813–1823 [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Cohen B. J., Richmond J. E. 1982. Electron microscopy of hepatitis B core antigen synthesized in E. coli. Nature 296: 677–679 [DOI] [PubMed] [Google Scholar]
12. Delius H., Gough N. M., Cameron C. H., Murray K. 1983. Structure of the hepatitis B virus genome. J. Virol. 47: 337–343 [DOI] [PMC free article] [PubMed] [Google Scholar]
13. Eble B. E., MacRae D. R., Lingappa V. R., Ganem D. 1987. Multiple topogenic sequences determine the transmembrane orientation of the hepatitis B surface antigen. Mol. Cell. Biol. 7: 3591–3601 [DOI] [PMC free article] [PubMed] [Google Scholar]
14. Fields B. N., Knipe D. M., Howley P. M. 2007. Fields virology, 5th ed. Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia, PA [Google Scholar]
15. Gao W., Hu J. 2007. Formation of hepatitis B virus covalently closed circular DNA: removal of genome-linked protein. J. Virol. 81: 6164–6174 [DOI] [PMC free article] [PubMed] [Google Scholar]
16. Garcia T., et al. 2009. Drastic reduction in the production of subviral particles does not impair hepatitis B virus virion secretion. J. Virol. 83: 11152–11165 [DOI] [PMC free article] [PubMed] [Google Scholar]
17. Gerlich W. H., Robinson W. S. 1980. Hepatitis B virus contains protein attached to the 5′ terminus of its complete DNA strand. Cell 21: 801–809 [DOI] [PubMed] [Google Scholar]
18. Guo H., et al. 2007. Characterization of the intracellular deproteinized relaxed circular DNA of hepatitis B virus: an intermediate of covalently closed circular DNA formation. J. Virol. 81: 12472–12484 [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Guo H., Mao R., Block T. M., Guo J. T. 2010. Production and function of the cytoplasmic deproteinized relaxed circular DNA of hepadnaviruses. J. Virol. 84: 387–396 [DOI] [PMC free article] [PubMed] [Google Scholar]
20. Hantz O., et al. 2009. Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells. J. Gen. Virol. 90: 127–135 [DOI] [PubMed] [Google Scholar]
21. Heermann K. H., et al. 1984. Large surface proteins of hepatitis B virus containing the pre-s sequence. J. Virol. 52: 396–402 [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Hirsch R. C., Lavine J. E., Chang L. J., Varmus H. E., Ganem D. 1990. Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as well as for reverse transcription. Nature 344: 552–555 [DOI] [PubMed] [Google Scholar]
23. Kaplan P. M., Greenman R. L., Gerin J. L., Purcell R. H., Robinson W. S. 1973. DNA polymerase associated with human hepatitis B antigen. J. Virol. 12: 995–1005 [DOI] [PMC free article] [PubMed] [Google Scholar]
24. Kock J., et al. 2010. Generation of covalently closed circular DNA of hepatitis B viruses via intracellular recycling is regulated in a virus specific manner. PLoS Pathog. 6: e1001082. [DOI] [PMC free article] [PubMed] [Google Scholar]
25. Koschel M., Oed D., Gerelsaikhan T., Thomssen R., Bruss V. 2000. Hepatitis B virus core gene mutations which block nucleocapsid envelopment. J. Virol. 74: 1–7 [DOI] [PMC free article] [PubMed] [Google Scholar]
26. Lambert C., Prange R. 2001. Dual topology of the hepatitis B virus large envelope protein: determinants influencing post-translational pre-S translocation. J. Biol. Chem. 276: 22265–22272 [DOI] [PubMed] [Google Scholar]
27. Landers T. A., Greenberg H. B., Robinson W. S. 1977. Structure of hepatitis B Dane particle DNA and nature of the endogenous DNA polymerase reaction. J. Virol. 23: 368–376 [DOI] [PMC free article] [PubMed] [Google Scholar]
28. Laras A., Koskinas J., Dimou E., Kostamena A., Hadziyannis S. J. 2006. Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients. Hepatology 44: 694–702 [DOI] [PubMed] [Google Scholar]
29. Laub O., et al. 1983. Synthesis of hepatitis B surface antigen in mammalian cells: expression of the entire gene and the coding region. J. Virol. 48: 271–280 [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Lenhoff R. J., Summers J. 1994. Construction of avian hepadnavirus variants with enhanced replication and cytopathicity in primary hepatocytes. J. Virol. 68: 5706–5713 [DOI] [PMC free article] [PubMed] [Google Scholar]
31. Lenhoff R. J., Summers J. 1994. Coordinate regulation of replication and virus assembly by the large envelope protein of an avian hepadnavirus. J. Virol. 68: 4565–4571 [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Lentz T. B., Loeb D. D. 2010. Development of cell cultures that express hepatitis B virus to high levels and accumulate cccDNA. J. Virol. Methods 169: 52–60 [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Lepere C., Regeard M., Le Seyec J., Gripon P. 2007. The translocation motif of hepatitis B virus envelope proteins is dispensable for infectivity. J. Virol. 81: 7816–7818 [DOI] [PMC free article] [PubMed] [Google Scholar]
34. Le Seyec J., Chouteau P., Cannie I., Guguen-Guillouzo C., Gripon P. 1999. Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain. J. Virol. 73: 2052–2057 [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Le Seyec J., Chouteau P., Cannie I., Guguen-Guillouzo C., Gripon P. 1998. Role of the pre-S2 domain of the large envelope protein in hepatitis B virus assembly and infectivity. J. Virol. 72: 5573–5578 [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Ling R., Harrison T. J. 1997. Production of hepatitis B virus covalently closed circular DNA in transfected cells is independent of surface antigen synthesis. J. Gen. Virol. 78: 1463–1467 [DOI] [PubMed] [Google Scholar]
37. Loffler-Mary H., Dumortier J., Klentsch-Zimmer C., Prange R. 2000. Hepatitis B virus assembly is sensitive to changes in the cytosolic S loop of the envelope proteins. Virology 270: 358–367 [DOI] [PubMed] [Google Scholar]
38. Mason W. S., et al. 1983. Experimental transmission of duck hepatitis B virus. Virology 131: 375–384 [DOI] [PubMed] [Google Scholar]
39. Mason W. S., Litwin S., Xu C., Jilbert A. R. 2007. Hepatocyte turnover in transient and chronic hepadnavirus infections. J. Viral Hepat. 14(Suppl. 1): 22–28 [DOI] [PubMed] [Google Scholar]
40. Miller R. H., Robinson W. S. 1984. Hepatitis B virus DNA forms in nuclear and cytoplasmic fractions of infected human liver. Virology 137: 390–399 [DOI] [PubMed] [Google Scholar]
41. Ni Y., Sonnabend J., Seitz S., Urban S. 2010. The pre-s2 domain of the hepatitis B virus is dispensable for infectivity but serves a spacer function for L-protein-connected virus assembly. J. Virol. 84: 3879–3888 [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Ostapchuk P., Hearing P., Ganem D. 1994. A dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis B viral morphogenesis. EMBO J. 13: 1048–1057 [DOI] [PMC free article] [PubMed] [Google Scholar]
43. Prange R., Streeck R. E. 1995. Novel transmembrane topology of the hepatitis B virus envelope proteins. EMBO J. 14: 247–256 [DOI] [PMC free article] [PubMed] [Google Scholar]
44. Robinson W. S., Clayton D. A., Greenman R. L. 1974. DNA of a human hepatitis B virus candidate. J. Virol. 14: 384–391 [DOI] [PMC free article] [PubMed] [Google Scholar]
45. Sattler F., Robinson W. S. 1979. Hepatitis B viral DNA molecules have cohesive ends. J. Virol. 32: 226–233 [DOI] [PMC free article] [PubMed] [Google Scholar]
46. Sprinzl M. F., Oberwinkler H., Schaller H., Protzer U. 2001. Transfer of hepatitis B virus genome by adenovirus vectors into cultured cells and mice: crossing the species barrier. J. Virol. 75: 5108–5118 [DOI] [PMC free article] [PubMed] [Google Scholar]
47. Summers J., Mason W. S. 1982. Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 29: 403–415 [DOI] [PubMed] [Google Scholar]
48. Summers J., O'Connell A., Millman I. 1975. Genome of hepatitis B virus: restriction enzyme cleavage and structure of DNA extracted from Dane particles. Proc. Natl. Acad. Sci. U. S. A. 72: 4597–4601 [DOI] [PMC free article] [PubMed] [Google Scholar]
49. Summers J., Smith P. M., Horwich A. L. 1990. Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification. J. Virol. 64: 2819–2824 [DOI] [PMC free article] [PubMed] [Google Scholar]
50. Summers J., Smith P. M., Huang M. J., Yu M. S. 1991. Morphogenetic and regulatory effects of mutations in the envelope proteins of an avian hepadnavirus. J. Virol. 65: 1310–1317 [DOI] [PMC free article] [PubMed] [Google Scholar]
51. Tuttleman J. S., Pourcel C., Summers J. 1986. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 47: 451–460 [DOI] [PubMed] [Google Scholar]
52. Ueda K., Tsurimoto T., Matsubara K. 1991. Three envelope proteins of hepatitis B virus: large S, middle S, and major S proteins needed for the formation of Dane particles. J. Virol. 65: 3521–3529 [DOI] [PMC free article] [PubMed] [Google Scholar]
53. Werle-Lapostolle B., et al. 2004. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology 126: 1750–1758 [DOI] [PubMed] [Google Scholar]
54. Wieland S. F., Spangenberg H. C., Thimme R., Purcell R. H., Chisari F. V. 2004. Expansion and contraction of the hepatitis B virus transcriptional template in infected chimpanzees. Proc. Natl. Acad. Sci. U. S. A. 101: 2129–2134 [DOI] [PMC free article] [PubMed] [Google Scholar]
55. Wong D. K., et al. 2004. Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients. Hepatology 40: 727–737 [DOI] [PubMed] [Google Scholar]
56. Wu T. T., Coates L., Aldrich C. E., Summers J., Mason W. S. 1990. In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway. Virology 175: 255–261 [DOI] [PubMed] [Google Scholar]
57. Zoulim F. 2005. New insight on hepatitis B virus persistence from the study of intrahepatic viral cccDNA. J. Hepatol. 42: 302–308 [DOI] [PubMed] [Google Scholar]

[B1] 1. Abraham T. M., Lewellyn E. B., Haines K. M., Loeb D. D. 2008. Characterization of the contribution of spliced RNAs of hepatitis B virus to DNA synthesis in transfected cultures of Huh7 and HepG2 cells. Virology 379: 30–37 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2. Bock C. T., Schranz P., Schroder C. H., Zentgraf H. 1994. Hepatitis B virus genome is organized into nucleosomes in the nucleus of the infected cell. Virus Genes 8: 215–229 [DOI] [PubMed] [Google Scholar]

[B3] 3. Bordier C. 1981. Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256: 1604–1607 [PubMed] [Google Scholar]

[B4] 4. Bourne E. J., et al. 2007. Quantitative analysis of HBV cccDNA from clinical specimens: correlation with clinical and virological response during antiviral therapy. J. Viral Hepat. 14: 55–63 [DOI] [PubMed] [Google Scholar]

[B5] 5. Bruss V. 2004. Envelopment of the hepatitis B virus nucleocapsid. Virus Res. 106: 199–209 [DOI] [PubMed] [Google Scholar]

[B6] 6. Bruss V. 1997. A short linear sequence in the pre-S domain of the large hepatitis B virus envelope protein required for virion formation. J. Virol. 71: 9350–9357 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7. Bruss V., Ganem D. 1991. The role of envelope proteins in hepatitis B virus assembly. Proc. Natl. Acad. Sci. U. S. A. 88: 1059–1063 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8. Bruss V., Lu X., Thomssen R., Gerlich W. H. 1994. Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein. EMBO J. 13: 2273–2279 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9. Bruss V., Thomssen R. 1994. Mapping a region of the large envelope protein required for hepatitis B virion maturation. J. Virol. 68: 1643–1650 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10. Chou Y. C., et al. 2005. Evaluation of transcriptional efficiency of hepatitis B virus covalently closed circular DNA by reverse transcription-PCR combined with the restriction enzyme digestion method. J. Virol. 79: 1813–1823 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11. Cohen B. J., Richmond J. E. 1982. Electron microscopy of hepatitis B core antigen synthesized in E. coli. Nature 296: 677–679 [DOI] [PubMed] [Google Scholar]

[B12] 12. Delius H., Gough N. M., Cameron C. H., Murray K. 1983. Structure of the hepatitis B virus genome. J. Virol. 47: 337–343 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13. Eble B. E., MacRae D. R., Lingappa V. R., Ganem D. 1987. Multiple topogenic sequences determine the transmembrane orientation of the hepatitis B surface antigen. Mol. Cell. Biol. 7: 3591–3601 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14. Fields B. N., Knipe D. M., Howley P. M. 2007. Fields virology, 5th ed. Wolters Kluwer Health/Lippincott Williams & Wilkins, Philadelphia, PA [Google Scholar]

[B15] 15. Gao W., Hu J. 2007. Formation of hepatitis B virus covalently closed circular DNA: removal of genome-linked protein. J. Virol. 81: 6164–6174 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16. Garcia T., et al. 2009. Drastic reduction in the production of subviral particles does not impair hepatitis B virus virion secretion. J. Virol. 83: 11152–11165 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17. Gerlich W. H., Robinson W. S. 1980. Hepatitis B virus contains protein attached to the 5′ terminus of its complete DNA strand. Cell 21: 801–809 [DOI] [PubMed] [Google Scholar]

[B18] 18. Guo H., et al. 2007. Characterization of the intracellular deproteinized relaxed circular DNA of hepatitis B virus: an intermediate of covalently closed circular DNA formation. J. Virol. 81: 12472–12484 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19. Guo H., Mao R., Block T. M., Guo J. T. 2010. Production and function of the cytoplasmic deproteinized relaxed circular DNA of hepadnaviruses. J. Virol. 84: 387–396 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20. Hantz O., et al. 2009. Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells. J. Gen. Virol. 90: 127–135 [DOI] [PubMed] [Google Scholar]

[B21] 21. Heermann K. H., et al. 1984. Large surface proteins of hepatitis B virus containing the pre-s sequence. J. Virol. 52: 396–402 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. Hirsch R. C., Lavine J. E., Chang L. J., Varmus H. E., Ganem D. 1990. Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as well as for reverse transcription. Nature 344: 552–555 [DOI] [PubMed] [Google Scholar]

[B23] 23. Kaplan P. M., Greenman R. L., Gerin J. L., Purcell R. H., Robinson W. S. 1973. DNA polymerase associated with human hepatitis B antigen. J. Virol. 12: 995–1005 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24. Kock J., et al. 2010. Generation of covalently closed circular DNA of hepatitis B viruses via intracellular recycling is regulated in a virus specific manner. PLoS Pathog. 6: e1001082. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B25] 25. Koschel M., Oed D., Gerelsaikhan T., Thomssen R., Bruss V. 2000. Hepatitis B virus core gene mutations which block nucleocapsid envelopment. J. Virol. 74: 1–7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26. Lambert C., Prange R. 2001. Dual topology of the hepatitis B virus large envelope protein: determinants influencing post-translational pre-S translocation. J. Biol. Chem. 276: 22265–22272 [DOI] [PubMed] [Google Scholar]

[B27] 27. Landers T. A., Greenberg H. B., Robinson W. S. 1977. Structure of hepatitis B Dane particle DNA and nature of the endogenous DNA polymerase reaction. J. Virol. 23: 368–376 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28. Laras A., Koskinas J., Dimou E., Kostamena A., Hadziyannis S. J. 2006. Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients. Hepatology 44: 694–702 [DOI] [PubMed] [Google Scholar]

[B29] 29. Laub O., et al. 1983. Synthesis of hepatitis B surface antigen in mammalian cells: expression of the entire gene and the coding region. J. Virol. 48: 271–280 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30. Lenhoff R. J., Summers J. 1994. Construction of avian hepadnavirus variants with enhanced replication and cytopathicity in primary hepatocytes. J. Virol. 68: 5706–5713 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31. Lenhoff R. J., Summers J. 1994. Coordinate regulation of replication and virus assembly by the large envelope protein of an avian hepadnavirus. J. Virol. 68: 4565–4571 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32. Lentz T. B., Loeb D. D. 2010. Development of cell cultures that express hepatitis B virus to high levels and accumulate cccDNA. J. Virol. Methods 169: 52–60 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33. Lepere C., Regeard M., Le Seyec J., Gripon P. 2007. The translocation motif of hepatitis B virus envelope proteins is dispensable for infectivity. J. Virol. 81: 7816–7818 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B34] 34. Le Seyec J., Chouteau P., Cannie I., Guguen-Guillouzo C., Gripon P. 1999. Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain. J. Virol. 73: 2052–2057 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B35] 35. Le Seyec J., Chouteau P., Cannie I., Guguen-Guillouzo C., Gripon P. 1998. Role of the pre-S2 domain of the large envelope protein in hepatitis B virus assembly and infectivity. J. Virol. 72: 5573–5578 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36. Ling R., Harrison T. J. 1997. Production of hepatitis B virus covalently closed circular DNA in transfected cells is independent of surface antigen synthesis. J. Gen. Virol. 78: 1463–1467 [DOI] [PubMed] [Google Scholar]

[B37] 37. Loffler-Mary H., Dumortier J., Klentsch-Zimmer C., Prange R. 2000. Hepatitis B virus assembly is sensitive to changes in the cytosolic S loop of the envelope proteins. Virology 270: 358–367 [DOI] [PubMed] [Google Scholar]

[B38] 38. Mason W. S., et al. 1983. Experimental transmission of duck hepatitis B virus. Virology 131: 375–384 [DOI] [PubMed] [Google Scholar]

[B39] 39. Mason W. S., Litwin S., Xu C., Jilbert A. R. 2007. Hepatocyte turnover in transient and chronic hepadnavirus infections. J. Viral Hepat. 14(Suppl. 1): 22–28 [DOI] [PubMed] [Google Scholar]

[B40] 40. Miller R. H., Robinson W. S. 1984. Hepatitis B virus DNA forms in nuclear and cytoplasmic fractions of infected human liver. Virology 137: 390–399 [DOI] [PubMed] [Google Scholar]

[B41] 41. Ni Y., Sonnabend J., Seitz S., Urban S. 2010. The pre-s2 domain of the hepatitis B virus is dispensable for infectivity but serves a spacer function for L-protein-connected virus assembly. J. Virol. 84: 3879–3888 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B42] 42. Ostapchuk P., Hearing P., Ganem D. 1994. A dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis B viral morphogenesis. EMBO J. 13: 1048–1057 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B43] 43. Prange R., Streeck R. E. 1995. Novel transmembrane topology of the hepatitis B virus envelope proteins. EMBO J. 14: 247–256 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B44] 44. Robinson W. S., Clayton D. A., Greenman R. L. 1974. DNA of a human hepatitis B virus candidate. J. Virol. 14: 384–391 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B45] 45. Sattler F., Robinson W. S. 1979. Hepatitis B viral DNA molecules have cohesive ends. J. Virol. 32: 226–233 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B46] 46. Sprinzl M. F., Oberwinkler H., Schaller H., Protzer U. 2001. Transfer of hepatitis B virus genome by adenovirus vectors into cultured cells and mice: crossing the species barrier. J. Virol. 75: 5108–5118 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B47] 47. Summers J., Mason W. S. 1982. Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 29: 403–415 [DOI] [PubMed] [Google Scholar]

[B48] 48. Summers J., O'Connell A., Millman I. 1975. Genome of hepatitis B virus: restriction enzyme cleavage and structure of DNA extracted from Dane particles. Proc. Natl. Acad. Sci. U. S. A. 72: 4597–4601 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B49] 49. Summers J., Smith P. M., Horwich A. L. 1990. Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification. J. Virol. 64: 2819–2824 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B50] 50. Summers J., Smith P. M., Huang M. J., Yu M. S. 1991. Morphogenetic and regulatory effects of mutations in the envelope proteins of an avian hepadnavirus. J. Virol. 65: 1310–1317 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B51] 51. Tuttleman J. S., Pourcel C., Summers J. 1986. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 47: 451–460 [DOI] [PubMed] [Google Scholar]

[B52] 52. Ueda K., Tsurimoto T., Matsubara K. 1991. Three envelope proteins of hepatitis B virus: large S, middle S, and major S proteins needed for the formation of Dane particles. J. Virol. 65: 3521–3529 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B53] 53. Werle-Lapostolle B., et al. 2004. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology 126: 1750–1758 [DOI] [PubMed] [Google Scholar]

[B54] 54. Wieland S. F., Spangenberg H. C., Thimme R., Purcell R. H., Chisari F. V. 2004. Expansion and contraction of the hepatitis B virus transcriptional template in infected chimpanzees. Proc. Natl. Acad. Sci. U. S. A. 101: 2129–2134 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B55] 55. Wong D. K., et al. 2004. Quantitation of covalently closed circular hepatitis B virus DNA in chronic hepatitis B patients. Hepatology 40: 727–737 [DOI] [PubMed] [Google Scholar]

[B56] 56. Wu T. T., Coates L., Aldrich C. E., Summers J., Mason W. S. 1990. In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway. Virology 175: 255–261 [DOI] [PubMed] [Google Scholar]

[B57] 57. Zoulim F. 2005. New insight on hepatitis B virus persistence from the study of intrahepatic viral cccDNA. J. Hepatol. 42: 302–308 [DOI] [PubMed] [Google Scholar]

PERMALINK

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 ^{^▿}

Thomas B Lentz

Daniel D Loeb

Abstract

INTRODUCTION