This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Garcia, M. L.
Right arrow Articles by Robek, M. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Garcia, M. L.
Right arrow Articles by Robek, M. D.

 Previous Article  |  Next Article 

Journal of Virology, May 2009, p. 4923-4933, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02644-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Hepatitis B Virus Replication and Release Are Independent of Core Lysine Ubiquitination{triangledown}

Mayra L. Garcia, Rushelle Byfield, and Michael D. Robek*

Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510

Received 23 December 2008/ Accepted 18 February 2009


arrow
ABSTRACT
 
Ubiquitin conjugation to lysine residues regulates a variety of protein functions, including endosomal trafficking and degradation. While ubiquitin plays an important role in the release of many viruses, the requirement for direct ubiquitin conjugation to viral structural proteins is less well understood. Some viral structural proteins require ubiquitin ligase activity, but not ubiquitin conjugation, for efficient release. Recent evidence has shown that, like other viruses, hepatitis B virus (HBV) requires a ubiquitin ligase for release from the infected cell. The HBV core protein contains two lysine residues (K7 and K96), and K96 has been suggested to function as a potential ubiquitin acceptor site based on the fact that previous studies have shown that mutation of this amino acid to alanine blocks HBV release. We therefore reexamined the potential connection between core lysine ubiquitination and HBV replication, protein trafficking, and virion release. In contrast to alanine substitution, we found that mutation of K96 to arginine, which compared to alanine is more conserved but also cannot mediate ubiquitin conjugation, does not affect either virus replication or virion release. We also found that the core lysine mutants display wild-type sensitivity to the antiviral activity of interferon, which demonstrates that ubiquitination of core lysines does not mediate the interferon-induced disruption of HBV capsids. However, mutation of K96 to arginine alters the nuclear-cytoplasmic distribution of core, leading to an accumulation in the nucleolus. In summary, these studies demonstrate that although ubiquitin may regulate the HBV replication cycle, these mechanisms function independently of direct lysine ubiquitination of core protein.


arrow
INTRODUCTION
 
The hepatitis B virus (HBV) particle consists of an enveloped nucleocapsid that contains the viral polymerase (Pol) and an incomplete 3.2-kb double-stranded DNA genome (9). In the cytoplasm, the viral core structural proteins interact to form homodimers, which further self-assemble into capsid particles that package Pol and the viral pregenomic RNA. Encapsidated Pol subsequently reverse transcribes pregenomic RNA to give rise to mature double-stranded relaxed circular DNA-containing capsids. HBV DNA-containing capsids are released from the cell as mature virions after acquiring an envelope consisting of cellular membrane lipids and the viral small, middle, and large envelope proteins (4, 9, 41). Due to the directed insertion of the envelope proteins in the endoplasmic reticulum and Golgi membrane, and the requirement of the large envelope protein for virion release, nucleocapsids are hypothesized to bud at intracellular membranes for release through the constitutive secretory pathway (5). Although the mechanism and site of HBV nucleocapsid envelopment and release remain poorly understood, emerging evidence indicates that the cellular ubiquitin pathway may play a role in this process.

Structural proteins of some enveloped RNA viruses contain highly conserved sequences [PPXY, P(T/S)AP, and YPXL] termed late (L) domains that mediate interactions with proteins of the endocytic pathway to facilitate virus budding and release (1). The P(T/S)AP motif binds Tsg101 (8, 10, 19, 27, 47), a key ESCRT (for endosomal sorting complex required for transport) component for the recognition and sorting of ubiquitinated proteins to internal vesicles of the multivesicular body (MVB), while the YPXL motif binds Alix, an ESCRT-associated protein (26, 44, 48). The PPXY motif binds proteins of the Nedd4 family ubiquitin ligases, which are responsible for ubiquitination of proteins targeted for endocytosis and sorting to the MVB (20), suggesting a link between ubiquitin and viral budding (3, 16, 17, 22, 43, 55). The observation that proteasome inhibition, which depletes free cellular ubiquitin by interfering with ubiquitin recycling, results in a viral budding defect similar to that seen in virus L domain mutants further supports the implication that ubiquitin plays a role in mediating virion release (15, 31, 40, 43). Furthermore, fusion of ubiquitin to the Rous sarcoma virus (RSV) PPPY-containing Gag protein and the equine infectious anemia virus (EIAV) Gag protein containing a heterologous PTAP or PPPY motif rescues the virus-like particle release defect induced by proteasome inhibition (18, 31). While the role of L domains in mediating virion release is relatively well established, it remains unclear whether direct ubiquitination of viral structural proteins is generally required for virion release. Mutation of ubiquitin acceptor lysine residues in the RSV Gag protein inhibits virus budding, but such mutations in human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus Gag protein exert no effect on virus release (29, 42). Recently, a retroviral (i.e., prototypic foamy virus) Gag protein engineered to lack ubiquitin acceptor lysines and encoding either the PSAP or PPXY motif of the L domain displayed no defect in viruslike particle release (58). Altogether, these results suggest that recruitment of host proteins to the L domain and ubiquitination of interacting proteins, but not the viral structural proteins, is required for ubiquitin-dependent virion release, at least for some viruses.

The HBV core structural protein contains two potential ubiquitin acceptor lysine residues (K7 and K96) and an L-domain-like PPAY motif (Fig. 1A). Structural studies indicate that residue K96 and the PPAY motif may be exposed on the surface of HBV capsid particles, at least transiently (4, 32, 37). Studies aimed at identifying interaction factors important for HBV particle release demonstrated a number of interesting findings. First, {gamma}2-adaptin, a cellular trafficking adaptor that contains a ubiquitin-interacting motif (UIM), interacts with both the viral large envelope protein and HBV core, and disruption of the HBV/{gamma}2-adaptin interaction inhibits virus secretion (14, 39). Second, core protein interacts with the Nedd4 ubiquitin ligase through the PPAY motif in core (39). Mutation of the tyrosine in the PPAY motif results in disrupted binding of Nedd4, and overexpression of a catalytically inactive Nedd4 mutant inhibits HBV particle secretion (39). Third, mutation of core K96, but not K7, to alanine results in a defective release phenotype, suggesting that K96 may serve as a ubiquitin conjugation site that aids virion release (32, 39). Recently, overexpression of dominant-negative proteins of the MVB machinery, such as the Vps4 ATPases and the ESCRT-III complex-forming CHMP proteins, were also shown to disrupt HBV budding and virion release, while subviral particles comprised only of envelope proteins were released efficiently (21, 24, 49). This suggests that nucleocapsids may release from the cell by a mechanism distinct from constitutive secretion. These studies show that similar to RNA viruses, HBV utilizes components of the cellular protein trafficking machinery to mediate virion release.


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 1. Generation of core lysine mutants. (A) The 21-kDa HBV core structural protein contains two lysine residues at positions 7 and 96 that serve as potential ubiquitin conjugation sites. These residues are highly conserved among the four major HBV genotypes (6). Core contains a late-domain-like PPXY motif that serves as a binding site for the Nedd4 E3 ubiquitin ligase. Core additionally contains a potential noncanonical SUMOylation motif at position 96. (B) Lysine mutations were generated by site-directed mutagenesis in the core gene contained within the HBV genome under the control of a CMV promoter. K7R contains a lysine-to-arginine mutation at position 7, K96R contains a lysine-to-arginine mutation at position 96, K96A contains a lysine-to-alanine mutation at position 96, and K7R/K96R contains arginine substituted at position 7 and position 96.

Although these findings imply that core ubiquitination may be necessary for HBV particle release, direct evidence of core ubiquitination has been elusive (33, 39; unpublished results). As suggested by previous Gag lysine mutagenesis studies, however, ubiquitin may instead indirectly be required through conjugation to an interacting protein that is essential for mediating HBV release (29, 58). Although core K7 and K96 have been previously assayed in the context of virion release by mutation of the lysine residues to alanine (32, 39), we expanded these studies by assaying core mutants with an arginine substitution at position K7 (K7R) and K96 (K96R), as well as a double lysine-to-arginine mutation (K7R/K96R). Compared to alanine, arginine serves as a more conserved mutation for lysine while still abolishing the potential ubiquitin conjugation site. In the present study, we utilized these mutants to comprehensively examine the role of the core lysines in HBV virus release, the formation of replication intermediates, intracellular localization of core, and the interferon (IFN)-mediated antiviral response.


arrow
MATERIALS AND METHODS
 
DNA constructs and mutagenesis. Plasmid pCMVayw, which encodes the HBV genome (ayw serotype) under the transcriptional control of the cytomegalovirus (CMV) immediate-early promoter, was used to express HBV in cells. To generate the expression constructs encoding the core lysine mutations, a SacI/XhoI HBV DNA fragment (nucleotide positions 534 to 2039) was subcloned into pBluescript II SK to produce the pBSKSII/ayw construct as a template for mutagenesis. Mutations were introduced by using a QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) and confirmed by DNA sequencing. The HBV DNA fragments containing the mutations were cloned into pCMVayw at the SacI and XhoI sites to produce pCMVayw/K7R, pCMVayw/K96R, pCMVayw/K96A, and pCMVayw/K7R.K96R.

Cell culture and transfection. Two mouse immortalized hepatocyte cell lines were used in the present study. The HBV-Met cell line was previously derived from HBV-transgenic mice (30) and replicates HBV from an endogenous transgene in a differentiation-dependent manner. Undifferentiated HBV-Met cells were used for transfection with the expression plasmids, so that virus replication is not initiated from the endogenous transgene. The TRE-HBV cell line is stably transfected with a doxycycline-inducible HBV expression cassette (50). HBV-Met and TRE-HBV cells were cultured on collagen-coated flasks (BD Biosciences) at 37°C in a humidified 5% CO2/air atmosphere in RPMI medium 1640 supplemented with 10% fetal bovine serum, 5 mM L-glutamine, 50 U of penicillin/ml, 50 µg of streptomycin/ml, 10 µg of epidermal growth factor/ml, 10 µg of insulin/ml, and 10 µg of insulin-like growth factor II/ml. Transfections of HBV-Met cells or the human hepatoma cell line Huh7 with plasmid DNA constructs were performed with ExGen 500 (Fermentas) or Lipofectin (Invitrogen), respectively, as recommended by the manufacturer.

Proteasome inhibition. TRE-HBV cells grown in collagen-coated Biocoat six-well plates were treated with doxycycline (10 µg/ml) for 4 days to induce HBV replication and achieve peak virion production. The cells were washed with 1x phosphate-buffered saline (PBS) and treated with 2 µM lactacystin (Calbiochem) for the indicated time. Media were collected and concentrated 10-fold by centrifugation through a Centricon 100 concentration unit (Amicon), and viral DNA was isolated by using a QIAmp DNA minikit (Qiagen, Valencia, CA). Virion DNA was resolved on a 1.6% 1x TAE agarose gel at 30 V for 16 h and transferred to a nylon membrane in 10x SSC buffer (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Nucleic acids were cross-linked on the membrane by UV irradiation and hybridized to probes prepared from 3.2-kb HBV DNA with 32P-labeled dCTP and a Random Primed DNA labeling kit (Roche). Virion-associated DNA was visualized with a PhosphorImager (Fuji) and quantified by using ImageGauge software version 4.22 (Fuji Film). At 8 h posttreatment, HBV DNA was alternatively assayed by quantitative PCR as previously described (54).

Detection of intracellular HBV replication intermediates. For isolation of intracellular nucleocapsids from the cellular cytoplasmic fraction, cells were lysed on ice for 10 min in 0.5 ml of lysis buffer (10 mM Tris [pH 8.0], 150 mM NaCl, 0.5% NP-40) 4 days after transfection. Nuclei and cell debris were removed by centrifugation at 14,000 rpm for 5 min at 4°C. After protein quantification by a BCA assay (Pierce), 50 µg of protein was resolved on a 0.6% agarose gel in 10 mM sodium phosphate buffer at 50 V for 1 h 30 min. The gel was transferred to a nitrocellulose membrane in TNE buffer (10 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA) and probed by immunoblotting with rabbit anti-HBcAg antibody (Dako Co.), followed by detection with secondary antibody using chemiluminescence. After protein detection, the same membrane was denatured in 0.2 M NaOH-1.5 M NaCl for 3 min and then neutralized in 0.2 M Tris-HCl-1.5 M NaCl for 5 min. Capsid-associated DNA was detected with radiolabeled HBV DNA probes and quantified as described above.

Detection of extracellular HBV virions. Media from HBV-Met cells transfected with the HBV expression plasmids were collected at 3 days posttransfection, concentrated 10-fold by centrifugation through a Centricon 100 concentration unit (Amicon), and treated with 10 mM MgCl2 and 100 µg of DNase I/ml for 1 h at 37°C. After protein quantification by using a Bradford assay (Sigma), 30 µg of protein was resolved on a 0.6% agarose gel in 10 mM sodium phosphate buffer at 50 V for 2 h 30 min. The gel was transferred to a nitrocellulose membrane in TNE buffer (10 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA) overnight. The membrane was denatured in 0.2 M NaOH-1.5 M NaCl for 3 min and then neutralized in 0.2 M Tris-HCl-1.5 M NaCl for 5 min. Virion-associated DNA was detected with a radiolabeled HBV DNA probe and quantified as described above.

Indirect immunofluorescence. For immunofluorescence microscopy, Huh7 cells grown on glass coverslips were transfected with the HBV expression plasmids. These cells were used for immunofluorescence because HBV-Met and TRE-HBV cells adhere poorly to coverslips, thus making visualization of core localization difficult. At 2 days posttransfection, the cells were fixed with 3% paraformaldehyde and then permeabilized with 0.5% Triton X-100 for 20 min at 25°C. After blocking for 1 h in PBS containing 10% fetal bovine serum, cells were incubated with rabbit anti-HBcAg antibody for 1 h at 25°C. Colocalization of HBcAg with nucleoli, SC-35 foci, or promyelotic leukemia protein (PML) bodies was determined by staining with mouse anti-nucleolin (Santa Cruz Biotechnology), mouse anti-splicing factor SC-35 (Sigma), or mouse anti-PML (Santa Cruz Biotechnology), respectively. Cells were rinsed with PBS and then incubated with Alexa Fluor 488-labeled goat anti-rabbit and Alexa Fluor 568-labeled goat anti-mouse secondary antibodies for 1 h at 25°C. After being washed, the cells were mounted onto microscope slides with ProLong Gold antifade reagent containing DAPI (4',6'-diamidino-2-phenylindole; Invitrogen) to stain nuclei. Images were captured with a SPOT digital camera using a Nikon Microphot-FX microscope equipped with a x40 or x60 oil immersion objective. ImageJ software (National Institutes of Health) was used for image processing and quantitative analysis. To calculate the amounts of core in the nucleolus, intensity of core signal in the entire nucleus and in each defined nucleolar compartment was determined. The sum of intensities of nucleolar-associated core was divided by the total intensity of nuclear compartment-associated core to obtain the percentage of core in the nucleolus.

MTT assay. Cell viability was determined by using the MTT colorimetric assay. Lactacystin-treated or untreated cells (105) at the indicated times were seeded in a 96-well plate. MTT was dissolved in PBS (5 mg/ml) and added to all wells, and the microplate was incubated at 37°C for 2 h. Viable cells with active mitochondria cleave the soluble tetrazolium salt into an insoluble colored formazan precipitate, which was dissolved in a 20% sodium dodecyl sulfate-50% dimethyl formamide lysis solution and quantified by spectrophotometry at 570 nm.


arrow
RESULTS
 
Proteasome inhibition represses HBV release. Depletion of free cellular ubiquitin by prevention of ubiquitin recycling via proteasome inhibition results in decreased release of viruses that utilize the cellular ubiquitin pathway to mediate budding (15, 31, 40, 43). We therefore sought to determine whether HBV release is sensitive to proteasome inhibition. TRE-HBV cells containing the HBV genome under the control of a tetracycline-inducible promoter were induced to produce HBV by treatment with 10 µg of doxycycline per ml. After 4 days of doxycycline treatment, which coincides with peak virus replication, 2 µM lactacystin was added to the cells for 8, 24, or 48 h. Cell culture media were collected for analysis of HBV DNA by Southern blot (24- and 48-h treatment groups) or quantitative PCR (8-h treatment group) (Fig. 2A). In the presence of lactacystin, HBV release was significantly lower at all three time points examined (Fig. 2A). This partial (rather than complete) inhibition of release is likely due to the fact that we intentionally utilized a relatively low concentration of lactacystin (~1x the 50% inhibitory concentration for proteasome inhibition in immortalized hepatocytes [data not shown]) to minimize potential toxic effects on the cells. To confirm that decreased cell viability was not a factor in the reduced levels of HBV release, the viability of HBV-replicating cells was measured in the absence or presence of lactacystin for the indicated time points by MTT assay (Fig. 2B). As expected, cell viability did not decrease with 2 µM lactacystin treatment compared to untreated cells at 8, 24, or 48 h. To determine whether the inhibition of HBV DNA release was due to a general inhibition of the constitutive secretory pathway, secretion of HBsAg from HBV-replicating cells was measured by enzyme-linked immunosorbent assay (Fig. 2C). HBsAg was efficiently released at 8, 24, and 48 h after lactacystin treatment compared to untreated cells. In fact, there was a slight increase in the amount of HBsAg in the medium of lactacystin-treated cells, which may be due to decreased cellular HBsAg turnover as a result of proteasome inhibition. Finally, because inhibition of HBV DNA release after lactacystin treatment may be a result of HBV replication inhibition, cytoplasmic extracts from HBV-replicating cells treated with 2 µM lactacystin for 24 h were harvested and subjected to native gel electrophoresis for Western and Southern blot analysis of intracellular HBV capsid formation (Fig. 2D). The level of capsid-associated core protein and HBV DNA in lactacystin-treated cells did not differ from the amount in the absence of lactacystin (Fig. 2D). Therefore, proteasome inhibition reduces HBV release in a manner independent of cellular viability, constitutive secretion, and intracellular replication.


Figure 2
View larger version (23K):
[in this window]
[in a new window]

  		FIG. 2. Proteasome inhibition suppresses HBV release. (A) Virions released in the media from TRE-HBV cells expressing HBV under a tetracycline-inducible promoter in the absence or presence of 2 µM lactacystin for 8, 24, and 48 h. HBV DNA in the medium was detected by PCR (8 h) or Southern blot (24 and 48 h). Quantifications of lactacystin-treated cells at the indicated time points were measured relative to untreated cells. (B) Cell viability as measured by MTT conversion assay. (C) HBsAg secretion into the media of lactacystin-treated cells. (D) Formation of cytoplasmic viral capsids as determined by detection of capsid-associated core protein or HBV DNA after 24 h of lactacystin treatment.

Core lysine residues are neither necessary nor sufficient for HBV release. To determine whether ubiquitin conjugation to core lysine residues is required for mediating virus release, codons encoding arginine were substituted for lysine by site-directed mutagenesis in the core gene of the HBV genome (Fig. 1B). Two single lysine-to-arginine mutants were generated, K7R and K96R, as well as a double lysine-to-arginine mutant (K7R/K96R) and the release-defective K96A mutant. Undifferentiated HBV-Met cells were transfected with plasmids encoding full-length replication competent wild-type HBV, K7R, K96R, K96A, or K7R/K96R, and media were harvested 3 days posttransfection and resolved by native gel electrophoresis to separate enveloped virions from released naked capsids prior to detection of virion-associated DNA by Southern blot analysis (Fig. 3A and B). As predicted from previous studies (32, 39), the K96A mutant released less efficiently than the wild-type virus (Fig. 3C). In contrast, release by the K96R mutant was similar to that observed for the wild type (Fig. 3C). Virion release by the K7R mutant was also identical to that by the wild type, as expected based on previous reports (32, 39). Finally, the K7R/K96R double mutant demonstrated virion release comparable to wild-type levels, indicating that K7 and K96 are neither necessary nor sufficient for mediating HBV release (Fig. 3C).


Figure 3
View larger version (41K):
[in this window]
[in a new window]

  		FIG. 3. Core lysines 7 and 96 are not required for HBV release. (A and B) Southern blot analysis of virions released in the media from transfected HBV-Met cells expressing replication competent HBV genomic DNA encoding wild-type core or core lysine mutations K7R, K96R, K96A, or K7R/K96R for 3 days. Enveloped virions were resolved from released naked capsids by native agarose gel electrophoresis and analyzed for virion-associated DNA. Duplicate transfections of each plasmid are shown in panel A. (C) Quantification of virion-associated DNA present in the medium of cells expressing HBV mutants measured relative to wild-type HBV release was determined by densitometry. Virion release and standard errors represent the averages of four independent experiments. WT, wild type.

Core lysine mutants demonstrate wild-type intracellular replication. To determine whether core lysine residues are required for mediating HBV replication, cells were transfected with expression constructs for wild type, K7R, K96R, K96A, or K7R/K96R and assayed for formation of HBV replication intermediates. Cytoplasmic extracts were harvested 3 days posttransfection and subjected to native gel electrophoresis prior to analysis of capsid formation by detecting capsid-associated core by Western blotting (Fig. 4A, top panel). Mutation of lysine to arginine at position 7 and/or position 96 in the core protein did not disrupt the formation of cytoplasmic HBV capsids compared to the wild type. The K96A mutant also demonstrated efficient formation of capsids; however, there was a slight shift in the migration of capsids compared to capsids formed by wild type, K7R, K96R, or K7R/K96R core (Fig. 4A and data not shown). To measure the formation of HBV nucleocapsid replication intermediates, capsids on the nylon membrane were denatured to release capsid-associated DNA for detection by Southern blotting (Fig. 4A, bottom panel). Formation of DNA-containing capsids by core lysine mutants expressed in the context of the HBV genome did not differ significantly from wild-type HBV (Fig. 4B).


Figure 4
View larger version (47K):
[in this window]
[in a new window]

  		FIG. 4. Core lysines 7 and 96 are not required for intracellular capsid formation. (A) HBV-Met cells expressing wild-type HBV or HBV core lysine mutants were harvested 3 days posttransfection and assayed for cytoplasmic capsid formation by native agarose gel electrophoresis and Western blot (WB) with anti-HBcAg polyclonal antibody (Dako; 1:1,000). Capsid-associated viral DNA was assayed by Southern blot (SB) analysis of disrupted capsids transferred to nitrocellulose. (B) Quantification of cytoplasmic capsid-associated DNA from HBV core lysine mutants measured relative to wild-type HBV was determined by densitometry. Nucleocapsid formation and standard errors represent averages of six independent experiments. The observed variations are not statistically significant (P > 0.05). WT, wild type.

Core lysine mutants retain sensitivity to IFN-{alpha}. Previous studies have demonstrated that HBV replication is inhibited by the IFN-{alpha}/β-mediated antiviral response and that IFN sensitivity by HBV requires proteasome activity but not the IFN-induced proteasome catalytic subunits (35, 36). Furthermore, IFN-{alpha}/β disrupts the formation of RNA-containing capsids at a posttranslational step, although the precise mechanism is not defined (50, 51). To determine whether core lysine residues are required for HBV sensitivity to IFN-{alpha}/β, HBV-Met cells were transfected with expression constructs for wild-type HBV, K7R, K96R, K96A, or K7R/K96R. At 3 days posttransfection, cells were treated with 500 U of IFN-{alpha} per ml for 24 h to inhibit HBV replication. Cytoplasmic extracts were harvested and subjected to native gel electrophoresis to detect the formation of capsids by Western blotting (Fig. 5A, middle panel). Capsid-associated DNA was subsequently detected by Southern blot after lysis of capsids embedded on the nitrocellulose membrane (Fig. 5A, top panel). Nucleocapsid formation by core lysine mutants was inhibited by IFN-{alpha} to an extent similar to that observed with wild-type HBV, indicating that core lysine mutants retain sensitivity to IFN-{alpha} (Fig. 5B). The inhibitory effect of nucleocapsid formation during IFN-{alpha} treatment was not due to downregulation of core protein expression as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of whole-cell lysates from HBV-expressing cells (Fig. 5A, bottom panel). Therefore, lysine ubiquitination of HBV core does not mediate the antiviral effect of IFN-{alpha}.


Figure 5
View larger version (63K):
[in this window]
[in a new window]

  		FIG. 5. Core lysines 7 and 96 are not required for the proteasome-dependent IFN-mediated inhibition of HBV replication. (A) HBV-Met cells were transfected with wild-type HBV or HBV core lysine mutants and 3 days later treated with 500 U of IFN-{alpha}/ml for 24 h. Cytoplasmic capsid formation was measured by native gel electrophoresis and Western blot (WB) with anti-core polyclonal antibody. Capsid-associated viral DNA was measured by Southern blot (SB) analysis of disrupted capsids transferred to nitrocellulose. Total core protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of equal amounts of cellular lysates and detected by Western blotting with an anti-HBcAg polyclonal antibody. (B) Quantification of cytoplasmic capsid-associated core and DNA from HBV-expressing cells treated with IFN-{alpha} measured relative to untreated HBV-expressing cells was determined by densitometry. Nucleocapsid formation and standard errors represent the averages of six independent experiments. Downregulation of core lysine mutant replication intermediates are not significantly different from that observed for the wild type (P > 0.05). WT, wild type.

Core K96R and K7R/K96R exhibit altered nucleocytoplasmic distribution. Core is found in both the nucleus and the cytoplasm of HBV replicating cells (13, 45). To determine whether lysine residues direct the intracellular localization of core, Huh7 cells transiently transfected with wild-type or lysine mutant expression constructs were fixed and stained for immunofluorescence microscopy. As previously described (28), the localization of wild-type core was both nuclear and cytoplasmic (Fig. 6A). The K7R mutant demonstrated a similar intracellular core distribution as HBV encoding wild-type core (Fig. 6B). In contrast, the K96R single mutant and the K7R/K96R double mutant demonstrated nuclear accumulation of core in a punctuate pattern indicating inclusion within nuclear bodies (Fig. 6C and D). Cells expressing the K96A mutant consistently exhibited an altered morphology and membrane blebbing, a finding consistent with cytotoxicity or cellular stress (data not shown). Random samples (1 cell per field for 20 fields) of wild-type-, K7R-, K96R-, or K7R/K96R-expressing cells were quantified for core localization in the nucleus and cytoplasm (Fig. 6M). Mutation of lysine 96 to arginine significantly altered the intracellular localization of core compared to the wild type, resulting in a predominant distribution of core in the nucleus. Furthermore, nuclear core in cells expressing the K96R single mutant or the K7R/K96R double mutant significantly exhibited an increased localization in nuclear bodies compared to the wild type (Fig. 6N).


Figure 6
View larger version (39K):
[in this window]
[in a new window]

  		FIG. 6. Core K96R and K7R/K96R demonstrate altered subcellular localization. At 2 days posttransfection, Huh7 cells expressing wild-type HBV (A), HBV core K7R mutant (B), HBV core K96R mutant (C), or HBV core K7R/K96R mutant (D) were fixed in 3% paraformaldehyde prior to permeabilization and staining. The core protein was detected using primary rabbit anti-core (Dako; 1:100) and secondary Alexa Fluor 488-labeled goat anti-rabbit (Invitrogen; 1:1,000) antibodies. (E to H) DAPI staining for nuclei of cells expressing wild type, K7R, K96R, or K7R/K96R, respectively. (I to L) Merged images. (M) Quantification of cytoplasmic core and nuclear core relative to total cellular core. (N) Quantification of core in nucleoplasm versus nuclear bodies relative to total core in the nucleus. Image analysis of core localization represents a sample of 20 cells imaged with a x40 objective lens and processed by using ImageJ software. *, P < 0.05; **, P < 0.005 (compared to the wild type). WT, wild type.

Core K96R colocalizes with the nucleolus but not with splicing factor SC-35 foci or PML bodies. Huh7 cells expressing wild type or the K96R mutant were stained with nuclear markers for immunofluorescence microscopy to identify the nuclear compartment(s) in which core localizes. The K96R mutant core did not colocalize with splicing factor SC-35 foci or PML bodies (Fig. 7) but predominantly colocalized with nucleolin in all of the cells expressing HBV (Fig. 8), indicating that core is in the nucleolus. Wild-type core expressed by HBV also did not colocalize with splicing factor SC-35 foci or PML bodies (Fig. 7) and was found to only occasionally localize in the nucleolus (Fig. 8). Quantification of the extent of colocalization with nucleolin demonstrated that the proportion of core in the nucleolus was enriched ~5-fold in the K96R mutant compared to the wild-type protein (Fig. 8I). Huh7 cells expressing a construct containing the wild type or K96R mutant within the HBV genome under an endogenous promoter demonstrated a similar core intracellular distribution, indicating that the accumulation of core in nuclear bodies was not a result of expression by the CMV promoter (data not shown). Therefore, lysine 96 directs the nucleocytoplasmic localization of core, and mutation of this residue alters the distribution of core between the nucleolus and nucleoplasm.


Figure 7
View larger version (61K):
[in this window]
[in a new window]

  		FIG. 7. Core K96R mutant does not colocalize with splicing factor SC-35 foci or PML bodies. Huh7 cells expressing wild-type HBV (A and B) or K96R (C and D) were fixed, permeabilized, and stained for core protein using primary rabbit anti-HBcAg. Colocalization of wild-type (E) or K96R (G) with PML bodies was determined by staining with primary mouse anti-PML antibody (Santa Cruz Biotechnology; 1:50). Colocalization of wild type (F) or K96R (H) with splicing factor SC-35 foci was determined by staining with primary mouse anti-SC-35 antibody (Sigma; 1:200). The secondary antibodies used were Alexa Fluor 488-labeled goat anti-rabbit antibodies and Alexa Fluor 568-labeled goat anti-mouse antibodies (Invitrogen; 1:1,000). (I to L) DAPI staining for nuclei of cells expressing wild type (I and J) or K96R (K and L). (M to P) Merged images. All images were acquired by using a x60 oil immersion objective lens. WT, wild type.


Figure 8
View larger version (25K):
[in this window]
[in a new window]

  		FIG. 8. Core K96R mutant accumulates in the nucleolus. Huh7 cells expressing wild-type (A) or K96R (E) HBV were fixed, permeabilized, and stained for core protein using primary rabbit anti-HBcAg. Colocalization of wild type (C) or K96R (G) with the nucleolus was determined by staining cells for nucleolin with primary mouse anti-nucleolin antibodies (Santa Cruz Biotechnology; 1:100). Secondary antibodies used were Alexa Fluor 488-labeled goat anti-rabbit antibodies and Alexa Fluor 568-labeled goat anti-mouse antibodies (Invitrogen; 1:1,000). (B and F) DAPI staining for nuclei of cells expressing wild type (B) or K96R (F). (D and H) Merged images of wild type (D) and K96R (H) with nucleolin and DAPI. (I) Quantification of core in nucleolus relative to total core in the nucleus. Image analysis of core localization represents a sample of 10 cells imaged with a x60 oil immersion objective and processed by using ImageJ software. WT, wild type.


arrow
DISCUSSION
 
In agreement with previous studies, mutation of lysine residue 96 to alanine in the core structural protein results in a significant virion release defect compared to HBV expressing wild-type core (32, 39). Furthermore, the K96A mutant is able to efficiently form intracellular nucleocapsids, indicating that the reduced efficiency in virion release is not due to inhibition of HBV replication. This has previously led to the suggestion that K96 may act as a potential site for ubiquitin conjugation, which would serve as a signal for virion envelopment and release from HBV replicating cells. However, the present study demonstrates that mutation of core lysine 96 to arginine did not decrease virion release compared to HBV expressing wild-type core. Mutation of lysine 7 to arginine also produced a virion release phenotype similar to that of wild-type HBV. Furthermore, efficient release by the double lysine mutant, K7R/K96R, demonstrates that not only are the individual lysines not necessary for release, but neither one alone is sufficient.

The previously reported release defect in the K96A mutant may be attributed to the substitution of a small neutral amino acid, alanine, for a relatively large basic amino acid, lysine, at a hydrophobic pocket formed at the interface of two core protein subunits that make a dimer (37, 53). The essential residues that form the hydrophobic pocket are P5, L95, K96, and I97 present in one subunit and residue L60 from the second subunit. Frequent natural mutations have been reported to occur at this pocket site within subtype ayw F97L and subtype adr I97L, resulting in the release of immature virions containing single-stranded DNA, and P5T, as well as L60V giving rise to low secretion of mature virions (25, 56, 57). In addition, this region of the core dimer when present in the context of the assembled capsid, demonstrates structural changes during double-stranded DNA synthesis, indicating that this pocket may mediate signals for mature double-stranded DNA-containing capsid envelopment and release from HBV-replicating cells (37). The possibility that the K96A mutation may unfavorably alter the capsid structure is supported by the observation that nucleocapsids formed by the K96A mutant migrated faster during native gel electrophoresis than nucleocapsids from cells expressing wild-type, K7R, K96R, or the K7R/K96R constructs. Imaging of cells expressing the K96A mutant by immunofluorescence microscopy also demonstrated an altered cellular morphology (i.e., membrane blebbing) indicative of cytotoxicity or stress, which could potentially be related to the defective release phenotype of that mutant (data not shown).

We attempted to detect ubiquitinated core in transfected cells expressing Flag-tagged core and His6-tagged ubiquitin or influenza virus hemagglutinin-tagged ubiquitin but were unsuccessful (unpublished results). Rost et al. and Puro et al. have made similar unsuccessful attempts utilizing epitope-tagged ubiquitin in the context of HBV replicating cells (33, 39). Of course, detection of ubiquitinated protein might be difficult if the modification occurs transiently or if only a small fraction of protein is modified at any one time. Nevertheless, the lack of evidence for core ubiquitination, along with the results from the present study, strongly suggests that lysine ubiquitination of core does not directly mediate virion release. However, HBV release has been implicated to be MVB dependent, as evidenced by the requirement of functional proteins from the ESCRT complex machinery such as VPS4A, VPS4B, AIP1/ALIX, and the CHMP proteins (24, 49). Furthermore, a functional Nedd4 ubiquitin ligase is required for virion release and interacts with core at a late domain-like PPAY motif (39). The MVB machinery and the Nedd4 ubiquitin ligase required for HBV capsid envelopment and release are the same cellular components necessitated by structural proteins of several RNA viruses that are dependent on the cellular ubiquitin pathway for release (1, 7). In support of the involvement of ubiquitin in virion release, inhibition of proteasome activity, which depletes free cellular ubiquitin available to the virus, significantly decreased HBV release from cells. The observation that proteasome inhibition repressed HBV release in a manner similar to that observed by ubiquitin-dependent release of several RNA viruses indicates that ubiquitin is required for HBV release but not through direct conjugation of ubiquitin to core lysine residues. It remains unclear whether direct ubiquitination of lysine residues encoded within viral structural proteins is generally required for virion release or if it is a bystander effect due to the recruitment of host proteins to the L domain (29, 42, 58). Recently, the Nedd4 ubiquitin ligase has been demonstrated to interact with the UIM of {gamma}2-adaptin in a ubiquitin-dependent manner (38). This observation raises the possibility that ubiquitin may instead indirectly interact with the core L domain through the Nedd4 ubiquitin ligase to facilitate recruitment of capsids to UIM-containing adapter proteins of the MVB machinery that are essential for mediating HBV release.

The ubiquitin-proteasome pathway has also been implicated to play a role in the downregulation of HBV replication during the IFN-induced antiviral response by acting as a downstream mediator of the IFN antiviral pathway (36). IFN-{alpha}/β and IFN-{gamma} treatment inhibits HBV replication by preventing assembly of cytoplasmic RNA-containing capsids through a posttranslational mechanism (50, 51). Robek et al. found that HBV DNA replication intermediates were insensitive to IFN treatment when HBV replicating cells were concurrently treated with inhibitors of proteasome activity. Sensitivity to IFN-{alpha}/β treatment was restored in HBV replicating cells with the subsequent removal of proteasome inhibitors. In the present study, hepatocytes expressing wild type or core lysine mutants were treated with IFN-{alpha} to determine whether nucleocapsids formed by core lysine mutants were insensitive to IFN. There was no significant difference in the IFN sensitivity of nucleocapsids formed by the core lysine mutants compared to the wild type, which suggests that conjugation of ubiquitin on core lysine residues are not required for the IFN-{alpha}/β-induced antiviral response.

The core lysine residues may also not be conjugated to other posttranslational modifiers that are induced by the IFN antiviral response, such as the ubiquitin-like protein ISG15. ISG15 and the ISG15-specific protease UBP43 are induced under conditions that correlate with the antiviral activity of IFN-{alpha}/β to HBV in transgenic mouse livers and immortalized transgenic hepatocytes (52). It is therefore possible that ISG15 may conjugate to core lysine residues to disrupt capsid formation during the IFN-mediated downregulation of HBV replication. However, our study indicates that this is unlikely due to the observation that capsids lacking the putative ISG15 acceptor sites at lysine 7 and 96 demonstrated a sensitivity to IFN-{alpha} similar to that of capsids formed by wild-type core. This result is consistent with the recent finding that whereas UBP43 influences the IFN-mediated antiviral response to HBV, this activity is independent of ISG15 (23). Interestingly, like IFN-{alpha}/β, tumor necrosis factor alpha also disrupts the formation of nucleocapsids by a posttranslational mechanism (2, 33). However, this process is also not mediated by ubiquitination, and the mechanism remains undefined (2, 33). It would be interesting to determine whether the antiviral response to HBV induced by IFN-{alpha} and tumor necrosis factor alpha converge to disrupt the formation of nucleocapsids by a similar mechanism.

Mature HBV DNA-containing capsids can either be released from the infected cell or recycle back to the nucleus to maintain covalently closed circular DNA production for continued viral replication (46). Although nuclear core has been described in hepatocytes derived from HBV transgenic mice, it remains unclear whether preformed capsids cross through the nuclear membrane during nuclear import or if nuclear capsids assemble only after transport of core subunits (12). Interestingly, the core protein containing the K96R mutation predominantly localized in the nuclear bodies of HBV-replicating cells. The nuclear bodies costained with nucleolin, which suggests that core K96R predominantly localizes at the nucleolus or that expression of K96R results in altered nuclear morphology, which directs localization of nucleolin to other nuclear bodies. The observation that core K96R does not localize to PML bodies or SC-35 foci suggests that organization of subnuclear compartments is not grossly altered during HBV replication and that core localization at the nucleolus compartment is specific. At closer examination, although HBV-expressing cells containing wild-type core did not have significant core in nuclear bodies, there were a few cells that did contain some nucleolar core. The localization of core in the nucleolus is independent of effects by the CMV promoter as evidenced by accumulation of nucleolar core in cells expressing HBV encoding either wild-type or K96R core under the endogenous HBV promoter (data not shown).

Similar to these results, Ning et al. reported that in HBV serotype adr, a small portion of core localizes to the nucleolus, and a core mutant containing a glutamate or tryptophan substitution for isoleucine at position 97 resulted in the accumulation of core in the nucleolus but not in splicing factor SC-35 foci (28). Extensive sequence analysis of core protein has revealed K7 and K96 to be highly conserved throughout the four HBV genotypes A, B, C, and D (6). The mutation of the adjacent ayw K96 residue results in a phenotype similar to that of the adr I97 mutants, which suggests that K96 may be situated in a motif that directs the nucleocytoplasmic trafficking of core and that this motif may be conserved throughout different HBV genotypes. Interestingly, the core K96 residue may be situated in a potential SUMOylation motif. SUMO is a ubiquitinlike protein that conjugates to lysine residues of target substrates to direct nucleocytoplasmic trafficking, as well as other nuclear activities such as gene transcription and cell cycle progression. Furthermore, SUMO is implicated to be involved in cellular processes that occur in the cytoplasm (11). The SUMO consensus motif is {Psi}KXE, where {Psi} is a large hydrophobic residue, X is any amino acid, and K acts as an acceptor site for SUMO conjugation. Although core does not contain an acidic residue downstream of K96 at position 98, it displays some similarities to a novel SUMOylation motif ({Psi}KKFR) found in the plasma membrane leak K+ channel K2P1 (34).

In summary, the present study indicates that while the cellular ubiquitin pathway may mediate HBV release, it is not through direct conjugation of ubiquitin on core K7 or K96. In addition, these lysine residues do not serve as sites for posttranslational modifications that decrease the stability of nucleocapsids during downregulation of HBV replication by the IFN-{alpha}/β-mediated antiviral effect. Although the core lysine residues do not regulate formation of nucleocapsid replication intermediates or virion release, K96 appears to be important for directing the trafficking of core between the nucleus and cytoplasm. Further studies on the K96 residue may provide some insight into the role nuclear core may play in facilitating covalently closed circular DNA replication for the maintenance of HBV replication in infected hepatocytes.


arrow
ACKNOWLEDGMENTS
 
This study was supported by Research Scholar Development Award K22 AI64757 (M.D.R.) from the National Institutes of Health. M.L.G. is supported by a Ford Foundation predoctoral fellowship. R.B. was supported by a Yale STARS II fellowship.

We thank Frank Chisari (The Scripps Research Institute) for providing the HBV-Met and TRE-HBV cells and Stefan Wieland for critical reading of the manuscript.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pathology, Yale University School of Medicine, P.O. Box 208023, 310 Cedar St., LH315A, New Haven, CT 06520-8023. Phone: (203) 785-6174. Fax: (203) 785-6127. E-mail: michael.robek{at}yale.edu Back

{triangledown} Published ahead of print on 25 February 2009. Back


arrow
REFERENCES
 
    1
  1. Bieniasz, P. D. 2006. Late budding domains and host proteins in enveloped virus release. Virology 344:55-63.[CrossRef][Medline]
    2. 2
  2. Biermer, M., R. Puro, and R. J. Schneider. 2003. Tumor necrosis factor alpha inhibition of hepatitis B virus replication involves disruption of capsid integrity through activation of NF-{kappa}B. J. Virol. 77:4033-4042.[Abstract/Free Full Text]
    3. 3
  3. Bouamr, F., J. A. Melillo, M. Q. Wang, K. Nagashima, M. de Los Santos, A. Rein, and S. P. Goff. 2003. PPPYVEPTAP motif is the late domain of human T-cell leukemia virus type 1 Gag and mediates its functional interaction with cellular proteins Nedd4 and Tsg101. J. Virol. 77:11882-11895.[Abstract/Free Full Text]
    4. 4
  4. Bruss, V. 2004. Envelopment of the hepatitis B virus nucleocapsid. Virus Res. 106:199-209.[CrossRef][Medline]
    5. 5
  5. Bruss, V., and D. Ganem. 1991. The role of envelope proteins in hepatitis B virus assembly. Proc. Natl. Acad. Sci. USA 88:1059-1063.[Abstract/Free Full Text]
    6. 6
  6. Chain, B. M., and R. Myers. 2005. Variability and conservation in hepatitis B virus core protein. BMC Microbiol. 5:33.[CrossRef][Medline]
    7. 7
  7. Demirov, D. G., and E. O. Freed. 2004. Retrovirus budding. Virus Res. 106:87-102.[CrossRef][Medline]
    8. 8
  8. Demirov, D. G., A. Ono, J. M. Orenstein, and E. O. Freed. 2002. Overexpression of the N-terminal domain of TSG101 inhibits HIV-1 budding by blocking late domain function. Proc. Natl. Acad. Sci. USA 99:955-960.[Abstract/Free Full Text]
    9. 9
  9. Ganem, D., and A. M. Prince. 2004. Hepatitis B virus infection: natural history and clinical consequences. N. Engl. J. Med. 350:1118-1129.[Free Full Text]
    10. 10
  10. Garrus, J. E., U. K. von Schwedler, O. W. Pornillos, S. G. Morham, K. H. Zavitz, H. E. Wang, D. A. Wettstein, K. M. Stray, M. Cote, R. L. Rich, D. G. Myszka, and W. I. Sundquist. 2001. Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding. Cell 107:55-65.[CrossRef][Medline]
    11. 11
  11. Geiss-Friedlander, R., and F. Melchior. 2007. Concepts in sumoylation: a decade on. Nat. Rev. Mol. Cell. Biol. 8:947-956.[CrossRef][Medline]
    12. 12
  12. Guidotti, L. G., V. Martinez, Y. T. Loh, C. E. Rogler, and F. V. Chisari. 1994. Hepatitis B virus nucleocapsid particles do not cross the hepatocyte nuclear membrane in transgenic mice. J. Virol. 68:5469-5475.[Abstract/Free Full Text]
    13. 13
  13. Guidotti, L. G., B. Matzke, H. Schaller, and F. V. Chisari. 1995. High-level hepatitis B virus replication in transgenic mice. J. Virol. 69:6158-6169.[Abstract]
    14. 14
  14. Hartmann-Stuhler, C., and R. Prange. 2001. Hepatitis B virus large envelope protein interacts with {gamma}2-adaptin, a clathrin adaptor-related protein. J. Virol. 75:5343-5351.[Abstract/Free Full Text]
    15. 15
  15. Harty, R. N., M. E. Brown, J. P. McGettigan, G. Wang, H. R. Jayakar, J. M. Huibregtse, M. A. Whitt, and M. J. Schnell. 2001. Rhabdoviruses and the cellular ubiquitin-proteasome system: a budding interaction. J. Virol. 75:10623-10629.[Abstract/Free Full Text]
    16. 16
  16. Harty, R. N., M. E. Brown, G. Wang, J. Huibregtse, and F. P. Hayes. 2000. A PPxY motif within the VP40 protein of Ebola virus interacts physically and functionally with a ubiquitin ligase: implications for filovirus budding. Proc. Natl. Acad. Sci. USA 97:13871-13876.[Abstract/Free Full Text]
    17. 17
  17. Harty, R. N., J. Paragas, M. Sudol, and P. Palese. 1999. A proline-rich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with WW domains of cellular proteins: implications for viral budding. J. Virol. 73:2921-2929.[Abstract/Free Full Text]
    18. 18
  18. Joshi, A., U. Munshi, S. D. Ablan, K. Nagashima, and E. O. Freed. 2008. Functional replacement of a retroviral late domain by ubiquitin fusion. Traffic 9:1972-1983.[CrossRef][Medline]
    19. 19
  19. Katzmann, D. J., M. Babst, and S. D. Emr. 2001. Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 106:145-155.[CrossRef][Medline]
    20. 20
  20. Katzmann, D. J., G. Odorizzi, and S. D. Emr. 2002. Receptor downregulation and multivesicular-body sorting. Nat. Rev. Mol. Cell. Biol. 3:893-905.[CrossRef][Medline]
    21. 21
  21. Kian Chua, P., M. H. Lin, and C. Shih. 2006. Potent inhibition of human hepatitis B virus replication by a host factor Vps4. Virology 354:1-6.[CrossRef][Medline]
    22. 22
  22. Kikonyogo, A., F. Bouamr, M. L. Vana, Y. Xiang, A. Aiyar, C. Carter, and J. Leis. 2001. Proteins related to the Nedd4 family of ubiquitin protein ligases interact with the L domain of Rous sarcoma virus and are required for gag budding from cells. Proc. Natl. Acad. Sci. USA 98:11199-11204.[Abstract/Free Full Text]
    23. 23
  23. Kim, J. H., J. K. Luo, and D. E. Zhang. 2008. The level of hepatitis B virus replication is not affected by protein ISG15 modification but is reduced by inhibition of UBP43 (USP18) expression. J. Immunol. 181:6467-6472.[Abstract/Free Full Text]
    24. 24
  24. Lambert, C., T. Doring, and R. Prange. 2007. Hepatitis B virus maturation is sensitive to functional inhibition of ESCRT-III, Vps4, and {gamma}2-adaptin. J. Virol. 81:9050-9060.[Abstract/Free Full Text]
    25. 25
  25. Le Pogam, S., T. T. Yuan, G. K. Sahu, S. Chatterjee, and C. Shih. 2000. Low-level secretion of human hepatitis B virus virions caused by two independent, naturally occurring mutations (P5T and L60V) in the capsid protein. J. Virol. 74:9099-9105.[Abstract/Free Full Text]
    26. 26
  26. Martin-Serrano, J., A. Yarovoy, D. Perez-Caballero, and P. D. Bieniasz. 2003. Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins. Proc. Natl. Acad. Sci. USA 100:12414-12419.[Abstract/Free Full Text]
    27. 27
  27. Martin-Serrano, J., T. Zang, and P. D. Bieniasz. 2001. HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Nat. Med. 7:1313-1319.[CrossRef][Medline]
    28. 28
  28. Ning, B., and C. Shih. 2004. Nucleolar localization of human hepatitis B virus capsid protein. J. Virol. 78:13653-13668.[Abstract/Free Full Text]
    29. 29
  29. Ott, D. E., L. V. Coren, E. N. Chertova, T. D. Gagliardi, and U. Schubert. 2000. Ubiquitination of HIV-1 and MuLV Gag. Virology 278:111-121.[CrossRef][Medline]
    30. 30
  30. Pasquetto, V., S. F. Wieland, S. L. Uprichard, M. Tripodi, and F. V. Chisari. 2002. Cytokine-sensitive replication of hepatitis B virus in immortalized mouse hepatocyte cultures. J. Virol. 76:5646-5653.[Abstract/Free Full Text]
    31. 31
  31. Patnaik, A., V. Chau, and J. W. Wills. 2000. Ubiquitin is part of the retrovirus budding machinery. Proc. Natl. Acad. Sci. USA 97:13069-13074.[Abstract/Free Full Text]
    32. 32
  32. Ponsel, D., and V. Bruss. 2003. Mapping of amino acid side chains on the surface of hepatitis B virus capsids required for envelopment and virion formation. J. Virol. 77:416-422.[CrossRef][Medline]
    33. 33
  33. Puro, R., and R. J. Schneider. 2007. Tumor necrosis factor activates a conserved innate antiviral response to hepatitis B virus that destabilizes nucleocapsids and reduces nuclear viral DNA. J. Virol. 81:7351-7362.[Abstract/Free Full Text]
    34. 34
  34. Rajan, S., L. D. Plant, M. L. Rabin, M. H. Butler, and S. A. Goldstein. 2005. Sumoylation silences the plasma membrane leak K+ channel K2P1. Cell 121:37-47.[CrossRef][Medline]
    35. 35
  35. Robek, M. D., M. L. Garcia, B. S. Boyd, and F. V. Chisari. 2007. Role of immunoproteasome catalytic subunits in the immune response to hepatitis B virus. J. Virol. 81:483-491.[Abstract/Free Full Text]
    36. 36
  36. Robek, M. D., S. F. Wieland, and F. V. Chisari. 2002. Inhibition of hepatitis B virus replication by interferon requires proteasome activity. J. Virol. 76:3570-3574.[Abstract/Free Full Text]
    37. 37
  37. Roseman, A. M., J. A. Berriman, S. A. Wynne, P. J. Butler, and R. A. Crowther. 2005. A structural model for maturation of the hepatitis B virus core. Proc. Natl. Acad. Sci. USA 102:15821-15826.[Abstract/Free Full Text]
    38. 38
  38. Rost, M., T. Doring, and R. Prange. 2008. {gamma}2-Adaptin, a ubiquitin-interacting adaptor, is a substrate to coupled ubiquitination by the ubiquitin ligase Nedd4 and functions in the endosomal pathway. J. Biol. Chem. 283:32119-32130.[Abstract/Free Full Text]
    39. 39
  39. Rost, M., S. Mann, C. Lambert, T. Doring, N. Thome, and R. Prange. 2006. Gamma-adaptin, a novel ubiquitin-interacting adaptor, and Nedd4 ubiquitin ligase control hepatitis B virus maturation. J. Biol. Chem. 281:29297-29308.[Abstract/Free Full Text]
    40. 40
  40. Schubert, U., D. E. Ott, E. N. Chertova, R. Welker, U. Tessmer, M. F. Princiotta, J. R. Bennink, H. G. Krausslich, and J. W. Yewdell. 2000. Proteasome inhibition interferes with gag polyprotein processing, release, and maturation of HIV-1 and HIV-2. Proc. Natl. Acad. Sci. USA 97:13057-13062.[Abstract/Free Full Text]
    41. 41
  41. Seeger, C., and W. S. Mason. 2000. Hepatitis B virus biology. Microbiol. Mol. Biol. Rev. 64:51-68.[Abstract/Free Full Text]
    42. 42
  42. Spidel, J. L., R. C. Craven, C. B. Wilson, A. Patnaik, H. Wang, L. M. Mansky, and J. W. Wills. 2004. Lysines close to the Rous sarcoma virus late domain critical for budding. J. Virol. 78:10606-10616.[Abstract/Free Full Text]
    43. 43
  43. Strack, B., A. Calistri, M. A. Accola, G. Palu, and H. G. Gottlinger. 2000. A role for ubiquitin ligase recruitment in retrovirus release. Proc. Natl. Acad. Sci. USA 97:13063-13068.[Abstract/Free Full Text]
    44. 44
  44. Strack, B., A. Calistri, S. Craig, E. Popova, and H. G. Gottlinger. 2003. AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell 114:689-699.[CrossRef][Medline]
    45. 45
  45. Sureau, C., J. L. Romet-Lemonne, J. I. Mullins, and M. Essex. 1986. Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. Cell 47:37-47.[CrossRef][Medline]
    46. 46
  46. Tuttleman, J. S., C. Pourcel, and J. Summers. 1986. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 47:451-460.[CrossRef][Medline]
    47. 47
  47. VerPlank, L., F. Bouamr, T. J. LaGrassa, B. Agresta, A. Kikonyogo, J. Leis, and C. A. Carter. 2001. Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55Gag. Proc. Natl. Acad. Sci. USA 98:7724-7729.[Abstract/Free Full Text]
    48. 48
  48. von Schwedler, U. K., M. Stuchell, B. Muller, D. M. Ward, H. Y. Chung, E. Morita, H. E. Wang, T. Davis, G. P. He, D. M. Cimbora, A. Scott, H. G. Krausslich, J. Kaplan, S. G. Morham, and W. I. Sundquist. 2003. The protein network of HIV budding. Cell 114:701-713.[CrossRef][Medline]
    49. 49
  49. Watanabe, T., E. M. Sorensen, A. Naito, M. Schott, S. Kim, and P. Ahlquist. 2007. Involvement of host cellular multivesicular body functions in hepatitis B virus budding. Proc. Natl. Acad. Sci. USA 104:10205-10210.[Abstract/Free Full Text]
    50. 50
  50. Wieland, S. F., A. Eustaquio, C. Whitten-Bauer, B. Boyd, and F. V. Chisari. 2005. Interferon prevents formation of replication-competent hepatitis B virus RNA-containing nucleocapsids. Proc. Natl. Acad. Sci. USA 102:9913-9917.[Abstract/Free Full Text]
    51. 51
  51. Wieland, S. F., L. G. Guidotti, and F. V. Chisari. 2000. Intrahepatic induction of alpha/beta interferon eliminates viral RNA-containing capsids in hepatitis B virus transgenic mice. J. Virol. 74:4165-4173.[Abstract/Free Full Text]
    52. 52
  52. Wieland, S. F., R. G. Vega, R. Muller, C. F. Evans, B. Hilbush, L. G. Guidotti, J. G. Sutcliffe, P. G. Schultz, and F. V. Chisari. 2003. Searching for interferon-induced genes that inhibit hepatitis B virus replication in transgenic mouse hepatocytes. J. Virol. 77:1227-1236.[CrossRef][Medline]
    53. 53
  53. Wynne, S. A., R. A. Crowther, and A. G. Leslie. 1999. The crystal structure of the human hepatitis B virus capsid. Mol. Cell 3:771-780.[CrossRef][Medline]
    54. 54
  54. Yang, P. L., A. Althage, J. Chung, and F. V. Chisari. 2002. Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection. Proc. Natl. Acad. Sci. USA 99:13825-13830.[Abstract/Free Full Text]
    55. 55
  55. Yasuda, J., E. Hunter, M. Nakao, and H. Shida. 2002. Functional involvement of a novel Nedd4-like ubiquitin ligase on retrovirus budding. EMBO Rep. 3:636-640.[CrossRef][Medline]
    56. 56
  56. Yuan, T. T., G. K. Sahu, W. E. Whitehead, R. Greenberg, and C. Shih. 1999. The mechanism of an immature secretion phenotype of a highly frequent naturally occurring missense mutation at codon 97 of human hepatitis B virus core antigen. J. Virol. 73:5731-5740.[Abstract/Free Full Text]
    57. 57
  57. Yuan, T. T., P. Tai, and C. Shih. 1999. Subtype-independent immature secretion and subtype-dependent replication deficiency of a highly frequent, naturally occurring mutation of human hepatitis B virus core antigen. J. Virol. 73:10122-10128.[Abstract/Free Full Text]
    58. 58
  58. Zhadina, M., M. O. McClure, M. C. Johnson, and P. D. Bieniasz. 2007. Ubiquitin-dependent virus particle budding without viral protein ubiquitination. Proc. Natl. Acad. Sci. USA 104:20031-20036.[Abstract/Free Full Text]

Journal of Virology, May 2009, p. 4923-4933, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02644-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Pairan, A., Bruss, V. (2009). Functional Surfaces of the Hepatitis B Virus Capsid. J. Virol. 83: 11616-11623 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Garcia, M. L.
Right arrow Articles by Robek, M. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Garcia, M. L.
Right arrow Articles by Robek, M. D.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»