JVI Accepts, published online ahead of print on 29 September 2010 J. Virol. doi:10.1128/JVI.01499-10 Copyright 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Impairment of hepatitis B virus virion secretion by single amino acid substitutions in the small envelope protein and rescue by a novel glycosylation site
Running title: S gene mutations modulate HBV secretion
Kiyoaki Ito,1, 4 Yanli Qin,1, 5# Michael Guarnieri,1, 6# Tamako Garcia,1, 7 Karen Kwei1, 8, Masashi Mizokami,2 Jiming Zhang,3 Jisu Li,1 Jack R. Wands,1 and Shuping Tong1*
The Liver Research Center, Rhode Island Hospital, Warren Alpert School of Medicine, Brown University, Providence, RI 1; Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan 2; and Department of Infectious Diseases, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China 3. *Corresponding author. Mailing address: Liver Research Center, 55 Claverick Street, Providence, RI 02906. Phone: (401) 444-7365. Fax: (401) 444-2939. E-mail:
Shuping_Tong_MD@Brown.edu.
#These authors contributed equally to this work. Current addresses: Research Center for Hepatitis and Immunology, National Center for Global Health and Medicine, Ichikawa, Japan 4; Department of Infectious Diseases, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China 5; University of Colorado at Denver, CO 6; Uniformed Services University of the Health Science, DC 7; Boston University School of Medicine, MA 8. Word count: Abstract: 244; Text: 4890.

Abstract

Mutations in the S region of hepatitis B virus (HBV) envelope gene are associated with immune escape, occult infection, and resistance to therapy. We previously identified naturally occurring mutations in the S gene that alter HBV virion secretion. Here we used transcomplementation assay to confirm that the I110M, G119E, and R169P mutations in the S domain of viral envelope proteins impair virion secretion, and that an M133T mutation rescues virion secretion of the I110M and G119E mutants. The G119E mutation impaired detection of secreted hepatitis B surface antigen (HBsAg), suggesting immune escape. The R169P mutant is defective in HBsAg secretion as well, and has dominant negative effect when coexpressed with wild-type envelope proteins. Although the S domain is present in all the three envelope proteins, the I110M, G119E, and R169P mutations impair virion secretion through the small envelope protein. Conversely, coexpression of just the small envelope protein of the M133T mutant could rescue virion secretion. The M133T mutation could also overcome secretion defect caused by the G145R immune escape mutation or mutation at N146, the site of N-linked glycosylation. In fact M133T creates a novel N-linked glycosylation site (131NST133). Destroying this site by N131Q/T mutation or preventing glycosylation by tunicamycin treatment of transfected cells abrogated effect of the M133T mutation. Our findings demonstrate that N-linked glycosylation of HBV envelope proteins is critical for virion secretion, and that secretion defect caused by mutations in the S protein can be rescued by an extra glycosylation site.

Introduction

The hepatitis B virus (HBV) is an enveloped DNA virus with a tropism for the liver. The 3.2-kb HBV genome harbors genes encoding core protein and its secreted version called HBeAg, DNA polymerase, the transcriptional transactivator HBx, and envelope proteins. The envelope gene, which is completely overlapped by the polymerase gene, has three in-frame AUG codons that can serve as alternative translation initiation sites. This leads to the expression of three coterminal envelope proteins: large (L), middle (M), and small (S). The sequence unique to the L protein is called the preS1 domain, while a downstream sequence shared with the M protein is called the preS2 domain. The S domain is present in all the three envelope proteins. The S and M proteins are translated from a 2.1-kb subgenomic RNA with a heterogeneous 5 end, while the L protein is expressed from a longer (2.4-kb) subgenomic RNA. The S protein is the most abundantly expressed envelope protein. The L and S proteins exist in nonglycosylated and monoglycosylated forms (L: p39 and gp42; S: p24 and gp27) due to a facultative N-linked glycosylation site (N-X-S/T) at N146 of the S domain. The M protein contains an extra, constitutive N-linked glycosylation site at position 4 in the preS2 domain and consequently exists in monoglycosylated (gp33) and diglycosylated (gp36) forms.

resistance. Nucleos(t)ide analogues currently licensed to treat chronic HBV infection target DNA polymerase, and prolonged therapy is associated with the selection of amino acid changes in the DNA polymerase that confer drug resistance (22, 42). Due to the overlap between the polymerase and envelope genes, drug resistant mutants can be accompanied by amino acid changes in the envelope proteins as well. So far, only a small fraction of mutations associated with immune escape or drug resistance has been functionally characterized. Notably the classic immune escape mutant G145R was reported to be severely impaired in virion secretion (17, 18).
We previously performed transfection experiments on twenty five full-length genotype A HBV genomes isolated from HBeAg positive French patients and identified clone 4B as highly efficient in virion secretion, clone 4C from the same patient as defective in virion secretion, and clone 3.4 from another patient as having poor virion secretion capacity (27). Through the analysis of chimeric constructs and site-directed mutants, we identified mutations in the envelope gene as responsible for impaired virion secretion. As summarized in Fig. 1A, three missense mutations in the S domain of viral envelope proteins could abolish or drastically reduce virion secretion: I110M (clones 4B and 4C), G119E (clone 3.4), and R169P (clone 4C). In contrast, an M133T mutation (clone 4B) could enhance virion secretion when present alone and rescue virion secretion when combined with the I110M or G119E mutation. These mutations are located in the immunodominant loops except for R169P, which is located in a transmembrane segment (Fig. 1B). In the present study, we explored the molecular mechanisms whereby the missense mutations in the envelope gene modulate virion secretion. Our results highlight the importance of N-linked glycosylation of envelope proteins for HBV virion secretion.

Materials and Methods

The 1.5mer and 0.7mer HBV constructs and site-directed mutants. The 1.5mer HBV construct has a 4.8-kb EcoRV ApaI fragment (nucleotides / 1 2600) of the HBV genome cloned to the pBluescript vector. As already described (7), the envelope-null mutant (LM-S-) has the S gene start codon converted to GCG to prevent S protein expression, and an additional G261A nonsense mutation at codon 36 of the S gene to prevent expression of fulllength L and M proteins (Fig. 1C). The 0.7mer construct has a 2.3-kb fragment covering nucleotides 2721- 3221/1-1770 cloned to the pBluescript vector (7). It could express all the three envelope proteins (L+M+S+) but not any other HBV proteins. Converting S gene start codon into GCG rendered the 0.7mer construct unable to express S protein (L+M+S-), while converting the 43rd codon of the preS2 region into a TAG stop codon truncated both L and M proteins (L-M-S+) (Fig. 1C). Missense mutations were introduced into the 0.7mer construct by overlap-extension PCR (20, 35). Plasmid DNA of the mutant constructs was purified by the high-speed plasmid midi kit (Qiagen).

Transient transfection and analysis of DNA replication and virion secretion. The Huh7 human hepatoma cells were seeded in six-well plates and transfected with LT1 (Mirus) as described (7, 15, 35). Each well of six-well plates received 2g HBV DNA and 5ng of cDNA encoding secreted alkaline phosphatase (SEAP) to serve as a control for transfection efficiency. Cells and culture supernatant were harvested at day 5 post-transfection. To abolish protein glycosylation, medium was changed two days post-transfection, with one well each supplemented with 20 l of DMSO (1% final concentration) or tunicamycin dissolved in DMSO
(final concentration 50 g/ml tunicamycin; 1% DMSO). Cells and culture supernatant were harvested at day 5 post-transfection. Cell lysis, core particle precipitation, and detection of HBV DNA replication by Southern blot analysis have been described (7, 20, 35). To concentrate virions from culture supernatant, antibodies were conjugated to protein G agarose beads (Roche) at a ratio of 2.8 l of horse anti-HBs (Ad/Ay, Abcam or Novus), 3 l of mouse anti-preS2 antibody (Virogen), and 10 l bed volume of beads. Next, 10 l of conjugated beads was incubated at 4oC for two days with 1.4 ml of precleared culture supernatant. The precipitate was digested with nucleases prior to virion DNA extraction and Southern blot analysis (7, 35).
Western blot analysis of cell-associated and secreted envelope proteins. Proteins from cell lysate were separated by SDS-polyacrylamide gel and transferred to membrane. Viral envelope proteins were detected by a horse or rabbit polyclonal anti-HBs antibody (Novus) as described (7). The blots were stripped and probed again for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to serve as a loading control (7). For in vitro deglycosylation of envelope proteins, 20 l of cell lysate was boiled with 2 l of denaturing buffer (5% SDS and 0.4M DTT) at 100oC. The samples were digested at 37oC with 40 U of endo H (New England Biolabs) prior to Western blot analysis. Secreted HBsAg was concentrated from 1.5ml of culture supernatant by ultracentrifugation at 46,000 rpm for 16 hrs using the AH65 rotor (Sorvall). The pellet was resuspended in protein loading buffer followed by Western blot analysis.
Measurement of SEAP and secreted HBsAg. SEAP was measured from 5 l of culture supernatant using the Great EscAPe SEAP reporter system (Clontech). HBsAg was detected from 4 l of precleared culture supernatant using the Auszyme monoclonal HBsAg Kit (Abbott

Laboratories), or 10-15 l of culture supernatant using Hepatitis B surface antigen ELISA Kit (Abazyme) when the Abbott kit discontinued.

Results

Rationale. The goal of the present study was to establish the molecular mechanisms whereby mutations in the envelope gene impair or restore virion secretion. The following specific questions were asked. First, do amino acid changes in envelope proteins or DNA polymerase, or nucleotide changes in the pregenomic RNA, alter virion secretion? In this regard the I110M, G119E, M133T, and R169P substitutions in the envelope proteins are caused by the T484G, G510A, T552C, and G660C point mutations in the viral genome (Fig. 1A), with the T484G mutation further inducing an S466A substitution in the DNA polymerase. Second, if mutations in the envelope proteins impair virion secretion, is the mutant L, M, or S protein responsible? Third, considering that the S protein is the morphogenic factor for the assembly of both virions and subviral particles, is the phenotype of virion secretion linked to the phenotype of HBsAg secretion? Finally, what is the mechanism whereby the M133T mutation rescues virion secretion of the I110M and G119E mutants? To answer these questions, we used a 1.5mer replication construct with wild-type sequence at nucleotide positions 484, 510, 552, and 660 as a source of genome replication. Its S gene AUG was mutated to GCG to prevent S protein expression; an additional nonsense mutation at the 5 end of the S gene prevented expression of full-length L and M proteins. By co-transfecting this envelope-null replication construct with a 0.7mer expression construct for the envelope proteins (wild-type or mutant) (Fig. 1C), we could
test the effect of mutant envelope proteins on virion secretion without complication of mutant pregenomic RNA or DNA polymerase. Furthermore, by employing two 0.7mer constructs for envelope protein expression, one for the L and M proteins and the other for the S protein, we could establish whether mutations in the L/M proteins or the S protein alter virion secretion.
The I110M, G119E, and R169P mutations in the envelope gene are sufficient to impair virion secretion. The human hepatoma cell line Huh7 grown in 6-well plates was cotransfected with 1.5 g of the 1.5mer replication construct and 0.5 g of 0.7mer expression construct for the wild-type (WT) envelope proteins or the I110M, G119E, or R169P mutant. Cells were harvested at day 5 post-transfection and core particles were extracted for Southern blot analysis of replicative HBV DNA. Similar levels of HBV DNA were detected although the R169P mutant displayed little mature (double stranded) viral DNA (Fig. 2A, lane 10). Considering that naked core particles are also released from transiently transfected Huh7 cells (35), we selectively immunoprecipitated virions from culture supernatant using a combination of monoclonal antibody against the pre-S2 domain and a polyclonal anti-HBs antibody. This approach also minimizes inefficient precipitation of the immune escape mutants such as G119E (see below). The results shown in Fig. 2B indicate that virion secretion is blocked by the I110M and R169P mutations and severely impaired by the G119E substitution in viral envelope proteins, despite the absence of T484G, G660C, and G510A mutations in the pregenomic RNA and S466A substitution in DNA polymerase.

The M133T mutation also rescues virion secretion of the G145R and R169H mutants and creates a novel N-linked glycosylation site. Introduction of the M133T mutation fully rescued virion secretion of the I110M mutant (Fig. 5B, lane 6), and partially rescued the
G119E mutant (Fig. 6B, lane 8). Although it failed to rescue the R169P mutant, it greatly improved virion secretion of an R169H mutant of (Fig. 6B, lanes 11 and 13). Furthermore, M133T could efficiently rescue virion secretion of the classic immune escape mutant, G145R (Fig. 5B, lane 17). Western blot analysis revealed that presence of the M133T mutation is associated with additional envelope proteins of higher molecular weights (Figs. 4G, 5C, 6C). In this regard, M133T is predicted to create a novel N-linked glycosylation site at N131 (131NST133), which should produce additional forms of S (gp30), M (gp39) and L (gp45) proteins. Indeed, endo H treatment of cell lysate simplified envelope proteins of all the constructs into p24, p30, and p39 corresponding to nonglycosylated S, M, and L proteins (Fig. 6F).
The M133T mutation works through the novel N-linked glycosylation site. A series of experiments were performed to ascertain whether the novel N-linked glycosylation site (N131), or the M133T mutation per se, improves the efficiency of virion secretion. We found that the M133I and M133Q mutations failed to significantly improve virion secretion of the I110M mutant (Fig. 5B, lanes 4 & 5) or the G119E mutant (Fig. 6B, lane 6). In contrast, an M133S mutation, which generates the same glycosylation site as M133T, partially rescued the G119E mutant (Fig. 6B&C, lane 7). Similarly, an artificial G112N mutation creating another novel N-linked glycosylation site (112NST114) could partially rescue the G119E mutant (Fig. 6B & C, lane 5), although not the I110M or G145R mutant (Fig. 5B, lanes 3, 19). Importantly, ability of the M133T mutation to rescue virion secretion of the I110M, G119E, G145R, and R169H mutations could be abrogated by an additional N131T or N131Q mutation (Figs. 5B, lanes 7, 8, 18; Fig. 6B, lanes 9, 14). As expected, none of the triple mutants expressed extra band of L (gp45), M (gp39), and S (gp30) proteins (Figs. 5C & 6C).
An alternative approach to prevent the M133T mediated glycosylation at N131 is to treat transfected cells with tunicamycin, which will also block N-linked glycosylation of N146 (for all the constructs) and N112 (for the G112N mutant). Cells were treated with 50 g/ml of tunicamycin two days post-transfection when cells were confluent, because tunicamycin added at earlier time points or higher concentration was associated with significant cell toxicity. Since tunicamycin was dissolved in DMSO solution, cells treated with same volume of DMSO (1% final concentration) served as a control. DMSO is known to maintain the differentiation status of primary duck hepatocytes and trigger differentiation of HepaRG cells leading to susceptibility to HBV infection (10, 31). DMSO treatment increased signals of virion DNA for all the secretion competent constructs, including wild type, M133T, I110M/M133T, M133T/G145R,

necessary although insufficient for efficient virion secretion of the I110M, G119E, and R169H mutants.

Discussion

Among the three envelope proteins, only L and S are essential for HBV virion formation / secretion (2, 6). Since the S but not L protein is also required for the secretion of subviral particles (HBsAg), it is the critical factor for particle assembly. The L protein suppresses the secretion of subviral particles in a dose-dependent manner (26, 29), thus channeling a fraction of the S protein towards virion formation. Moreover, the L protein directly interacts with core particles via its preS domains. We previously characterized several patient-derived HBV clones and mapped the determinant for virion secretion to the S region of envelope gene (20, 27). In the present study, we used transcomplementation assay to confirm that the amino acid changes in the S domain: I110M, G119E, M133T, and R169P, modulate the efficiency of virion secretion. Triple transfection experiments established the mutant S protein, rather than L or M protein, as the determinant for altered virion secretion. Consistent with our findings, mutant S protein was found by other investigators to cause virion secretion defect of the G145R immune escape mutant and W172* mutant (conversion of a tryptophan codon into a stop codon) associated with adefovir resistance (17, 36). These findings, together with the HBsAg secretion defect of the R169P mutant, reinforce the S protein as the driving force in particle formation and secretion.
The exact mechanisms whereby the I110M, G119E, and R169P mutations impair virion
secretion remain to be worked out. Residues 110 and G119 are located inside ER lumen during virion morphogenesis, while residue 169 is membrane associated (Fig. 1B). Since none of these residues are accessible for interaction with the core particle located in cytosol, they probably affect a later step of S protein cross-linking essential for particle assembly. Indeed the R169P, R169G, and R169L mutations blocked secretion of both virions and subviral particles. The continued HBsAg secretion for the other mutants with virion secretion defect (I110M, G119E, G119V, and R169Q) may reflect less dramatic structural changes and a better tolerance of subviral particles, which use a different route for secretion (37). The I110M, G145R, R169P, R169G, and R169L mutants all displayed reduced levels of L protein. Thus, reduced L/S protein ratios may partly contribute to impaired virion secretion of these mutants. From the same figures, it is also evident that the G145R and R169L mutants had a lower ratio of gp27/p24, suggesting less efficient S protein glycosylation.

HBsAg secreted by the G119E mutant was detected poorly by the ELISA assay. Therefore, G119E represents an immune escape mutant. In this regard, G119 is juxtaposed to a cysteine residue (C121). The charged glutamic acid probably interferes with the maintenance of proper intramolecular disulfide bond between C121 and another cysteine residue, thus altering the conformation of the determinant. Indeed, a G119R mutation has been associated with occult HBV infection (41).
Several R169 mutants share similarities with the W172* mutant, which has the Cterminal 55 residues of the S domain deleted (36). These include a block in the secretion of both viral and subviral particles (R169P/G/L), reduced intracellular level of L protein (R169P/G/L)
and the glycosylated form of S protein (R169L), reduced intracellular level of the mature (double stranded) genome (R169P/G/L), and dominant negative effect on virion secretion (R169P/G). We hypothesize that core particles with mature genome could be enveloped for R169P/G/L mutants, but such particles are degraded intracellularly, thus depleting both L protein and mature genome. Why the lack of S protein secretion in the case of either R169P/G/L or W172* mutant (36) fail to cause intracellular retention remains to be elucidated. The W172* mutant can transform NIH3T3 cells and has been found in patients with hepatocellular carcinoma (21). It will be interesting to determine whether the R169 mutants, which share many properties with W172* but probably does not have the transactivation function of truncated envelope proteins, can promote malignant transformation.
The M133T mutants showed reduced levels of both intracellular S protein (based on Western blot analysis) and secreted HBsAg (based on ELISA). This is most likely due to interference of extra polysaccharides on antibody binding, because this phenotype was reversed by either an N131 mutation to destroy the new glycosylation site or tunicamycin treatment of transfected cells to prevent glycosylation at this site. Similar phenotype has been reported for other mutations that create novel N-linked glycosylation site (40). The M133T mutation efficiently rescues virion secretion of the G145R mutant without altering its immune escape phenotype (Fig. 5E), thus arguing against a mechanism through restoration of S protein folding or intra-molecular interactions mediated by disulfide bonds. Consistent with this finding, the M133T mutation could rescue virion secretion of the I110M mutant when present on a different molecule of the S protein. Probably the M133T mutation restores correct intermolecular interactions mediated by disulfide bonds (24). Increased virion secretion as a result of the

M133T mutation was correlated with moderate reduction of double stranded DNA inside cells (Fig. 5A, lanes 6 & 13; Fig. 7A, lane 3). Our interpretation is that the M133T mutation increases the envelopment and export of core particles with mature genome, thus depleting the intracellular pool.
The ability of the M133T mutation to rescue virion secretion of a wide range of mutants (I110M, G145R, N146Q, N146S, R169H, and to a lesser extent, G119E) argues for a general mechanism of action. Three types of evidence suggest the novel N-linked glycosylation site (most likely in the S protein) as the basis for rescue. First, M133S but not M133I or M133Q mutation could achieve a similar effect, and the rescuing capacity of M133T was abrogated by an additional N131T/Q mutation. Second, either an N146Q or N146S mutation, which prevents Nlinked glycosylation of the S and L proteins of wild-type HBV, could block the secretion of HBV viral albeit not subviral particles. Such a secretion defect could be overcome by introduction of the M133T mutation. Third, virion secretion of the M133T/N146S or M133T/N146Q mutant was markedly reduced by tunicamycin treatment, which nearly completely abolished glycosylated forms of the S protein for these mutants. Secretion of subviral particles was not reduced by tunicamycin treatment. Similarly, others reported that tunicamycin dose-dependently inhibited virion secretion of wild-type HBV from a HepG2 cell line, but not the secretion of subviral particles (1, 23, 28, 32). We observed a modest reduction in virion secretion for the wild-type virus (Fig. 8B, lanes 3, 4), possibly because of incomplete block to the glycosylation of the wild-type envelope proteins at the concentration used (Fig. 8C, lane 3). Taken together, our findings and earlier studies by others demonstrate a critical role of N-linked glycosylation for HBV virion secretion, but not for the secretion of subviral particles. In this
regard, N-linked glycans mediate interaction with lectin chaperones such as calnexin and calreticulin to guarantee proper folding of the glycoprotein (11). Interestingly, the potential Nlinked glycosylation site in the duck hepatitis B virus envelope proteins is inefficiently used, suggesting a different mechanism for virion formation (33).
In conclusion, immune escape mutants such as G119E and G145R, and other mutants such as I110M and R169P are impaired in virion secretion. A novel N-linked glycosylation site created by M133T mutation could rescue virion secretion of the immune escape mutants without restoring virus recognition by the neutralizing antibodies. Therefore, combination of these mutations will restore viral fitness under the selective pressure of neutralizing antibodies. Indeed, the M133T mutation is found highly prevalent in HBsAg negative patients (12). In patients infected with the T126I or G145K immune escape mutant, viral persistence correlated with the simultaneous emergence of the M133T mutation (19). This scenario is reminiscent of HBV resistance to nucleotide or nucleoside therapy, where the primary mutation in the P gene reduces viral replication capacity, which is nevertheless compensated for by a second-site mutation that arises later (42). In short, plasticity of the HBV genome poses serious challenge to our efforts to prevent and treat HBV infection.

Figure 5. Modulation of virion secretion of the I110M and G145R mutants by the G112 and M133 mutations. The 1.5mer replication construct (1.5 g) was co-transfected with 0.5 g of expression construct of single, double, or triple mutant as indicated. (A) Replicative DNA. (B) Virion DNA. (C) Intracellular envelope proteins. (D) GAPDH as a loading control. (E) ELISA for secreted HBsAg (Abazyme kit for lanes 1-14 and Abbott kit for lanes 15-20).
Figure 6. The M133T mutation rescues virion secretion of the G119E and R169H mutants and creates a novel N-linked glycosylation site. The 1.5mer replication construct (1.5 g) was cotransfected with 0.5 g of the expression construct of the G119E, R169P, and R169H mutant, with or without additional mutations at positions 112, 131, and 133. (A) Replicative DNA. (B) Virion DNA. (C) Intracellular envelope proteins. (D) Intracellular GAPDH. (E) ELISA for secreted HBsAg using the Abazyme kit. (F) Effect of endo H treatment on envelope protein migration. The cell lysate was either treated with endo H (+) or not (-) prior to gel electrophoresis. The p39, p30, and p24 denote nonglycosylated L, M, and S proteins, respectively.
Figure 7. Impacts of tunicamycin treatment on envelope protein glycosylation and virion secretion of the WT virus, I110M, M133T, I110M/M133T, M133T/G145R, and N146Q mutants. Huh-7 cells in 6-well plates were co-transfected with the replication construct (1.5 g), expression construct (0.5 g), and SEAP plasmid (5ng) in triplicate. Medium was changed two days post-transfection, with one well supplemented with 20 l of DMSO, and another well with 20 l of tunicamycin (5 mg/ml sock solution in DMSO). Cells and culture supernatant were harvested 3 days later. (A) Replicative DNA. (B) Virion DNA. (C) Intracellular envelope
proteins detected by the rabbit and horse antibodies, respectively. (D) GAPDH as a loading control. (E) ELISA for secreted HBsAg (Abazyme kit). (F) SEAP activities as a measurement of ER stress.
Figure 8. Impact of tunicamycin treatment on envelope protein glycosylation and virion secretion of the G112N/G119E, M133T/N146Q, M133T/N146S, and M133T/R169P mutants. Huh-7 cells in 6-well plates were co-transfected with the replication construct (1.5 g), expression construct (0.5 g), and SEAP plasmid (5ng) in triplicates. Medium was changed two days post-transfection, with one well each supplemented with 20 l of DMSO or tunicamycin (5 mg/ml stock in DMSO). Cells and culture supernatant were harvested 3 days later. (A) Replicative DNA. (B) Virion DNA. (C) Intracellular envelope proteins detected by the rabbit and horse antibodies. (D) GAPDH as a loading control. (E) ELISA for secreted HBsAg (Abazyme). (F) SEAP activities as a measurement of ER stress.

Figure 9. Requirement of at least one N-linked glycosylation site for virion secretion. The replication construct (1.5 g) was co-transfected with 0.5 g of expression construct. (A) Replicative DNA. (B) Virion DNA. (C) Intracellular envelope proteins. (D) ELISA for secreted HBsAg using the Abazyme kit.

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nucleotide

DNA polymerase S466A silent silent silent

S domain

virion secretion impair impair impair enhance

4B, 4C 3.4 4C 4B, 4C

T484G G510A G660C T552C

I110M G119E R169P M133T

polymerase

X core

envelope

core 2600 (ApaI)

1.5mer 1040 (EcoRV) (replication+)

131 N S 133 M

1.5mer

0.7mer

119 G 101

- L M S+

Immunodominant loops
COOH (226) ER lumen (virion surface)

163 Membrane

Cytosol (virion interior)

0.7mer (replication- )

Cytosolic loop

Figure 1

marker

P G H L Q 15 DS

WT M D G T E A T V

1.7/1.5kb SS

Replicative DNA

Virion DNA

1.7/1.5kb

L protein

gp42 Intracellular p39 envelope proteins

horse anti-HBs

S protein 1 S protein gp27 rabbit anti-HBs p24 gp27 p24

2.0 1.5 1.0 0.5 0

gp27 p24
Secreted S protein horse anti-HBs

Secreted HBsAg

Figure 2

WT mutant 0 1

Figure 3

LM proteins S protein

WT (0.6) I110M (0.6) WT + I110M (0.3+0.3) I110M + M133T (0.3+0.3) I110M + WT LM (0.5+0.1) I110M + M133T LM (0.5+0.1) I110M + WT S (0.3+0.3) I110M /+ M133T S (0.3+0.3) mock

G119E R169P

I110M WT

R169P WT WT WT -