Hepatitis B virus core antigen determines viral

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May 18, 2010 - These results indicate that the immune response trig- gered in mice by HBcAg ..... Because HBcAg functions as a potent im- munogen ..... Milich DR, et al. (1997) Role of B cells in antigen presentation of the hepatitis B core.
Hepatitis B virus core antigen determines viral persistence in a C57BL/6 mouse model Yi-Jiun Lina,1, Li-Rung Huangb,1, Hung-Chih Yangc, Horng-Tay Tzengd, Ping-Ning Hsud, Hui-Lin Wue, Pei-Jer Chena,f,2, and Ding-Shinn Chene,2 a

Graduate Institute of Microbiology, dGraduate Institute of Immunology, fGraduate Institute of Clinical Medicine, cDepartment of Medical Research, and Hepatitis Research Center, National Taiwan University College of Medicine and National Taiwan University Hospital, Taipei 10002, Taiwan; and bInstitute of Molecular Medicine and Experimental Immunology, Bonn University Hospital, Sigmund Freud, 53105 Bonn, Germany e

Contributed by Ding-Shinn Chen, April 8, 2010 (sent for review November 24, 2009)

We recently developed a mouse model of hepatitis B virus (HBV) persistence, in which a single i.v. hydrodynamic injection of HBV DNA to C57BL/6 mice allows HBV replication and induces a partial immune response, so that about 20–30% of the mice carry HBV for more than 6 months. The model was used to identify the viral antigen crucial for HBV persistence. We knocked out individual HBV genes by introducing a premature termination codon to the HBV core, HBeAg, HBx, and polymerase ORFs. The specific-genedeficient HBV mutants were hydrodynamically injected into mice and the HBV profiles of the mice were monitored. About 90% of the mice that received the HBcAg-mutated HBV plasmid exhibited high levels of hepatitis B surface antigenemia and maintained HBsAg expression for more than 6 months after injection. To map the region of HBcAg essential for viral clearance, we constructed a set of serial HBcAg deletion mutants for hydrodynamic injection. We localized the essential region of HBcAg to the carboxyl terminus, specifically to the 10 terminal amino acids (HBcAg176–185). The majority of mice receiving this HBV mutant DNA did not elicit a proper HBcAg-specific IFN-γ response and expressed HBV virions for 6 months. These results indicate that the immune response triggered in mice by HBcAg during exposure to HBV is important in determining HBV persistence. hepatitis B surface antigenemia

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ersistent hepatitis B virus (HBV) infections affect about 350 million people worldwide and are a major health problem. Factors directing the infection toward chronicity have been studied extensively. The exposure of neonates or young children to a high HBV viral load, together with hepatitis B e antigen (HBeAg), predicts a high rate of persistent HBV infection. A recent genomewide association study identified HLA-DP polymorphisms as another factor in the persistence of HBV infections (1). Nevertheless, the immune mechanisms that lead to HBV persistence have not been resolved. To address this issue, a commonly used mouse model, such as the HBV transgenic mouse, has been used to study the possible mechanisms. However, the main drawback of HBV transgenic mouse models is that they are immunologically tolerant of viral antigens. Therefore, to explore the issue of HBV persistence, the adoptive transfer of HBV-primed immune cells or other manipulations must be used to overcome this tolerance. Another alternative is to introduce the HBV genome into the mouse liver by hydrodynamic injection through the tail vein. With this approach, HBV was shown to replicate in the mouse liver, and the immune responses against HBV proteins to clear the HBV infection could be documented in 1–2 weeks after the injection (2). Recently, we improved this approach by modifying the HBV DNA plasmid and injecting the plasmid into C57BL/6 mice and succeeded in delaying the mouse immune clearance of HBV (3). Clearance could be postponed for 6–8 weeks and about 10–30% of the injected mice maintained HBV persistence even up to 6 months after injection. This animal model provides a platform from which to study the mechanisms of HBV persistence.

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As a first step in a systematical investigation of the HBV genes that influence viral persistence in this model, we knocked out each of the ORFs in the HBV genome by site-directed mutagenesis. Each mutant HBV plasmid, deficient in the expression of only one viral gene, was then injected into C57BL/6 mice and HBV persistence was determined by serum HBV surface antigen (HBsAg) and HBV DNA detection. We found that the mutant HBV DNA that did not express the HBV core (HBc) gene established HBsAg persistence for more than 6 months in about 90% of the injected animals. More interestingly, the HBV mutant expressing a shortened core antigen of 175 amino acids established not only HBsAg but also HBV virion persistence for more than 6 months in 70% of the injected animals. Here, we demonstrate the accessibility of this model for investigating the contribution of various HBV mutants to HBV persistence. These results should allow us to understand and explore the mechanisms involved in HBV persistence. Results Influence of Individual HBV Antigens on Viral Persistence in C57BL/6 Mice. Although some lines of evidence suggest that fetal or peri-

natal exposure to HBeAg contributes to the persistence of HBV, a systematic investigation of the role of each individual HBV gene had not been conducted. Our mouse model provides a platform for this reverse genetic analysis (3). Therefore, we constructed an array of viral mutants that contained premature stop codons in the core, HBe, HBx, or polymerase (pol) ORFs of a replicationcompetent HBV plasmid pAAV/HBV1.2 using in situ sitedirected mutagenesis (Fig. S1 A and B and Table S1). The mutations were carefully selected and did not affect the translation of other overlapping frames. Ten micrograms of wild-type (WT) or mutant pAAV/HBV1.2 were injected hydrodynamically into the tail veins of male C57BL/6 mice, as described previously (3). After injection, the mice were regularly bled to monitor the serum levels of HBsAg, HBeAg, and HBV DNA or were killed to evaluate the intrahepatic viral transcription and replication, and HBcAg expression. Of the mice receiving WT pAAV/HBV1.2, ≈50% remained HBsAg positive at 12 weeks postinjection (wpi) (Fig. 1 A–D). The serum levels and the persistence rates of HBsAg in mice receiving HBeAg-null or pol-null mutants were similar to those of the mice receiving WT DNA, from the first day to the 12th week after hydrodynamic injection (Fig. 1 A and B). These results indicate that the absence of HBeAg or polymerase did

Author contributions: Y.-J.L., L.-R.H., P.-N.H., P.-J.C., and D.-S.C. designed research; Y.-J.L., L.-R.H., and H.-T.T. performed research; Y.-J.L., L.-R.H., and P.-J.C. analyzed data; and Y.-J. L., L.-R.H., H.-C.Y., H.-L.W., and P.-J.C. wrote the paper. The authors declare no conflict of interest. 1

Y.-J.L. and L.-R.H. contributed equally to this work.

2

To whom correspondence may be addressed. E-mail: [email protected] or chends@ ntu.edu.tw.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1004762107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1004762107

Fig. 2. HBV DNA in the sera of mice receiving WT or mutant pAAV/HBV1.2. The serum HBV DNA in the hydrodynamically injected mice at the indicated time points was quantified by real-time PCR. The titers of serum HBV DNA observed in the mice receiving WT pAAV/HBV1.2 (●) were compared with those of mice receiving each individual mutant DNA (○), including the HBeAg-null (A), HBeAg/core-null (B), pol-null (C), and HBx-null mutants (D). The detection limit for HBV DNA in our system was 100 copies per milliliter.

not alter the HBsAg persistence rate in mice after hydrodynamic injection. In contrast, almost all of the mice receiving HBeAg/core-null pAAV/HBV1.2 expressed high levels of HBsAg in their sera and 93.3% of them even remained HBsAg positive at 12 wpi (Fig. 1C). The mice receiving the HBx-null mutant also exhibited nearly 90% persistence at 12 wpi, although the HBsAg levels in these mice were lower in the beginning than those of the mice receiving WT HBV (Fig. 1D). The levels of HBsAg then increased in the mice receiving the HBx-null mutant, whereas those of the mice receiving the WT HBV DNA declined rapidly. These data suggest that the expression of HBcAg and HBx during HBV exposure influences viral persistence in C57BL/6 mice. The levels of serum HBV DNA in the hydrodynamically injected mice were also quantified using real-time PCR. In the mice receiving WT pAAV/HBV1.2, the average titer of serum HBV DNA in 15 injected animals was below 1 × 104 copies per milliliter at 1 day postinjection (dpi) and reached 2.16 × 106 copies per milliliter at 7 dpi (Fig. 2A). At later time points, some mice lost both serum HBsAg (Fig. 1D) and HBV DNA, whereas others remained positive for both. The average titer of serum HBV DNA in all 15 injected mice dropped below 1 × 105 copies per milliliter after 7 dpi (Fig. 2A). At all time points, the average titers of serum HBV DNA in the mice receiving HBeAg-null pAAV/HBV1.2 were similar to those of the mice receiving WT Lin et al.

Negative Correlation Between Intrahepatic Expression of HBcAg and HBV Persistence. To further characterize the viral transcription,

replication, and protein expression patterns of the various mutants, we collected the liver tissues of the mice receiving WT pAAV/HBV1.2 or each individual mutant at 3 and 33 dpi and assayed for HBV replication intermediates and transcripts using Southern and Northern hybridization. The episomal input DNA was detected in the livers of all mice receiving either WT or mutant pAAV/HBV1.2 at 3 dpi (Fig. 3A Top). However, HBV replication intermediates, including relaxed circular (RC) DNA and single-stranded DNA (ssDNA), were only detected in the livers of mice receiving WT, HBx-null, or HBeAg-null pAAV/ HBV1.2. For unknown reasons, most of the replication intermediates in the mice receiving the HBeAg-null mutant were ssDNA (Fig. 3A Top, lanes 3 and 4). In this hydrodynamics-based HBV model, most of the HBV transcripts were derived from the input plasmid. Therefore, as long as the input DNA remained present, the HBV transcripts could be detected in the liver (Fig. 3A Middle). However, only low levels of HBV transcripts were observed in the livers of the mice receiving mutants that did not express HBx protein, including the HBx-null mutant and tripleknockout (tko) pAAV/HBV1.2 (Fig. 3A Middle, lanes 7 and 8). We also observed a reduction in both the HBV transcription/ replication efficiency (Fig. 3A Top, lanes 7 and 8) and HBcAg expression (Fig. 3A Bottom, lanes 7 and 8). The intrahepatic expression of HBcAg was only observed in the mice receiving WT, HBeAg-null, or pol-null pAAV/HBV1.2, but not in those receiving the HBeAg/core-null, HBx-null, or tko mutant (Fig. 3A Bottom). Serum HBeAg was undetectable in the mice receiving the HBeAg/core-null, HBeAg-null, or tko mutant at 2 and 32 dpi (Fig. 3). All mice expressed high levels of serum HBsAg at 2 dpi (Figs. 1 and 3). PNAS | May 18, 2010 | vol. 107 | no. 20 | 9341

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Fig. 1. HBsAg persistence rates in mice receiving mutants that do not express HBcAg or HBx are higher than those in the mice receiving WT pAAV/HBV1.2. C57BL/6 mice were injected hydrodynamically with 10 μg of pAAV/HBV1.2 or a mutant construct. The serum HBsAg titers (Left) and the rates of positive serum HBsAg (Right) in the mice receiving WT (●) pAAV/HBV1.2 were compared with those in mice receiving each individual mutant DNA (○), including the HBeAg-null (A), pol-null (B), HBeAg/core-null (C), and HBx-null mutants (D). The serum HBsAg titers were determined at the indicated time points with an enzyme immunoassay [calculated as signal/noise (S/N) ratios]. Positivity for HBsAg was defined as S/N ≧ 2. N = number of mice in each experiment. Error bars indicate SD here and in the other figures. The statistical P values were analyzed by Kaplan–Meier analysis and significant differences were observed in the Right panels of C (P = 0.0079) and D (P = 0.0224).

DNA. On the contrary, in the absence of HBcAg or pol, the replication cycle of HBV was disrupted, and no HBV DNA was detected at any time point after the first two time points, when the presence of HBV DNA could be attributable to residual injected DNA (Fig. 2 B and C). In the absence of HBx, the replication efficiency of HBV was reduced. The serum HBV DNA of the mice receiving the HBx-null mutant was detectable but lower than that of the mice receiving WT HBV (Fig. 2D). This observation is consistent with the previous study, which showed that HBx is required for HBV replication in vivo (4).

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Fig. 3. HBV replication and transcription, and intrahepatic HBcAg expression in mice receiving WT or mutant pAAV/HBV1.2 injections. Liver samples were collected at 3 dpi (A) or 33 dpi (B) after the hydrodynamic injection of 10 μg of WT or mutant DNA. The intrahepatic levels of the input HBV DNA, RC, ssDNAs, and the 3.5-kb pregenomic and 2.1/2.4-kb surface mRNAs were determined using Southern and Northern blotting. The intrahepatic levels of glyceraldehyde-3-phosphate dehydrogenase mRNA (mGAPDH) were used as the loading control for Northern blotting. The levels of β-actin were used as the loading control for Western blotting. One day before the mice were killed, serum samples were collected from the mice and HBsAg and HBeAg were measured. The titers of HBsAg (S/N) and HBeAg (signal/cutoff, S/CO) are shown beneath the figures.

The serum HBsAg levels and the intrahepatic levels of the input DNA, HBV replication intermediates, transcripts, and HBcAg in some mice receiving WT, HBeAg-null, or pol-null pAAV/ HBV1.2 declined to undetectable levels at 33 dpi (Fig. 3B). In contrast, the input DNA persisted in the livers of all mice receiving the HBeAg/core-null, HBx-null, or tko mutant at 33 dpi (Fig. 3B Top). Despite the lack of detectable HBV replication in the livers of these mice, high to moderate levels of HBV transcripts and HBsAg were readily detected in either the livers or sera (Fig. 3B). Therefore, only the HBV mutants that failed to express a detectable amount of intrahepatic HBcAg could persist in hepatocytes and maintain a continuous expression of HBV proteins such as HBsAg. To further verify the inverse correlation between HBcAg and HBV persistence, we coinjected the HBcAg-expressing plasmid with HBeAg/core-null pAAV/HBV1.2, which promotes HBV clearance in mice (SI Text, and Fig. S2 A–C). These results together indicate that HBcAg plays a vital role in intrahepatic HBV DNA persistence. The presence of HBcAg may trigger intrahepatic antiviral responses and facilitate the clearance of both infected hepatocytes and the input HBV DNA in this hydrodynamics-based mouse model. Consistently, our previous study demonstrated that the HBcAg-specific cellular immunity generated by a DNA vaccine encoding HBcAg was crucial for the clearance of HBV during both the acute phase and the chronic carriage phase in this model (3). C-Terminal Domain of HBcAg Is Critical for HBV Clearance. Because

HBcAg plays a crucial role in the persistence of HBsAg, we tried to identify the region of HBcAg that is important for HBsAg persistence. HBcAg consists of two domains: the assembly domain (amino acids 1–149), which forms the contiguous spherical shell, and the protamine-like domain (amino acids 150–183/185), which is responsible for RNA packaging and DNA synthesis. We generated a series of C-terminally truncated HBcAg mutants derived from pAAV/HBV1.2, which resulted in the differential deletion of these domains (Fig. 4A). Each of the HBcAg mutants contained a premature stop codon at a unique site within the HBc ORF, which did not alter the amino acid sequence of the overlapping pol ORF, although the mutations may have unavoidably affected the overlapping precore protein sequence and subsequently expressed HBeAg (Fig. 4A, see legend for details). However, from the above data, viral HBeAg is not critical for HBsAg persistence in this 9342 | www.pnas.org/cgi/doi/10.1073/pnas.1004762107

Fig. 4. pAAV/HBV1.2 mutants expressing C-terminal-truncated HBcAg enhanced HBsAg persistence in mice. (A) A schematic map of the different pAAV/HBV1.2 mutants with a variety of C-terminal truncations in HBcAg. Each individual HBcAg mutant contains a unique single-nucleotide mutation (marked as a match with a yellow head), which results in a premature stop codon (*) within the HBc coding region. The integrity of HBeAg corresponding to each mutant is shown on the Right. The titers of serum HBsAg (B) and HBeAg (D) and the positive rates of serum HBsAg (C) and HBeAg (E) in the mice receiving WT (●), HBeAg/core-null (○), HBc118 (▲), HBc150 (△), HBc166 (▼), or HBc175 (□) pAAV/HBV1.2 are shown. The differences in the HBsAg persistence rates in the mice receiving WT DNA or the individual mutants were analyzed by Kaplan–Meier analysis and were shown to be significant (P < 0.01).

mouse model. Hence we mainly focused on the role of HBcAg in the persistence of HBsAg. Each individual HBcAg-truncated mutant, WT, and HBeAg/core-null pAAV/HBV1.2 was hydrodynamically injected into mice and the serum levels of HBsAg and HBeAg were examined (Fig. 4 B–E). The significant difference in the HBsAg persistence rates of the mice receiving WT DNA and those receiving the HBeAg/core-null mutant is consistent with the above data (Figs. 1C and 4 B and C). The mice receiving HBc118 pAAV/HBV1.2 showed a pattern of HBsAg persistence very similar to that of the mice receiving the HBeAg/core-null mutant. It is noteworthy that the HBc118 mutant retained only a partial assembly domain of HBcAg and therefore could not form core particles. The mice receiving HBc150 pAAV/HBV1.2, which contains the intact assembly domain but no protamine-like domain of HBcAg, and the HBeAg/core-null mutant exhibited identical patterns of HBsAg persistence. Furthermore, the persistence rates in the mice receiving the HBc166 or HBc175 pAAV/ Lin et al.

HBV1.2 mutant, both of which result in truncated HBcAg with a partially impaired protamine-like domain, were slightly lower than those of mice receiving HBeAg/core-null mutant but significantly higher than those of mice receiving WT DNA. At 12 wpi, 84.5% of the mice receiving the HBc175 mutant DNA were HBsAg positive, whereas only 20% of the mice receiving WT HBV DNA remained HBsAg positive. In addition to HBsAg, HBe antigenemia was also present in the mice receiving WT, HBc150, HBc166, or HBc175 pAAV/HBV1.2, with markedly higher levels of serum HBeAg in the mice receiving the HBc150 mutant DNA (Fig. 4 D and E). The persistence of HBeAg and HBsAg correlated well in the mice receiving these DNAs. As expected (5), HBeAg could not be detected in the sera of either the mice injected with the HBeAg/core-null mutant DNA or those receiving the HBc118 mutant DNA. Deletion of the 10 Amino Acids at the C Terminus of HBcAg Promotes HBV Persistence. Because HBcAg is critical for the HBV life cycle,

HBcAg mutations may affect HBV replication and subsequently influence HBV persistence rates. Therefore, we examined the intrahepatic viral replication, transcription, and protein expression in mice receiving the WT or different HBcAg mutants. Previous studies have shown that the HBV mutant with truncated HBcAg1–173, equivalent to HBcAg1–175 of genotype A used in our study, behaves like WT HBV, whereas mutant HBcAgs with less than 173 residues exhibit impaired DNA synthesis during viral replication in vitro (6, 7). In the mice receiving HBc175 pAAV/ HBV1.2, the intrahepatic levels of viral replication were similar to those of the mice receiving the WT HBV DNA at both 3 dpi (Fig. 5A) and 41 dpi (Fig. 5B). Consistent with intrahepatic viral replication, serum viral DNA was readily detected in these mice [WT: (1.77 ± 0.33) × 106 and (2.89 ± 2.05) × 105; HBc175 pAAV/ HBV1.2: (7.43 ± 1.09) × 106 and (3.43 ± 3.77) × 105 at 1 wpi and 4 wpi, respectively]. All other HBc mutants, including HBeAg/ core-null, HBc118, HBc150, and HBc166 pAAV/HBV1.2, showed impaired viral replication, which is in agreement with the results of previous studies (Fig. 5 A and B). The levels of viral transcription from the input DNA, which was used as the template, were fairly similar in the livers of the mice receiving the WT and HBc mutant DNAs. Despite the similar transcription levels among the mice receiving different mutants, the expression of the viral proteins was not equal. Among the truncated forms of

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HBcAg, only HBcAg1–166 and HBcAg1–175 were detected with either immunohistochemical staining (Fig. 5D xv and xviii) or Western blotting analysis (Fig. 5C lanes 6 and 7). However, the expression of HBsAg was similar in the mice receiving the WT or mutant DNA (Fig. 5D Middle). Our failure to detect the truncated HBcAgs was not attributable to the failure of the antibody to recognize them because this antibody has been proven functional. Therefore, even in the presence of HBcAg and HBV replication, the mice receiving HBc175 pAAV/HBV1.2 exhibited a dramatically elevated HBsAg persistence rate compared with that of mice receiving WT DNA (Fig. 4C). Taken together, our observations suggest that a stretch of 10 amino acids at the C terminus of HBcAg (HBcAg176–185) is critical for the clearance of not only HBsAg but also HBV. Impaired Immune Response to HBV Core Antigen in Mice Receiving HBc175 pAAV/HBV1.2. Because HBcAg functions as a potent im-

munogen, inducing both the humoral and cellular immune responses, we first examined the capacity of WT HBV and the different HBc mutants to induce anti-HBc antibodies in mice. We observed that all of the mice receiving WT pAAV/HBV1.2 produced anti-HBc at 1 wpi (Fig. 6 A), consistent with the fact that HBV-infected patients produce anti-HBc. In contrast, none of the mice receiving the HBeAg/core-null, HBc118, or HBc150 mutant generated anti-HBc even at 13 wpi. The absence of antiHBc in these mice correlated well with their undetectable HBcAg expression (Fig. 5 C and D). Interestingly, the kinetics of anti-HBc production were slow in the mice receiving the HBc166 or HBc175 mutant. At 1 wpi, anti-HBc was detected in 33.3% and 53.8% of mice receiving the HBc166 and HBc175 mutants, respectively. However, anti-HBc positivity reached 100% in the mice receiving the HBc175 mutant at 8 wpi, whereas 41.7% of the mice receiving the HBc166 mutant remained anti-HBc negative at 13 wpi (Fig. 6A). Even though its major immunodominant region is located around residue 80 (8), HBcAg1-166 was still unable to elicit a strong B-cell response in some of the mice. These results indicate that the C terminus of HBcAg promotes the proper B-cell response to HBcAg. It has been shown that IFN-γ production in T cells in response to HBcAg stimulation correlates with the resolution of HBV infections (9). Therefore, we investigated the ability of different HBc mutants to elicit IFN-γ secretion using an enzyme-linked

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Fig. 5. HBV replication, transcription, and translation in mice receiving WT or mutant pAAV/HBV1.2. Liver samples were collected from mice receiving WT, HBeAg/core-null, HBc118, HBc150, HBc166, or HBc175 pAAV/HBV1.2 at 3 dpi (A) and 41 dpi (B) to examine viral replication and transcription, as described in Fig. 3. (C) Western blot analysis of the intrahepatic expression of HBcAg (arrows) in mice receiving PBS only (lane 1, the negative control), WT (lane 2), HBeAg/core-null (lane 3), HBc118 (lane 4), HBc150 (lane 5), HBc166 (lane 6), or HBc175 (lane 7) pAAV/HBV1.2 at 41 dpi. Intrahepatic β-actin expression was used as the loading control. (D) Immunohistochemical staining at 3 dpi for HBsAg (ii, v, viii, xi, xiv, and xvii), HBcAg (iii, vi, ix, xii, xv, and xviii), and the negative control (i, iv, vii, x, xiii, and xvi) in the livers of mice receiving WT (i–iii), HBeAg/core-null (iv–vi), HBc118 (vii–ix), HBc150 (x–xii), HBc166 (xiii–xv), or HBc175 (xvi–xviii) pAAV/HBV1.2.

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Fig. 6. Humoral immune responses and IFN-γ responses stimulated by WT or individual mutant forms of HBcAg. (A) The percentage of anti-HBc-positive mice receiving WT or individual HBc mutant pAAV/HBV1.2 at different time points after hydrodynamic injection. The blood of the mice receiving WT (n = 10), HBeAg/core-null (n = 12), HBc118 (n = 12), HBc150 (n = 12), HBc166 (n = 12), or HBc175 (n = 13) DNA was collected at 1, 4, 8, and 13 wpi to examine the production of anti-HBc antibodies. The bars representing HBeAg/core-null, HBc118, and HBc150 mutants are absent because none of the corresponding mice produced anti-HBc. (B) The IFN-γ responses of splenocytes from mice receiving a mock vector (negative control, NC), WT, HBeAg/core-null, HBc150, or HBc175 pAAV/HBV1.2 (n = 3). Each sample was collected at 10 dpi and subjected to an ELISPOT assay to measure the frequency of HBcAg-specific IFN-γ-secreting cells. The results are expressed as spot-forming cells (SFCs) per million splenocytes. The asterisk symbolizes significant differences between the WT group and each of the other groups (P < 0.001).

immunospot (ELISPOT) assay. Splenocytes isolated from mice receiving WT HBV or the HBc mutants were cocultured with recombinant HBcAg and the numbers of responding IFNγ-secreting cells were measured. In the mice receiving WT HBV DNA, the frequency of HBcAg-specific IFN-γ-secreting cells was 346/106 splenocytes (Fig. 6B). However, none of the mice receiving the HBc mutants, including HBeAg/core-null, HBc150, and HBc175 pAAV/HBV1.2, induced IFN-γ-secreting cells at a similar level to that observed in mice receiving WT HBV DNA. Significant differences (P < 0.001) were noticed between the mice injected with WT HBV and those receiving each individual HBc mutant DNA. Deletion of HBcAg residues 176–185 seemed sufficient to prevent HBcAg from triggering the IFN-γ response in mice. Using an ELISPOT assay, we excluded the contribution of a critical T-cell epitope located in this region to this phenomenon (Fig. S3). Together, our data suggest that the increased HBV persistence in mice receiving the HBc mutant DNAs (Fig. 4) resulted from the inability of these truncated HBcAgs to induce an efficient protective immune response, including the production of IFN-γ. Further studies are required to investigate the underlying mechanisms. Discussion In this study, we systematically investigated the effect of each individual HBV gene on viral persistence using a reverse genetics strategy and a hydrodynamics-based immunocompetent mouse model. We found that knocking out HBcAg or HBx, but not HBeAg or pol, led to HBV persistence in mice. We further localized the region of HBcAg critical for viral clearance to the 10 residues at its C terminus, and deletion of a mere 10 residues at the HBcAg C terminus mitigated the host immune response to HBV. The outcome of HBV infection, recovery or persistence, is controlled by the interplay between viral proteins and host factors. A previous study revealed that the HBcAg-induced immune response is associated with recovery from hepatitis B (10), and IFN-γ production is associated with the resolution of HBV infections (11). Moreover, HBcAg-stimulated IFN-γ responses were detected in patients with acute hepatitis rather than chronic hepatitis (12). Our study consistently identified HBcAg as the most immunogenic viral antigen during the early course of HBV infection. Knockout of HBcAg significantly promoted HBV persistence in C57BL/6 mice receiving HBV DNAs. Whereas nonneutralizing anti-HBc antibodies do not appear to play a critical role in viral clearance, the robust IFN-γ response induced by WT HBcAg is linked to the resolution of HBV. In contrast, all of the mice receiving HBcAg mutants, including the HBc175 mutant, only exhibited weak IFN-γ responses and failed to purge the HBV. 9344 | www.pnas.org/cgi/doi/10.1073/pnas.1004762107

Interestingly, the 10 residues at the C terminus of HBcAg are critical for inducing robust immune responses against HBV. It remains unclear whether the primary sequence of HBcAg176–185 or a possible conformational change resulting from the deletion of this region plays the key role. Capsid formation, the main feature of HBcAg, would not be disrupted in mice receiving HBc150, HBc166, or HBc175 pAAV/HBV1.2 because all of the mutants contain the essential assembly domain (13). However, the prolonged HBV persistence in mice receiving either the HBc166 or the HBc175 mutant suggests that these two C-terminally truncated HBcAgs and WT HBcAg behave differently in the induction of the antiviral immune responses. We were unable to detect the intrahepatic expression of HBcAg1–150 probably because this truncated protein was unstable and subsequently degraded by proteasome. Alternatively, it might be rapidly secreted. The highly conserved C-terminal domain of HBcAg controls HBV pregenomic RNA (pgRNA) binding and viral DNA synthesis. HBcAg1–166 failed to support viral replication, whereas HBcAg1–175 could. HBsAg persistence can occur irrespective of HBV replication, which suggests that the deletion of HBcAg176–185 promotes HBV persistence but retains HBV replication almost intact. So far no B-cell or T-cell epitopes have yet been identified in the C terminus of HBcAg. Experimentally, we also did not find a candidate T-cell epitope in this region (Fig. S3). Therefore, it is very unlikely that these 10 residues directly induce strong adaptive immune responses against HBV. Alternatively, HBcAg may influence HBV persistence by evoking innate immunity. Because it is likely that HBcAg1–166 still has the ability to bind pgRNA without further reverse transcription, it is possible that the C-terminal truncation of HBcAg prevents the exposure of the pgRNA, which may function as a ligand for intracellular pattern recognition receptors. The encapsidated ssRNA, rather than HBcAg itself, stimulates Toll-like receptor 7 signaling in mice (14). Moreover, the binding of the C-terminal domain of HBcAg to membrane heparin sulfate on the surfaces of macrophages, B cells, and dendritic cells mediates the breakdown of the viral capsid (15–17). This interaction can stimulate proinflammatory cytokine production. Therefore, the lack of the 10 HBcAg C-terminal residues causes a failure to elicit successful immune responses to clear the HBV infection. In addition to HBcAg, we also observed increased HBV persistence in the absence of HBx. It has been demonstrated that HBx transactivates the transcription of the major HBV genes, including HBcAg (4, 18). Furthermore, some patients in the inactive viral replication phase of chronic hepatitis B infections do not express HBcAg in their liver (19), suggesting that the lack of HBcAg may contribute to HBV persistence. In this model, knockout of the HBx gene dramatically reduced the HBV tranLin et al.

1. Kamatani Y, et al. (2009) A genome-wide association study identifies variants in the HLA-DP locus associated with chronic hepatitis B in Asians. Nat Genet 41:591–595. 2. Yang PL, Althage A, Chung J, Chisari FV (2002) Hydrodynamic injection of viral DNA: A mouse model of acute hepatitis B virus infection. Proc Natl Acad Sci USA 99:13825–13830. 3. Huang LR, Wu HL, Chen PJ, Chen DS (2006) An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection. Proc Natl Acad Sci USA 103: 17862–17867. 4. Keasler VV, Hodgson AJ, Madden CR, Slagle BL (2007) Enhancement of hepatitis B virus replication by the regulatory X protein in vitro and in vivo. J Virol 81:2656–2662. 5. Carlier D, Jean-Jean O, Fouillot N, Will H, Rossignol JM (1995) Importance of the C terminus of the hepatitis B virus precore protein in secretion of HBe antigen. J Gen Virol 76:1041–1045. 6. Nassal M (1992) The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly. J Virol 66:4107–4116. 7. Le Pogam S, Chua PK, Newman M, Shih C (2005) Exposure of RNA templates and encapsidation of spliced viral RNA are influenced by the arginine-rich domain of human hepatitis B virus core antigen (HBcAg 165-173). J Virol 79:1871–1887. 8. Salfeld J, Pfaff E, Noah M, Schaller H (1989) Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus. J Virol 63:798–808. 9. Jung MC, et al. (1991) Hepatitis B virus antigen-specific T-cell activation in patients with acute and chronic hepatitis B. J Hepatol 13:310–317. 10. Lau GK, et al. (2002) Resolution of chronic hepatitis B and anti-HBs seroconversion in humans by adoptive transfer of immunity to hepatitis B core antigen. Gastroenterology 122:614–624. 11. Wieland S, Thimme R, Purcell RH, Chisari FV (2004) Genomic analysis of the host response to hepatitis B virus infection. Proc Natl Acad Sci USA 101:6669–6674. 12. Szkaradkiewicz A, et al. (2003) HBcAg-specific cytokine production by CD4 T lymphocytes of children with acute and chronic hepatitis B. Virus Res 97:127–133. 13. Gallina A, et al. (1989) A recombinant hepatitis B core antigen polypeptide with the protamine-like domain deleted self-assembles into capsid particles but fails to bind nucleic acids. J Virol 63:4645–4652.

Lin et al.

host immunity induced by the 10-residue-long C-terminal truncation of HBcAg. The underlying mechanism responsible for this phenomenon remains unclear and is currently under investigation. Materials and Methods Plasmids. Site-directed mutagenesis for generating all pAAV/HBV1.2 mutants was performed by QuickChange II site-directed mutagenesis kit (Stratagene) following the manufacturer’s protocol. For more details, see SI Text. Animal Study. C57BL/6 mice (male, 6 to 7 weeks old) were anesthetized with ketamine and xylazine. Ten micrograms of HBV plasmid DNA were injected into the tail veins of mice within 5 s in a volume of PBS equivalent to 8% of the mouse body weight (SI Text). Detection of HBV Antigen, Antibody, and HBV DNA. Serum levels of HBsAg, HBeAg, anti-HBc of the mice were determined using the AXSYM system kits (Abbott), and the reporting unit is S/N or S/CO ratio. The cutoff value for determining HBsAg-, HBeAg-, and anti-HBc-positivity is S/N ratio ≧ 2, S/CO ≧ 1, and S/CO < 1, respectively. For detection of serum HBV DNA, each serum sample was pretreated with 25 units of DNase I (Roche) at 37 °C for overnight and total DNA was extracted and detected for HBV DNA by real-time PCR as previously described (25) (SI Text) Southern and Northern Hybridization. HBV viral RNA and replicative DNA intermediates were detected by Northern and Southern blot analysis of total liver RNA and DNA, respectively, as previously described (26). IFN-γ Enzyme-Linked Immunospot Assay. Splenocytes were prepared from the mice receiving hydrodynamic injection at 10 dpi. The IFN-γ enzyme-linked immunospot (ELISPOT) assay was performed as described in SI Text. ACKNOWLEDGMENTS. This work was supported by National Science Council Grants NSC97-2321-B-002-025 and NSC98-2321-B-002-003.

14. Lee BO, et al. (2009) Interaction of the hepatitis B core antigen and the innate immune system. J Immunol 182:6670–6681. 15. Vanlandschoot P, Van Houtte F, Serruys B, Leroux-Roels G (2005) The arginine-rich carboxy-terminal domain of the hepatitis B virus core protein mediates attachment of nucleocapsids to cell-surface-expressed heparan sulfate. J Gen Virol 86:75–84. 16. Cooper A, Tal G, Lider O, Shaul Y (2005) Cytokine induction by the hepatitis B virus capsid in macrophages is facilitated by membrane heparan sulfate and involves TLR2. J Immunol 175:3165–3176. 17. Cooper A, Shaul Y (2006) Clathrin-mediated endocytosis and lysosomal cleavage of hepatitis B virus capsid-like core particles. J Biol Chem 281:16563–16569. 18. Reifenberg K, et al. (1999) The hepatitis B virus X protein transactivates viral core gene expression in vivo. J Virol 73:10399–10405. 19. Hsu HC, et al. (1987) Biologic and prognostic significance of hepatocyte hepatitis B core antigen expressions in the natural course of chronic hepatitis B virus infection. J Hepatol 5:45–50. 20. Milich DR, et al. (1997) Role of B cells in antigen presentation of the hepatitis B core. Proc Natl Acad Sci USA 94:14648–14653. 21. Chen MT, et al. (2004) A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen. Proc Natl Acad Sci USA 101:14913–14918. 22. Milich DR, et al. (1990) Is a function of the secreted hepatitis B e antigen to induce immunologic tolerance in utero? Proc Natl Acad Sci USA 87:6599–6603. 23. Stevens CE, Neurath RA, Beasley RP, Szmuness W (1979) HBeAg and anti-HBe detection by radioimmunoassay: Correlation with vertical transmission of hepatitis B virus in Taiwan. J Med Virol 3:237–241. 24. Chen M, et al. (2005) Immune tolerance split between hepatitis B virus precore and core proteins. J Virol 79:3016–3027. 25. Yeh SH, et al. (2004) Quantification and genotyping of hepatitis B virus in a single reaction by real-time PCR and melting curve analysis. J Hepatol 41:659–666. 26. Wu HL, et al. (2005) RNA interference-mediated control of hepatitis B virus and emergence of resistant mutant. Gastroenterology 128:708–716.

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MICROBIOLOGY

scripts. Therefore, we surmise that the increase in the HBsAg persistence rate in mice receiving HBx-null pAAV/HBV1.2 was attributable to the low expression of HBcAg in their liver. This indicates that the expression of HBcAg might be tightly controlled by HBV itself, such as HBx, to ensure persistent infections. Although HBeAg and HBcAg share 149 residues and are crossreactive at the T-cell level during infection and immunization, they apparently elicit different patterns of immune responses, as inferred from the comparison of the HBV persistence rates in mice receiving WT or mutant pAAV/HBV1.2 (Fig. 1 A and C) and other systems (20). Previous studies have demonstrated that HBeAg exhibits some immunoregulatory functions (20, 21). However, we failed to observe any immunoregulatory effect of HBeAg in our model (Fig. 1A). There are two plausible explanations of this discrepancy. First, the timing for HBeAg to execute its immunoregulatory function may be critical. Secreted HBeAg may have an immunoregulatory function in utero and may induce a T-cell tolerance of HBeAg/HBcAg in exposed neonates (22). Infants born to HBeAg-seropositive mothers often develop chronic hepatitis rather than acute hepatitis after infection, supporting the above argument (23). Second, the levels of serum HBeAg in the mice receiving hydrodynamic injections of pAAV/HBV1.2 and the HBeAg-transgenic mice may not be equivalent. Indeed, it is clear that the levels of serum HBeAg influence the potency of HBeAg in inducing T-cell tolerance (24). Taken together, these data suggest that some mechanisms other than the immunoregulatory functions of HBeAg contribute to the establishment of HBV persistence in this mouse model. In conclusion, HBcAg is a major viral determinant of HBV persistence, and the region HBcAg176–185 is critical for this persistence. Enhanced viral persistence resulted from the impaired