JVI Accepts, published online ahead of print on 21 May 2014 J. Virol. doi:10.1128/JVI.01123-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Cao et al., HBV quasispecies and viral pathogenesis
1
Coexistence of hepatitis B virus quasispecies enhances viral
2
replication and the ability to induce host antibody and cellular
3
immune responses
4 5
Liang Cao1,2, Chunchen Wu1#, Hui Shi1, Zuojiong Gong3, Ejuan Zhang1, Hui Wang1,2,
6
Kaitao Zhao1,2, Shuhui Liu1,2, Songxia Li1, Xiuzhu Gao4, Yun Wang1, Rongjuan Pei1,
7
Mengji Lu1, 5, 6, Xinwen Chen1#
8 9
1.
State Key Lab of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
10 11
2.
University of Chinese Academy of Sciences, Beijing, China
12
3.
Department of Infectious Diseases, Renmin Hospital of Wuhan University, Wuhan, China
13 14
4.
Department of Hepatology, First Hospital of Jilin University, Changchun, China
15
5.
Institute of Virology, University Hospital of Essen, Essen, Germany
16
6.
Department of Microbiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
17 18 19
Running title: HBV quasispecies and viral pathogenesis
20 21
#
Corresponding author:
1
Cao et al., HBV quasispecies and viral pathogenesis 22
Prof. Dr. Xinwen Chen
23
Wuhan Institute of Virology, Chinese Academy of Science
24
Wuhan 430071, People’s Republic of China
25
Phone/fax: 86-027-87199106; E-mail: chenxw@ wh.iov.cn
26 27
Dr. Chunchen Wu
28
Wuhan Institute of Virology,
29
Chinese Academy of Science
30
Wuhan 430071, People’s Republic of China
31
Phone: 86-027-87197575; E-mail: [email protected]
32 33
Number of figures and tables: there are 7 figures in the manuscript
34 35
Electronic word count: abstract: 236; importance paragraph: 129; main text: 4150
36 37
2
Cao et al., HBV quasispecies and viral pathogenesis 38
ABSTRACT
39
Hepatitis B virus (HBV) quasispecies contain a large number of variants that serve as
40
a reservoir for viral selection under antiviral treatment and the immune response,
41
leading to the acute exacerbation and subsequent development of liver failure.
42
However, there is no clear experimental evidence for a significant role of HBV
43
quasispecies in viral pathogenesis. In the present study, HBV sequences were
44
amplified from a patient with severe liver disease and constructed into HBV
45
replication-competent plasmids. Western blot, Enzyme-linked immunosorbent assay
46
(ELISA), and immunofluorescence staining were performed to analyze the expression,
47
secretion and subcellular localization of viral proteins in vitro. Viral replication
48
intermediates were detected by Southern blot. HBV gene expression and replication
49
and the induction of specific immune responses in an HBV hydrodynamic injection
50
(HI) mouse model were investigated. The results demonstrated that two naturally
51
occurring HBV variants, SH and SH-DPS, were identified. The variant SH-DPS
52
expressed only a non-exportable hepatitis B surface antigen (HBsAg) with abnormal
53
intracellular accumulation. The coexistence of the HBV variants at a ratio of 1 to 4
54
(SH to SH-DPS) increased HBV replication. Significantly stronger intrahepatic
55
cytotoxic T lymphocyte (CTL) responses and antibody responses specific to HBsAg
56
were induced in mice by the HBV variants when co-applied by HI. These findings
57
uncovered an unexpected aspect of HBV quasispecies: that the coexistence of
58
different variants can significantly modulate specific host immune responses,
59
representing a novel mechanism for the immunopathogenesis of HBV infection.
3
Cao et al., HBV quasispecies and viral pathogenesis 60
IMPORTANCE
61
Hepatitis B virus (HBV) is a important human pathogen. HBV quasispecies with
62
genetically heterogenous variants are thought to play a role in the progression of
63
HBV-associated liver diseases. So far, direct evidence is only available in few cases to
64
confirm the proposed role of HBV variants in the pathogenesis. We report here that
65
the coexistence of two naturally occurring HBV variants at a ratio of 1 to 4 increased
66
HBV replication and induced significantly stronger intrahepatic cytotoxic T
67
lymphocyte responses and antibody responses specific to HBsAg in mice. Our
68
discovery uncovered an unexpected aspect of HBV quasispecies that the coexistence
69
of different variants can significantly modulate specific host immune responses and
70
may enhance immune-mediated liver damage under some circumstances, representing
71
a novel mechanism for the immunopathogenesis of HBV infection.
72 73
KEYWORDS: Hepatitis B virus, acute liver failure, quasispecies, immune response
74
4
Cao et al., HBV quasispecies and viral pathogenesis INTRODUCTION
75 76
Hepatitis B virus (HBV) infection causes a wide spectrum of clinical
77
manifestations ranging from an asymptomatic carrier state to acute or chronic
78
hepatitis, with progression to liver cirrhosis, hepatocellular carcinoma, and other
79
severe liver diseases (1-4).
80 81
The HBV population in the host consists of genetically heterologous variants and
82
exists in the form of quasispecies. It is proposed that quasispecies may contribute to
83
viral persistence and pathogenesis because quasispecies contain a large number of
84
mutated genes that serve as a reservoir for viral selection under antiviral treatment and
85
the immune response (5). It has been previously reported that co-infection with human
86
immunodeficiency virus (HIV) and HBV results in low quasispecies complexity (4).
87
In patients with HBV infection-related liver failure (HB-LF), the quasispecies showed
88
distinct characteristics with higher complexity and diversity within the HBV precore
89
(preC)/core gene (6). However, there is no clear experimental evidence for a
90
significant role of HBV quasispecies in the pathogenesis of HB-LF.
91 92
A number of publications have suggested that the emergence of HBV variants
93
leads to acute exacerbation and subsequently contributes to the development of liver
94
failure (LF). Some mutations in HBV genomes, such as the A1762T/G1764A double
95
mutation in the basal core promoter (BCP) and the G1896A mutation in the preC
96
region, are thought to play a role in the progression of liver diseases (1, 6-8). These
97
mutations were found to enhance HBV replication in vitro and to abrogate expression
98
of hepatitis B e antigen (HBeAg), which is suspected to interrupt immune tolerance in
99
the host and to contribute to the development of LF (1, 7, 8). However, no direct 5
100
Cao et al., HBV quasispecies and viral pathogenesis evidence is available to confirm the proposed role of HBV mutations in the
101
pathogenesis of HB-LF so far. In addition, other studies have reported contradictory
102
findings, indicating that there is no obvious link between HBV BCP/preC mutations
103
and the development of LF (9, 10). It also remains mechanistically unclear how HBV
104
BCP/preC mutations affect the development of HB-LF.
105 106
In general, HBV variants may cause liver damage by a direct cytopathic effect or
107
by indirectly promoting immunopathology. There are a few examples of exacerbation
108
of liver diseases associated with cytopathic HBV variants (11-15). However, it is
109
currently unknown whether the appearance of HBV variants has any influence on host
110
immune responses which would in turn cause liver damage.
111 112
In the present study, we characterized HBV isolates from a patient with severe
113
liver disease and identified two major HBV variants, HBV-SH (SH) and
114
HBV-SH-DPS (SH-DPS), which harbored a number of mutations including two
115
deletions within the preS regions and hepatitis B surface antigen (HBsAg) sequences.
116
The variant SH-DPS expressed only a non-exportable SHBsAg with abnormal
117
intracellular accumulation. Both SH and SH-DPS coexisted at a ratio of 1 to 4. These
118
two isolates were phenotypically characterized alone or together in different ratios by
119
transient transfection. The results demonstrated that the coexistence of SH and
120
SH-DPS at a ratio of 1 to 4 increased HBV replication and led to a predominant
121
nuclear localization of HBV core antigen (HBcAg). Using an HBV hydrodynamic
122
injection (HI) mouse model, we found that mice mounted significantly stronger
123
antibody and cytotoxic T lymphocyte (CTL) responses to HBsAg only if SH and
124
SH-DPS were co-applied. Thus, the coexistence of different variants may
6
125
Cao et al., HBV quasispecies and viral pathogenesis significantly modulate specific host immune responses and may enhance
126
immune-mediated liver damage under some circumstances, representing a novel
127
mechanism for the immunopathogenesis of HBV infection.
128
7
Cao et al., HBV quasispecies and viral pathogenesis MATERIALS AND METHODS
129 130
Patient. A 38-year-old male patient from China had a history of chronic hepatitis
131
B infection for over 30 years. He was positive for HBsAg and the antibody to the
132
hepatitis B e antigen (anti-HBe) and was negative for HBeAg and the antibody to
133
HBsAg (anti-HBs).
134 135
The patient was diagnosed with HB-LF manifesting as a rise in
136
alanine aminotransferase (ALT) to 283 U/L along with HBV DNA levels > 106
137
copies/ml, jaundice (bilirubin, 7.9 mg/dL) and coagulopathy (grade II), complicated
138
within 4 weeks by ascites and encephalopathy. The patient received artificial liver
139
support 3 times as well as other treatments, but the illness worsened precipitously,
140
complicated by hepatic encephalopathy, infection and hepatorenal syndrome (Fig. 1).
141 142
The patient gave signed, informed consent. Sample collection, processing, and
143
storage conformed to the ethical guidelines of the 1975 Declaration of Helsinki as
144
reflected in a prior approval by the institution's human research committee.
145 146
Characterization of HBV isolates from patient serum samples and cloning.
147
Isolation of HBV viral DNA from patient serum samples was performed as described
148
previously with minor modifications (16, 17). A polymerase chain reaction (PCR) was
149
performed to amplify 2.1 kb fragment (1821 -699 bp) and 1.2 kb fragment (669 -1825
150
bp) with the primer pairs P1/P3 and P2/P4, respectively: P1, 5’-CCGGC
8
Cao et al., HBV quasispecies and viral pathogenesis 151
GTCGACGAGCTCTTCTTTTTCACCTCTGCCTAATCA-3’ (nt1821-1841); P2, 5’-
152
CCGGCGTCGACGAGCTCTTCAAAAAGTTGCATGGTGCTGG-3’ (nt1825-1806);
153
P3, 5’-CACTGAACAAATGGCACTAGTAAACTGAGCC-3’ (nt699-669); P4, 5’-G
154
GCTCAGTTTACTAGTGCCATTTGTTCAGTG-3’ (nt669-699). To reduce the
155
possibility of error-prone amplification, an enzyme with excellent high PCR fidelity
156
and efficiency, KOD-PLUS (Toyobo) was used in PCR. The two PCR products were
157
cloned into the pGEM®-T Easy vector (Promega, Madison, Wisconsin, USA) for
158
sequence analysis (ten clones of each fragment). The sequences of the 1.2 kb
159
fragments were consistent, while the 2.1 kb fragment had two types with either
160
entirely HBsAg gene (2 clones) or 2 deletions within HBsAg gene (8 clones) (Fig.
161
2A). To get full-length HBV genomes, the 1.2 kb SacI-SpeI fragment and a 2.1 kb
162
SalI-SpeI fragment were released from the pGEM®-T Easy vector and cloned into the
163
cloning vector pUC19 pre-digested with SalI and SacI. The two head-to-tail fragments
164
were sub-cloned into the pUC19 vector and resulted in pUC19-HBV1-SH and
165
pUC19-HBV1-SH-DPS harboring a complete HBV genome and a type of mutant
166
genome with two deletions within the HBV preS region (GenBank accession number:
167
SH: KC492739.1 and SH-DPS KC492740.1) (Fig. 2A).
168 169
Two plasmids with replication-competent, 1.3-fold HBV genomes, pUC19-
170
HBV1.3-SH (pSH) and pUC19-HBV1.3-SH-DPS (pSH-DPS) (18), and a positive
171
control (PC) pUC19-HBV1.3-B were constructed as described previously (19). A
172
previously described replication deficient HBV clone pHBV1.3-rtG244Y (20) was
9
Cao et al., HBV quasispecies and viral pathogenesis 173
used to serve as a negative control (NC) in Southern blot experiment. Additionally,
174
the 1.3-fold over-length HBV genomes were sub-cloned into the pAAV vector to
175
produce pAAV-SH, pAAV-SH-DPS and pAAV-PC for HI in mice.
176 177
Cells and mice. The human hepatoma cell line Huh7 (provided by American
178
Type Culture Collection, Manassas, VA) was maintained and transfected as described
179
previously (20). Male C57BL/6 (H-2b) mice (6 to 8 weeks of age) were kept under
180
specific-pathogen-free (SPF) conditions in the Central Animal Laboratory of Wuhan
181
Institute of Virology, Chinese Academy of Sciences and treated by following the
182
guidelines of the institutional animal ethical standard.
183 184
Western blot analysis. Western blot analysis was performed as described
185
previously (21, 22). The following antibodies were used: anti-HBs (Abcam,
186
Cambridge, UK), anti-HBc (Santa Cruz, Santa Cruz, CA) and anti-beta-actin (Santa
187
Cruz, Santa Cruz, CA). Relative band intensities for viral proteins were quantified
188
using NIH ImageJ software.
189 190
Enzyme-linked immunosorbent assay (ELISA). HBsAg, HBeAg, antibodies to
191
HBsAg in mouse sera, culture supernatants, and cell lysates of transfected cells were
192
detected as described previously (23-25).
193 194
Immunofluorescence (IF) staining and confocal laser scanning microscopy.
10
Cao et al., HBV quasispecies and viral pathogenesis 195
Indirect IF staining of transfected cells was performed as described previously (21, 23,
196
24). Anti-HBc (Dako, Carpinteria, CA) and anti-HBs (S1, kindly provided by Yan Bin)
197
were used as primary antibodies and Alexa Fluor 488-conjugated and Alexa Fluor
198
568-conjugated antibodies (Life Technologies, Carlsbad, CA) were used for
199
secondary detection.
200 201
Southern Blot Analysis. Huh7 cells were transiently transfected with 2.5 μg of
202
pHBV-B，pSH or pSH-DPS alone or co-transfected with 2.5 μg of mixtures of pSH
203
and pSH-DPS, or pHBV-B and pSH-DPS at the indicated ratios. Encapsidated HBV
204
replication intermediates were extracted and subjected to Southern blot analysis as
205
described previously (20). The hybridization signals were quantified with ImageJ
206
software (National Institutes of Health).
207 208
HBV challenge by hydrodynamic injection. Mice in each group were
209
challenged by hydrodynamic injection (HI) as described previously with minor
210
modifications (17). In brief, 10 μg of HBV plasmid DNA was incubated with 20 μl
211
Lipofectamine 2000 (Life Technologies, Carlsbad, CA) for 20 min at room
212
temperature to form the DNA–Lipofectamine 2000 complex. Then, the complexes
213
were injected into the tail veins of mice in a volume of PBS equivalent to 8% of the
214
mouse body weight within 5 seconds.
215 216
Detection of serum HBV DNA and intrahepatic core-associated HBV DNA.
11
Cao et al., HBV quasispecies and viral pathogenesis 217
Purification
of
218
quantification of HBV DNA were performed as described previously (23, 26). The
219
real-time
220
5’-AAATCTCCAGTCACTCACCAACC-3’ (nt321-343); P6, 5’-CATAGCAGCAGG
221
ATGCAGAGG-3’ (nt423-403); TaqMan probe, 5’-FAM-TCCTCCAATTTGTCCT
222
GGTTATCGCT-MGB-3’ (nt349-374). The plasmid pHBV-B was used in 10-fold
223
serial dilutions ranging from 101 to 109 copies per reaction as a standard curve.
PCR
core-associated
primers
P5/P6
HBV
and
the
DNA
TaqMan
in
liver
probe
tissue
are
used:
and
P5,
224 225
Immunohistochemistry (IHC). Liver tissues were collected from mice
226
sacrificed at 5 days post-hydrodynamic injection (dpi) and subjected to IHC staining
227
as described previously (20). Intrahepatic HBcAg and HBsAg were detected by IHC
228
staining of formalin-fixed, paraffin-embedded liver tissue sections using anti-HBc
229
antibody (Dako, Carpinteria, CA) and anti-HBs antibody (Thermo Scientific Pierce，
230
Rockford, IL) with an appropriate HRP-conjugated secondary antibody and visualized
231
by the Envision System.
232 233
Intracellular cytokines staining and flow cytometry. Splenocytes and
234
intrahepatic lymphocytes were isolated from mice at 14 and 28 days and subjected to
235
flow cytometry analysis (27). Lymphocytes were supplemented with CD28 (1:1000,
236
ebioscience，San Diego, CA) and Brefeldin A (1:1000, ebioscience，San Diego, CA),
237
stimulated with or without peptide derived from HBcAg and HBsAg corresponding to
238
S protein CD8+ T cells epitope (Kb/S190–197, VWLSVIWM), core protein CD8+ T
12
Cao et al., HBV quasispecies and viral pathogenesis 239
cells epitope (Kb/C93–100, VWLSVIWM), S protein CD4+ T cells epitope (S182-196,
240
FFLLTFULTIFQSLD)
241
TPPAYRPPNAPIL), respectively, and then stained for surface marker CD4 and CD8
242
(BD Biosciences, San Jose, CA). Lymphocytes were fixed and permeabilized in
243
Cytofix/Cytoperm solution (Cytofix/CytopermTM kit, BD Biosciences, San Jose, CA)
244
followed by intracellular staining for IFN-γ (BD Biosciences, San Jose, CA). Dead
245
cells were excluded by staining with 7-aminoactinomycin D (7AAD, Biolegend, San
246
Diego, CA).
and
core
protein
CD4+
T
cells
epitope
(C128-140,
247 248
Statistical
analysis.
The
statistical
analysis
was
carried
out
using
249
GraphPadPrism 5.0 software (Graphpad Software). Two tailed t-test was used to
250
determine the differences in multiple comparisons. A P value < 0.05 was considered
251
statistically significant. Results were presented as mean ± standard deviation (SD).
252
13
Cao et al., HBV quasispecies and viral pathogenesis RESULTS
253 254
Sequence analysis of HBV genomes. First, HBV DNA was isolated from the
255
serum of the patient and subjected to sequence analysis. A comparison of the cloned
256
HBV DNA sequences with published HBV sequences in Genbank indicated that the
257
isolates belong to the genotype B and serotype adw2 and harbor the A1762T/G1764A
258
double mutation in the BCP region and the G1896A mutation in the preC region (Fig.
259
2A). The A1762T/G1764A double mutation has been reported to reduce the
260
production of HBeAg and to enhance the HBV replication level in vitro (28, 29),
261
whereas the G1896A stop codon mutation abolishes HBeAg expression (30). In 8 of
262
10 sequenced HBV genomes, 2 deletions in the preS1 region (nt 2976-3102) and in
263
the preS2 promoter and preS2 open reading frame (ORF) region (nt 3203-3215, nt
264
1-31) were found (Fig. 2A), resulting in a stop codon mutation in the ORF of large
265
HBsAg (LHBsAg) and the deletion of the start codon in the ORF of middle HBsAg
266
(MHBsAg), respectively. In addition, two typical point mutations, N146S and P120S,
267
were present. The mutation N146S prevents the glycosylation of HBV envelope
268
proteins (31). The mutation P120S may be associated with immune escape (Fig. 2A)
269
(32). Thus, two major HBV variants coexisted in this patient; the one with complete
270
HBsAg was designated as SH, and the other one with deletion mutations in HBsAg
271
ORFs was designated as SH-DPS. The ratio of SH to SH-DPS was 1 to 4 based on the
272
numbers of the sequenced clones.
273 274
Phenotypic characterization of SH and SH-DPS in vitro. The plasmids pSH
14
Cao et al., HBV quasispecies and viral pathogenesis 275
and pSH-DPS containing the respective 1.3-fold over-length HBV genomes were
276
transfected into Huh7 cells. PSH produced much less intracellular and supernatant
277
HBsAg (52% and 30%, respectively) than PC (32) (Fig. 2B). PSH-DPS did not
278
produce detectable HBsAg in supernatant but produced a low level of intracellular
279
HBsAg. These results suggested that HBsAg with deletions and mutations encoded by
280
SH-DPS was secretion deficient. No HBeAg was detected in culture supernatants,
281
consistent with the presence of the G1896A mutation in the preC region that abolishes
282
HBeAg expression (Fig. 2C) (1, 2). WB with anti-HBs antibodies indicated that
283
glycosylated and un-glycosylated bands corresponding to L, M and SHBsAg were
284
detected for both SH and PC as reported previously (33), while only the
285
non-glycosylated form of SHBsAg could be detected for SH-DPS, consistent with the
286
sequence information that included the termination of L and MHBsAg and the loss of
287
the glycosylation site by N146S substitution (Fig. 2D). Based on immunofluorescence
288
(IF) staining with anti-HBs antibodies, HBsAg was evenly distributed in the
289
cytoplasm of cells transfected with pSH or PC (Fig. 2E) (23). In contrast, a dot-like
290
distribution of HBsAg was observed in the cytoplasm of cells transfected with
291
pSH-DPS (Fig. 1E). Thus, the mutations in HBsAg expressed from pSH-DPS caused
292
abnormal localization of HBsAg and prevented its secretion.
293 294
The coexistence of SH and SH-DPS at a ratio of 1 to 4 increased HBV
295
replication. We co-transfected pSH and pSH-DPS at different ratios and analyzed
296
HBV gene expression and replication by enzyme linked immunosorbent assay
15
Cao et al., HBV quasispecies and viral pathogenesis 297
(ELISA), Western blot, Southern blot, and IF staining (Fig. 3). Increasing amounts of
298
pSH-DPS led to a decrease of HBsAg production in supernatant (Fig. 3A), as well as
299
a decrease of HBcAg expression level in cell lysates (Fig.3B). Both the pSH and
300
pSH-DPS clones were replication competent. However, pSH-DPS produced much
301
more core-associated DNA than pSH (Fig. 3B and C). Interestingly, when pSH and
302
pSH-DPS were co-transfected at different ratios, the highest amount of
303
core-associated HBV replication intermediates was detected at a ratio of 1 to 4, which
304
yielded a 15% increase compared to pSH-DPS alone (Fig. 3B and C). This
305
demonstrated a synergistic effect of HBV quasispecies on genomic replication.
306 307
The coexistence of SH and SH-DPS led to a predominant nuclear
308
localization of HBcAg. In Fig. 3D, HBcAg evenly distributed in both the cytoplasm
309
and nucleus in cells transfected with pSH-DPS but only in the cytoplasm in cells
310
transfected with pSH and PC. When the ratio of pSH and pSH-DPS was 4 to 1, both
311
HBsAg and HBcAg were evenly distributed in the cytoplasm, similar to pSH
312
transfection. At a ratio of 1 to 4, HBsAg aggregated in the cytoplasm and HBcAg was
313
located both in the cytoplasm and nucleus similar to the distribution with pSH-DPS
314
transfection.
315 316
Phenotypic characterization of SH and SH-DPS in vivo. We further
317
characterized these two HBV variants using an HBV HI mouse model (23, 25).
318
pAAV-SH and pAAV-SH-DPS were generated by cloning the 1.3-fold over-length
16
Cao et al., HBV quasispecies and viral pathogenesis 319
HBV genomes into the pAAV plasmid. After HI of HBV constructs into C57BL/6
320
mice, serum HBsAg levels were monitored from day 1 on and up to day 63 post-HI. A
321
high serum HBsAg level was detected in all mice receiving pAAV-PC and pAAV-SH
322
at 1 dpi to 7 dpi, then declined to an undetectable level at 28 dpi (Fig. 4A). HBsAg
323
was not detected in mice receiving pAAV-SH-DPS, consistent with the secretion
324
deficiency of mutated HBsAg (Fig. 4A). For the pAAV-SH and pAAV-SH-DPS
325
groups co-injected at a ratio of 4 to 1, HBsAg was detected at 1 dpi and
326
seroconversion occurred at 21 dpi (Fig. 4A). However, when pAAV-SH and
327
pAAV-SH-DPS were co-injected at a ratio of 1 to 4, no HBsAg was detected (Fig.
328
4A).
329 330
Further, we detected serum and hepatic HBV DNA in mice by real-time PCR at
331
the indicated time points after HI. In pAAV-PC-injected mice, the serum HBV DNA
332
level was 1.4×106 copies/ml at 3 dpi and increased to 2.4×107 copies/ml at 5 dpi (Fig.
333
4B). HI with pAAV-SH in mice resulted in significantly lower serum HBV DNA
334
levels, at approximately 6% of the levels in the control with pAAV-HBV-B. The
335
serum HBV DNA levels were below the detection limit in other mice receiving
336
pAAV-SH-DPS
337
intrahepatic HBV DNA levels at 5 dpi were positive in all mice injected with
338
pAAV-SH, pAAV-SH-DPS, and pAAV-SH/pAAV-SH-DPS but were less than 10% of
339
that of the PC group (Fig. 4C), indicating that both SH and SH-DPS were replication
340
competent in vivo but at a lower level. However, there were no significant differences
and
pAAV-SH/pAAV-SH-DPS
co-injection
(Fig.
4B).
The
17
Cao et al., HBV quasispecies and viral pathogenesis 341
among these groups. Southern blot hybridization detected HBV replication
342
intermediates from the PC group but significantly lower levels of intermediates from
343
the liver samples from the other groups (Fig. 4C).
344 345
Immunohistochemistry (IHC) staining for HBsAg and HBcAg with liver tissue
346
sections collected at 5 dpi showed a cytoplasmic distribution of HBsAg in mice
347
injected
348
pAAV-SH-DPS-injected mice (Fig. 5). The staining of HBcAg showed both
349
cytoplasmic and nuclear distribution (Fig. 5B). When SH and SH-DPS were
350
co-injected, both diffused distribution and dot-like distribution of HBsAg in the
351
cytoplasm were observed (Fig. 5A). Additionally, the percentage of HBsAg- or
352
HBcAg-positive hepatocytes was calculated and positively related to the intrahepatic
353
HBV DNA levels of each group (Figs. 5A and B).
with
pAAV-PC
and
pAAV-SH
and
a
dot-like
distribution
in
354 355
HBV-specific antibody- and cell-mediated immune responses. The levels of
356
HBsAg in the sera of all mice receiving pAAV-PC and pAAV-SH declined to an
357
undetectable level at 28 dpi. Inversely, anti-HBs antibodies were initially undetectable
358
but became positive at 21 dpi and then were maintained at a high level through the
359
experiment period in mice of both groups (Fig. 6A). However, like HBsAg, anti-HBs
360
antibodies were not detected in mice receiving pAAV-SH-DPS, consistent with the
361
secretion deficiency of mutated HBsAg (Fig. 4A). Strikingly, when pAAV-SH and
362
pAAV-SH-DPS were co-injected at a ratio of 1 to 4, a significantly higher anti-HBs
18
Cao et al., HBV quasispecies and viral pathogenesis 363
antibody response was induced in the mice of this group, although we could not detect
364
HBsAg previously (Figs. 4A, 6A and B). When mice were co-injected with pAAV-SH
365
and pAAV-SH-DPS at a ratio of 4 to 1, the HBsAg levels decreased before 21 dpi and
366
anti-HBs antibody levels increased after 21 dpi (Figs. 4A and 6A). Therefore, the
367
coexistence of SH and SH-DPS variants enhanced the HBsAg-specific immune
368
responses.
369 370
The IgG1/IgG2a antibody responses could serve as an indication for the Th bias
371
of specific immune responses. Thus, we determined the subtypes of HBsAg-specific
372
IgGs. The IgG1 response to HBsAg was comparable in all mice receiving SH,
373
SH/SH-DPS at the ratio of 1 to 4 and PC. Interestingly, HBsAg-specific IgG2a
374
antibodies were only detected in mice receiving SH/SH-DPS (Figs. 6C and D),
375
suggesting that the coexistence of SH and SH-DPS induced a significantly stronger
376
Th1 response.
377 378
Next, we further examined the induction of cell-mediated, specific immune
379
responses to HBsAg and HBcAg. Splenocytes and intrahepatic lymphocytes were
380
isolated at 14 and 28 dpi, respectively, and stimulated with two H-2kb-restricted CTL
381
epitope peptides derived from HBsAg and HBcAg, as well as a CD4+ T cell epitope
382
peptide. Flow cytometric analysis was performed for the detection of CD4+ and
383
CD8+ IFN-γ-producing cells. CD8+ IFN-γ-producing cell populations specific to
384
HBsAg were detected in mice from all groups except the mock group at 14 dpi and 28
19
Cao et al., HBV quasispecies and viral pathogenesis 385
dpi. Notably, at 28 dpi, the frequencies of CD8+ IFN-γ-producing cells were the
386
highest if pAAV-SH and pAAV-SH-DPS were co-injected at a ratio of 1:4 (Figs. 7A
387
and B), consistent with the production of anti-HBs antibody. However, the HBsAg-
388
and HBcAg-specific CD4+ T cell response and the HBcAg-specific CD8+ T cell
389
response in all mice were not detected in both splenocytes and intrahepatic
390
lymphocytes (Figs. 7C, D, E, F, G and H).
391 392
DISCUSSION
393
HBV mutants are thought to play a role in the progression of HBV-associated
394
liver diseases (1, 6-8). The HBV population in the host exists in the form of
395
quasispecies and contains a great spectrum of mutants (5). The role of HBV
396
quasispecies in the pathogenesis of HBV infection is currently not understood. In the
397
present study, we found that the coexistence of two naturally occurring HBV variants
398
at a ratio of 1 to 4 increased HBV replication and led to a predominant nuclear
399
localization of HBcAg. More importantly, significantly stronger intrahepatic CTL
400
responses and antibody responses specific to HBsAg were induced in mice by these
401
HBV variants when co-applied by HI. These findings revealed the potential role of
402
HBV quasispecies and their mutations in the pathogenesis of HBV infection.
403 404
The cell-mediated immune response is considered to contribute to both viral
405
clearance and liver injury in HBV infection (34). In mice receiving HI with HBV
406
genomes, a higher frequency of HBsAg-specific CD8+ T cells was detected in the
407
liver than in the spleen. Strikingly, the coexistence of SH and SH-DPS at a ratio of 1
20
Cao et al., HBV quasispecies and viral pathogenesis 408
to 4 induced a stronger CTL response than each HBV vector alone (Figs. 7A and B).
409
At the same time, the magnitude and the IgG subtype distribution of anti-HBs
410
antibody responses were significantly changed by the coexistence of the HBV
411
quasispecies (Figs. 6B, C and D). Both higher CTL frequencies and the appearance of
412
the IgG2a subtype of anti-HBs antibodies indicated a shift of HBV-specific immune
413
responses toward the Th1 type. The enhancement of HBV-specific immune response
414
may be due to secretion defect of HBsAg. In addition, increased HBV replication
415
activity when both HBV clones were co-injected and changed subcellular localization
416
of HBsAg may also contribute to the induction of stronger specific immune responses.
417 418
Though our study does not prove a direct link between the HBV quasispecies and
419
HBV-associated severe liver diseases, it hints at a possible explanation for the nearly
420
obligatory presence of HBV variants in patients suffering from acute LF. It also gives
421
an idea for why the characterization of HBV variants in cell culture systems seldom
422
delivers a clear answer about their pathogenic potential. We could hypothesize that
423
only the combination of different WT and mutant proteins effectively trigger specific
424
host immune responses and leads to observed immunopathology in LF patients. This
425
hypothesis implies that HBV variants are not innocent bystanders selected by
426
decreased liver functions but are the major causes for LF, and thus may need attention
427
in clinical monitoring.
428 429
Previously, the A1762T/G1764A and G1896A mutations in BCP/preC were
21
Cao et al., HBV quasispecies and viral pathogenesis 430
found to affect HBV replication and to abrogate expression of HBeAg. It was
431
suspected that these mutations may contribute to the development of LF (1, 6-8).
432
However, most of these results were based on in vitro transfection of single HBV
433
replication-competent plasmids with such mutations. Here, co-transfection of two
434
HBV replication-competent plasmids increased viral replication in the ratio of 1 to 4,
435
suggesting that the HBV quasispecies should be paid more attention in clinical
436
monitoring.
437 438
HBcAg tended to localize in the cytoplasm in the presence of the
439
A1762T/G1764A double mutation (35, 36). However, the localization of HBcAg from
440
a fulminant HBV strain was changed to the nucleus when both G1862T and G1896A
441
were also present (3). Early studies on clinical samples indicated that the subcellular
442
localization of HBcAg was closely correlated with hepatitis activity in
443
HBeAg-positive patients (37, 38). However, the relationship between intracellular
444
distribution of HBcAg and hepatitis activity is unknown in HBeAg-negative or
445
anti-HBe-positive patients. Here, we observed a predominant nuclear localization of
446
HBcAg when SH DNA and SH-DPS DNA were transfected into cells at a ratio of 1 to
447
4 (Fig. 3C), suggesting that the coexistence of SH and SH-DPS changed the
448
preferential localization of HBcAg from the cytoplasmic to nuclear compartment. To
449
clarify the mechanism, we performed Western blot and found that increasing amounts
450
of pSH-DPS led to a decrease of HBcAg expression level (Fig.3B). Low level of
451
HBcAg could not drive capsid assembly and virion formation in cytoplasm for
22
Cao et al., HBV quasispecies and viral pathogenesis 452
budding (39) but could freely enter the nucleus by nuclear pore complex (40).
453
Moreover, mature nucleocapsids could be targeted back to the nucleus and amplify the
454
pool of cccDNA, or be targeted to the Endoplasmic reticulum to bud and exit the cell.
455
Envelope proteins play a regulatory role in the process (41). However, SH-DPS
456
produced a low level of intracellular HBsAg (Fig.2). Therefore the difference in core
457
protein localization between SH, SH-DPS and the co-existence of SH and SH-DPS
458
was observed. However, the mechanism determining the shuttle of HBcAg between
459
the nucleus and cytoplasm is not completely understood. Nuclear localization/export
460
signals and some host factors may play a role in this process (42). The presence of
461
different HBV proteins including mutated HBsAg may change the expression or
462
function of such viral and host factors. Future investigation is required to identify the
463
factors involved in the regulation of HBcAg localization.
464 465
To our knowledge, the present study demonstrated for the first time that the
466
interaction of HBV quasispecies may enhance HBV replication and induce stronger
467
host immune responses, thereby potentially contributing to acute exacerbation of liver
468
diseases and the development of liver failure.
469 470 471 472
ACKNOWLEDGMENTS We are grateful to Bin Yan, Xuefang An, Yuan Zhou, and Xue Hu for excellent technical support. We also thank Pei-Jer Chen for helpful suggestions.
473
This work was supported by the National Basic Research Priorities Program of
474
China (2012CB519001) and the National Nature Science Foundation of China 23
Cao et al., HBV quasispecies and viral pathogenesis 475
(31200699).
476
REFERENCES
477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515
1.
Ren X, Xu Z, Liu Y, Li X, Bai S, Ding N, Zhong Y, Wang L, Mao P, Zoulim F, Xu D. 2010. Hepatitis B virus genotype and basal core promoter/precore mutations are associated with hepatitis B-related acute-on-chronic liver failure without pre-existing liver cirrhosis. J Viral Hepat 17:887-895.
2.
Kay A, Zoulim F. 2007. Hepatitis B virus genetic variability and evolution. Virus Res
3.
Inoue J, Ueno Y, Nagasaki F, Wakui Y, Kondo Y, Fukushima K, Niitsuma H,
127:164-176. Shimosegawa T. 2009. Enhanced intracellular retention of a hepatitis B virus strain associated with fulminant hepatitis. Virology 395:202-209. 4.
Bernal W, Auzinger G, Dhawan A, Wendon J. 2010. Acute liver failure. Lancet
5.
Pawlotsky JM. 2005. The concept of hepatitis B virus mutant escape. J Clin Virol 34 Suppl
6.
Chen L, Zheng CX, Lin MH, Huang ZX, Chen RH, Li QG, Li Q, Chen P. 2013. Distinct
376:190-201. 1:S125-129. quasispecies characteristics and positive selection within precore/core gene in hepatitis B virus HBV associated acute-on-chronic liver failure. J Gastroenterol Hepatol 28:1040-1046. 7.
Baumert TF, Rogers SA, Hasegawa K, Liang TJ. 1996. Two core promotor mutations identified in a hepatitis B virus strain associated with fulminant hepatitis result in enhanced viral replication. J Clin Invest 98:2268-2276.
8.
Tong S, Kim KH, Chante C, Wands J, Li J. 2005. Hepatitis B Virus e Antigen Variants. Int J
9.
Sterneck M, Gunther S, Santantonio T, Fischer L, Broelsch CE, Greten H, Will H. 1996.
Med Sci 2:2-7. Hepatitis B virus genomes of patients with fulminant hepatitis do not share a specific mutation. Hepatology 24:300-306. 10.
Chun YK, Kim JY, Woo HJ, Oh SM, Kang I, Ha J, Kim SS. 2000. No significant correlation exists between core promoter mutations, viral replication, and liver damage in chronic hepatitis B infection. Hepatology 32:1154-1162.
11.
Bock CT, Tillmann HL, Maschek HJ, Manns MP, Trautwein C. 1997. A preS mutation isolated from a patient with chronic hepatitis B infection leads to virus retention and misassembly. Gastroenterology 113:1976-1982.
12.
Chisari FV, Filippi P, Buras J, McLachlan A, Popper H, Pinkert CA, Palmiter RD, Brinster RL. 1987. Structural and pathological effects of synthesis of hepatitis B virus large envelope polypeptide in transgenic mice. Proc Natl Acad Sci U S A 84:6909-6913.
13.
Fan YF, Lu CC, Chang YC, Chang TT, Lin PW, Lei HY, Su IJ. 2000. Identification of a pre-S2 mutant in hepatocytes expressing a novel marginal pattern of surface antigen in advanced diseases of chronic hepatitis B virus infection. J Gastroenterol Hepatol 15:519-528.
14.
Gilles PN, Guerrette DL, Ulevitch RJ, Schreiber RD, Chisari FV. 1992. HBsAg retention sensitizes the hepatocyte to injury by physiological concentrations of interferon-gamma.
24
Cao et al., HBV quasispecies and viral pathogenesis 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559
Hepatology 16:655-663. 15.
Wang HC, Wu HC, Chen CF, Fausto N, Lei HY, Su IJ. 2003. Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress. Am J Pathol 163:2441-2449.
16.
Gunther S, Li BC, Miska S, Kruger DH, Meisel H, Will H. 1995. A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients. J Virol 69:5437-5444.
17.
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 U S A 103:17862-17867.
18.
Alcantara FF, Tang H, McLachlan A. 2002. Functional characterization of the interferon regulatory element in the enhancer 1 region of the hepatitis B virus genome. Nucleic Acids Res 30:2068-2075.
19.
Li L, Shen H, Li A, Zhang Z, Wang B, Wang J, Zheng X, Wu J, Yang D, Lu M, Song J. 2013. Inhibition of hepatitis B virus (HBV) gene expression and replication by HBx gene silencing in a hydrodynamic injection mouse model with a new clone of HBV genotype B. Virol J 10:214.
20.
Qin B, Budeus B, Cao L, Wu C, Wang Y, Zhang X, Rayner S, Hoffmann D, Lu M, Chen X. 2013. The amino acid substitutions rtP177G and rtF249A in the reverse transcriptase domain of hepatitis B virus polymerase reduce the susceptibility to tenofovir. Antiviral Res 97:93-100.
21.
Xu S, Pei R, Guo M, Han Q, Lai J, Wang Y, Wu C, Zhou Y, Lu M, Chen X. 2012. Cytosolic phospholipase A2 gamma is involved in hepatitis C virus replication and assembly. J Virol 86:13025-13037.
22.
Pei R, Zhang X, Xu S, Meng Z, Roggendorf M, Lu M, Chen X. 2012. Regulation of hepatitis C virus replication and gene expression by the MAPK-ERK pathway. Virol Sin 27:278-285.
23.
Wu C, Deng W, Deng L, Cao L, Qin B, Li S, Wang Y, Pei R, Yang D, Lu M, Chen X. 2012. Amino acid substitutions at positions 122 and 145 of hepatitis B virus surface antigen (HBsAg) determine the antigenicity and immunogenicity of HBsAg and influence in vivo HBsAg clearance. J Virol 86:4658-4669.
24.
Wu C, Zhang X, Tian Y, Song J, Yang D, Roggendorf M, Lu M, Chen X. 2010. Biological significance of amino acid substitutions in hepatitis B surface antigen (HBsAg) for glycosylation, secretion, antigenicity and immunogenicity of HBsAg and hepatitis B virus replication. J Gen Virol 91:483-492.
25.
Yin Y, Wu C, Song J, Wang J, Zhang E, Liu H, Yang D, Chen X, Lu M, Xu Y. 2011. DNA immunization with fusion of CTLA-4 to hepatitis B virus (HBV) core protein enhanced Th2 type responses and cleared HBV with an accelerated kinetic. PLoS One 6:e22524.
26.
Weinberger KM, Wiedenmann E, Bohm S, Jilg W. 2000. Sensitive and accurate quantitation of hepatitis B virus DNA using a kinetic fluorescence detection system (TaqMan PCR). J Virol Methods 85:75-82.
27.
Qu D, Lanier G, Yuan ZH, Wen YM, Howard CR, Ahmed R. 2008. Localization of CD8+ cells specific for hepatitis B virus surface protein in the liver of immunized mice. J Med Virol 80:225-232.
25
Cao et al., HBV quasispecies and viral pathogenesis 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603
28.
Buckwold VE, Xu Z, Chen M, Yen TS, Ou JH. 1996. Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication. J Virol 70:5845-5851.
29.
Moriyama K, Okamoto H, Tsuda F, Mayumi M. 1996. Reduced precore transcription and enhanced core-pregenome transcription of hepatitis B virus DNA after replacement of the precore-core promoter with sequences associated with e antigen-seronegative persistent infections. Virology 226:269-280.
30.
Carman WF, Jacyna MR, Hadziyannis S, Karayiannis P, McGarvey MJ, Makris A, Thomas HC. 1989. Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet 2:588-591.
31.
Ito K, Qin Y, Guarnieri M, Garcia T, Kwei K, Mizokami M, Zhang J, Li J, Wands JR, Tong S. 2010. Impairment of hepatitis B virus virion secretion by single-amino-acid substitutions in the small envelope protein and rescue by a novel glycosylation site. J Virol 84:12850-12861.
32.
Shin D, Kim SI, Kim M, Park M. 2006. Efficient inhibition of hepatitis B virus replication
33.
Lepere-Douard C, Trotard M, Le Seyec J, Gripon P. 2009. The first transmembrane
by small interfering RNAs targeted to the viral X gene in mice. Virus Res 119:146-153. domain of the hepatitis B virus large envelope protein is crucial for infectivity. J Virol 83:11819-11829. 34.
Chisari FV. 2000. Rous-Whipple Award Lecture. Viruses, immunity, and cancer: lessons from hepatitis B. Am J Pathol 156:1117-1132.
35.
Kawai K, Horiike N, Michitaka K, Onji M. 2003. The effects of hepatitis B virus core promoter mutations on hepatitis B core antigen distribution in hepatocytes as detected by laser-assisted microdissection. J Hepatol 38:635-641.
36.
Liu CJ, Jeng YM, Chen CL, Cheng HR, Chen PJ, Chen TC, Liu CH, Lai MY, Chen DS, Kao JH. 2009. Hepatitis B virus basal core promoter mutation and DNA load correlate with expression of hepatitis B core antigen in patients with chronic hepatitis B. J Infect Dis 199:742-749.
37.
Chu CM, Liaw YF. 1987. Intrahepatic distribution of hepatitis B surface and core antigens in chronic hepatitis B virus infection. Hepatocyte with cytoplasmic/membranous hepatitis B core antigen as a possible target for immune hepatocytolysis. Gastroenterology 92:220-225.
38.
Chu CM, Yeh CT, Sheen IS, Liaw YF. 1995. Subcellular localization of hepatitis B core antigen in relation to hepatocyte regeneration in chronic hepatitis B. Gastroenterology 109:1926-1932.
39.
Seifer M, Standring DN. 1995. Assembly and antigenicity of hepatitis B virus core particles.
40.
Rabe B, Delaleau M, Bischof A, Foss M, Sominskaya I, Pumpens P, Cazenave C,
Intervirology 38:47-62. Castroviejo M, Kann M. 2009. Nuclear entry of hepatitis B virus capsids involves disintegration to protein dimers followed by nuclear reassociation to capsids. PLoS Pathog 5:e1000563. 41.
Lentz TB, Loeb DD. 2011. Roles of the envelope proteins in the amplification of covalently closed circular DNA and completion of synthesis of the plus-strand DNA in hepatitis B virus. J Virol 85:11916-11927.
42.
Li HC, Huang EY, Su PY, Wu SY, Yang CC, Lin YS, Chang WC, Shih C. 2010. Nuclear
26
Cao et al., HBV quasispecies and viral pathogenesis 604 605 606
export and import of human hepatitis B virus capsid protein and particles. PLoS Pathog 6:e1001162.
607 608
FIGURE LEGENDS
609
Fig. 1. Clinical course of the patient with HB-LF. (A) The level of serum
610
transaminase (ALT and AST), total bilirubin (TBIL) and direct bilirubin (DBIL) level.
611
(B) The level of PT (prothrombin time), PTA (prothrombin time activity percentage),
612
APTT (activated partial thromboplastin time) and FIB (fibriogen).
613 614
Fig. 2. Expression, secretion and subcellular localization of viral proteins. (A)
615
Schematic representation of SH and SH-DPS. Stop codon in the preS1 ORF is
616
indicated by an asterisk. The mutations and deletions are marked by dark arrows. (B,
617
C, D and E) Huh7 cells were transfected with PC, pSH or pSH-DPS. At 72 hours (h)
618
post transfection, HBsAg (B) and HBeAg (C) in culture supernatants and cell lysates
619
were detected by ELISA. Surface proteins (D) were detected by Western blot. (E)
620
Cells were fixed 48 h after transfection and stained for HBsAg. The nuclei were
621
stained with Hoechst 33258. Higher-magnification images of the selected area are
622
also shown. PC, Huh7 cells transfected with a HBV replication-competent plasmid
623
pHBV-B originated from genotype B as a positive control. Mock, Huh7 cells
624
transfected with a mock plasmid.
625 626
Fig. 3. SH and SH-DPS synergistically enhance viral replication and change
627
subcellular localization of the core protein. Huh7 cells were transfected with pSH, 27
Cao et al., HBV quasispecies and viral pathogenesis 628
pSH-DPS or pSH and pSH-DPS at the indicated ratios. (A) HBsAg in culture
629
supernatants was detected by ELISA. (B) HBV replication intermediates were
630
detected by Southern blot. The positions of relaxed circular (RC), double stranded
631
linear (DL), and single stranded (SS) DNA are indicated (upper panel). HBcAg were
632
detected by Western blot. The last lane was mock (Middle panel). The levels of
633
β-actin served as a loading control (lower panel). Relative band intensities for HBV
634
replication intermediates or HBcAg were quantified using NIH ImageJ software and
635
presented as the percentage of DNA or protein in positive control (PC). (C) Viral
636
DNA levels of three independent Southern blot experiments were quantified and
637
plotted as relative level (mean±SD) of PC samples. (D) Cells were fixed and stained
638
for HBsAg and HBcAg. The nuclei were stained with Hoechst 33258.
639
Higher-magnification images of the selected area are also shown. PC, Huh7 cells
640
transfected with a HBV replication-competent plasmid pHBV-B originated from
641
genotype B as a positive control. NC, Huh7 cells transfected with a HBV
642
replication-deficient plasmid pHBV1.3-rtG244Y as a negative control. Mock, Huh7
643
cells transfected with a mock plasmid.
644 645
Fig. 4. Phenotypic characterization of SH and SH-DPS in vivo. Hydrodynamic
646
injection (HI) was performed with HBV constructs at the indicated ratios in C57BL/6
647
mice (n=5). (A) The levels of HBsAg in the serum were detected by ELISA. Results
648
were presented as the OD 450 value. HBV DNA expression in mouse sera (B) or liver
649
samples (C) collected at 5 days post HI (dpi) was determined by real-time PCR and
28
Cao et al., HBV quasispecies and viral pathogenesis 650
Southern blot. PC, mice HI with a HBV replication-competent plasmid pHBV-B
651
originated from genotype B as a positive control. Mock, mice HI with PBS as a
652
negative control. Two tailed t-test was used to determine the differences in multiple
653
comparisons (*, P
Cao et al., HBV quasispecies and viral pathogenesis
1
Coexistence of hepatitis B virus quasispecies enhances viral
2
replication and the ability to induce host antibody and cellular
3
immune responses
4 5
Liang Cao1,2, Chunchen Wu1#, Hui Shi1, Zuojiong Gong3, Ejuan Zhang1, Hui Wang1,2,
6
Kaitao Zhao1,2, Shuhui Liu1,2, Songxia Li1, Xiuzhu Gao4, Yun Wang1, Rongjuan Pei1,
7
Mengji Lu1, 5, 6, Xinwen Chen1#
8 9
1.
State Key Lab of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China
10 11
2.
University of Chinese Academy of Sciences, Beijing, China
12
3.
Department of Infectious Diseases, Renmin Hospital of Wuhan University, Wuhan, China
13 14
4.
Department of Hepatology, First Hospital of Jilin University, Changchun, China
15
5.
Institute of Virology, University Hospital of Essen, Essen, Germany
16
6.
Department of Microbiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
17 18 19
Running title: HBV quasispecies and viral pathogenesis
20 21
#
Corresponding author:
1
Cao et al., HBV quasispecies and viral pathogenesis 22
Prof. Dr. Xinwen Chen
23
Wuhan Institute of Virology, Chinese Academy of Science
24
Wuhan 430071, People’s Republic of China
25
Phone/fax: 86-027-87199106; E-mail: chenxw@ wh.iov.cn
26 27
Dr. Chunchen Wu
28
Wuhan Institute of Virology,
29
Chinese Academy of Science
30
Wuhan 430071, People’s Republic of China
31
Phone: 86-027-87197575; E-mail: [email protected]
32 33
Number of figures and tables: there are 7 figures in the manuscript
34 35
Electronic word count: abstract: 236; importance paragraph: 129; main text: 4150
36 37
2
Cao et al., HBV quasispecies and viral pathogenesis 38
ABSTRACT
39
Hepatitis B virus (HBV) quasispecies contain a large number of variants that serve as
40
a reservoir for viral selection under antiviral treatment and the immune response,
41
leading to the acute exacerbation and subsequent development of liver failure.
42
However, there is no clear experimental evidence for a significant role of HBV
43
quasispecies in viral pathogenesis. In the present study, HBV sequences were
44
amplified from a patient with severe liver disease and constructed into HBV
45
replication-competent plasmids. Western blot, Enzyme-linked immunosorbent assay
46
(ELISA), and immunofluorescence staining were performed to analyze the expression,
47
secretion and subcellular localization of viral proteins in vitro. Viral replication
48
intermediates were detected by Southern blot. HBV gene expression and replication
49
and the induction of specific immune responses in an HBV hydrodynamic injection
50
(HI) mouse model were investigated. The results demonstrated that two naturally
51
occurring HBV variants, SH and SH-DPS, were identified. The variant SH-DPS
52
expressed only a non-exportable hepatitis B surface antigen (HBsAg) with abnormal
53
intracellular accumulation. The coexistence of the HBV variants at a ratio of 1 to 4
54
(SH to SH-DPS) increased HBV replication. Significantly stronger intrahepatic
55
cytotoxic T lymphocyte (CTL) responses and antibody responses specific to HBsAg
56
were induced in mice by the HBV variants when co-applied by HI. These findings
57
uncovered an unexpected aspect of HBV quasispecies: that the coexistence of
58
different variants can significantly modulate specific host immune responses,
59
representing a novel mechanism for the immunopathogenesis of HBV infection.
3
Cao et al., HBV quasispecies and viral pathogenesis 60
IMPORTANCE
61
Hepatitis B virus (HBV) is a important human pathogen. HBV quasispecies with
62
genetically heterogenous variants are thought to play a role in the progression of
63
HBV-associated liver diseases. So far, direct evidence is only available in few cases to
64
confirm the proposed role of HBV variants in the pathogenesis. We report here that
65
the coexistence of two naturally occurring HBV variants at a ratio of 1 to 4 increased
66
HBV replication and induced significantly stronger intrahepatic cytotoxic T
67
lymphocyte responses and antibody responses specific to HBsAg in mice. Our
68
discovery uncovered an unexpected aspect of HBV quasispecies that the coexistence
69
of different variants can significantly modulate specific host immune responses and
70
may enhance immune-mediated liver damage under some circumstances, representing
71
a novel mechanism for the immunopathogenesis of HBV infection.
72 73
KEYWORDS: Hepatitis B virus, acute liver failure, quasispecies, immune response
74
4
Cao et al., HBV quasispecies and viral pathogenesis INTRODUCTION
75 76
Hepatitis B virus (HBV) infection causes a wide spectrum of clinical
77
manifestations ranging from an asymptomatic carrier state to acute or chronic
78
hepatitis, with progression to liver cirrhosis, hepatocellular carcinoma, and other
79
severe liver diseases (1-4).
80 81
The HBV population in the host consists of genetically heterologous variants and
82
exists in the form of quasispecies. It is proposed that quasispecies may contribute to
83
viral persistence and pathogenesis because quasispecies contain a large number of
84
mutated genes that serve as a reservoir for viral selection under antiviral treatment and
85
the immune response (5). It has been previously reported that co-infection with human
86
immunodeficiency virus (HIV) and HBV results in low quasispecies complexity (4).
87
In patients with HBV infection-related liver failure (HB-LF), the quasispecies showed
88
distinct characteristics with higher complexity and diversity within the HBV precore
89
(preC)/core gene (6). However, there is no clear experimental evidence for a
90
significant role of HBV quasispecies in the pathogenesis of HB-LF.
91 92
A number of publications have suggested that the emergence of HBV variants
93
leads to acute exacerbation and subsequently contributes to the development of liver
94
failure (LF). Some mutations in HBV genomes, such as the A1762T/G1764A double
95
mutation in the basal core promoter (BCP) and the G1896A mutation in the preC
96
region, are thought to play a role in the progression of liver diseases (1, 6-8). These
97
mutations were found to enhance HBV replication in vitro and to abrogate expression
98
of hepatitis B e antigen (HBeAg), which is suspected to interrupt immune tolerance in
99
the host and to contribute to the development of LF (1, 7, 8). However, no direct 5
100
Cao et al., HBV quasispecies and viral pathogenesis evidence is available to confirm the proposed role of HBV mutations in the
101
pathogenesis of HB-LF so far. In addition, other studies have reported contradictory
102
findings, indicating that there is no obvious link between HBV BCP/preC mutations
103
and the development of LF (9, 10). It also remains mechanistically unclear how HBV
104
BCP/preC mutations affect the development of HB-LF.
105 106
In general, HBV variants may cause liver damage by a direct cytopathic effect or
107
by indirectly promoting immunopathology. There are a few examples of exacerbation
108
of liver diseases associated with cytopathic HBV variants (11-15). However, it is
109
currently unknown whether the appearance of HBV variants has any influence on host
110
immune responses which would in turn cause liver damage.
111 112
In the present study, we characterized HBV isolates from a patient with severe
113
liver disease and identified two major HBV variants, HBV-SH (SH) and
114
HBV-SH-DPS (SH-DPS), which harbored a number of mutations including two
115
deletions within the preS regions and hepatitis B surface antigen (HBsAg) sequences.
116
The variant SH-DPS expressed only a non-exportable SHBsAg with abnormal
117
intracellular accumulation. Both SH and SH-DPS coexisted at a ratio of 1 to 4. These
118
two isolates were phenotypically characterized alone or together in different ratios by
119
transient transfection. The results demonstrated that the coexistence of SH and
120
SH-DPS at a ratio of 1 to 4 increased HBV replication and led to a predominant
121
nuclear localization of HBV core antigen (HBcAg). Using an HBV hydrodynamic
122
injection (HI) mouse model, we found that mice mounted significantly stronger
123
antibody and cytotoxic T lymphocyte (CTL) responses to HBsAg only if SH and
124
SH-DPS were co-applied. Thus, the coexistence of different variants may
6
125
Cao et al., HBV quasispecies and viral pathogenesis significantly modulate specific host immune responses and may enhance
126
immune-mediated liver damage under some circumstances, representing a novel
127
mechanism for the immunopathogenesis of HBV infection.
128
7
Cao et al., HBV quasispecies and viral pathogenesis MATERIALS AND METHODS
129 130
Patient. A 38-year-old male patient from China had a history of chronic hepatitis
131
B infection for over 30 years. He was positive for HBsAg and the antibody to the
132
hepatitis B e antigen (anti-HBe) and was negative for HBeAg and the antibody to
133
HBsAg (anti-HBs).
134 135
The patient was diagnosed with HB-LF manifesting as a rise in
136
alanine aminotransferase (ALT) to 283 U/L along with HBV DNA levels > 106
137
copies/ml, jaundice (bilirubin, 7.9 mg/dL) and coagulopathy (grade II), complicated
138
within 4 weeks by ascites and encephalopathy. The patient received artificial liver
139
support 3 times as well as other treatments, but the illness worsened precipitously,
140
complicated by hepatic encephalopathy, infection and hepatorenal syndrome (Fig. 1).
141 142
The patient gave signed, informed consent. Sample collection, processing, and
143
storage conformed to the ethical guidelines of the 1975 Declaration of Helsinki as
144
reflected in a prior approval by the institution's human research committee.
145 146
Characterization of HBV isolates from patient serum samples and cloning.
147
Isolation of HBV viral DNA from patient serum samples was performed as described
148
previously with minor modifications (16, 17). A polymerase chain reaction (PCR) was
149
performed to amplify 2.1 kb fragment (1821 -699 bp) and 1.2 kb fragment (669 -1825
150
bp) with the primer pairs P1/P3 and P2/P4, respectively: P1, 5’-CCGGC
8
Cao et al., HBV quasispecies and viral pathogenesis 151
GTCGACGAGCTCTTCTTTTTCACCTCTGCCTAATCA-3’ (nt1821-1841); P2, 5’-
152
CCGGCGTCGACGAGCTCTTCAAAAAGTTGCATGGTGCTGG-3’ (nt1825-1806);
153
P3, 5’-CACTGAACAAATGGCACTAGTAAACTGAGCC-3’ (nt699-669); P4, 5’-G
154
GCTCAGTTTACTAGTGCCATTTGTTCAGTG-3’ (nt669-699). To reduce the
155
possibility of error-prone amplification, an enzyme with excellent high PCR fidelity
156
and efficiency, KOD-PLUS (Toyobo) was used in PCR. The two PCR products were
157
cloned into the pGEM®-T Easy vector (Promega, Madison, Wisconsin, USA) for
158
sequence analysis (ten clones of each fragment). The sequences of the 1.2 kb
159
fragments were consistent, while the 2.1 kb fragment had two types with either
160
entirely HBsAg gene (2 clones) or 2 deletions within HBsAg gene (8 clones) (Fig.
161
2A). To get full-length HBV genomes, the 1.2 kb SacI-SpeI fragment and a 2.1 kb
162
SalI-SpeI fragment were released from the pGEM®-T Easy vector and cloned into the
163
cloning vector pUC19 pre-digested with SalI and SacI. The two head-to-tail fragments
164
were sub-cloned into the pUC19 vector and resulted in pUC19-HBV1-SH and
165
pUC19-HBV1-SH-DPS harboring a complete HBV genome and a type of mutant
166
genome with two deletions within the HBV preS region (GenBank accession number:
167
SH: KC492739.1 and SH-DPS KC492740.1) (Fig. 2A).
168 169
Two plasmids with replication-competent, 1.3-fold HBV genomes, pUC19-
170
HBV1.3-SH (pSH) and pUC19-HBV1.3-SH-DPS (pSH-DPS) (18), and a positive
171
control (PC) pUC19-HBV1.3-B were constructed as described previously (19). A
172
previously described replication deficient HBV clone pHBV1.3-rtG244Y (20) was
9
Cao et al., HBV quasispecies and viral pathogenesis 173
used to serve as a negative control (NC) in Southern blot experiment. Additionally,
174
the 1.3-fold over-length HBV genomes were sub-cloned into the pAAV vector to
175
produce pAAV-SH, pAAV-SH-DPS and pAAV-PC for HI in mice.
176 177
Cells and mice. The human hepatoma cell line Huh7 (provided by American
178
Type Culture Collection, Manassas, VA) was maintained and transfected as described
179
previously (20). Male C57BL/6 (H-2b) mice (6 to 8 weeks of age) were kept under
180
specific-pathogen-free (SPF) conditions in the Central Animal Laboratory of Wuhan
181
Institute of Virology, Chinese Academy of Sciences and treated by following the
182
guidelines of the institutional animal ethical standard.
183 184
Western blot analysis. Western blot analysis was performed as described
185
previously (21, 22). The following antibodies were used: anti-HBs (Abcam,
186
Cambridge, UK), anti-HBc (Santa Cruz, Santa Cruz, CA) and anti-beta-actin (Santa
187
Cruz, Santa Cruz, CA). Relative band intensities for viral proteins were quantified
188
using NIH ImageJ software.
189 190
Enzyme-linked immunosorbent assay (ELISA). HBsAg, HBeAg, antibodies to
191
HBsAg in mouse sera, culture supernatants, and cell lysates of transfected cells were
192
detected as described previously (23-25).
193 194
Immunofluorescence (IF) staining and confocal laser scanning microscopy.
10
Cao et al., HBV quasispecies and viral pathogenesis 195
Indirect IF staining of transfected cells was performed as described previously (21, 23,
196
24). Anti-HBc (Dako, Carpinteria, CA) and anti-HBs (S1, kindly provided by Yan Bin)
197
were used as primary antibodies and Alexa Fluor 488-conjugated and Alexa Fluor
198
568-conjugated antibodies (Life Technologies, Carlsbad, CA) were used for
199
secondary detection.
200 201
Southern Blot Analysis. Huh7 cells were transiently transfected with 2.5 μg of
202
pHBV-B，pSH or pSH-DPS alone or co-transfected with 2.5 μg of mixtures of pSH
203
and pSH-DPS, or pHBV-B and pSH-DPS at the indicated ratios. Encapsidated HBV
204
replication intermediates were extracted and subjected to Southern blot analysis as
205
described previously (20). The hybridization signals were quantified with ImageJ
206
software (National Institutes of Health).
207 208
HBV challenge by hydrodynamic injection. Mice in each group were
209
challenged by hydrodynamic injection (HI) as described previously with minor
210
modifications (17). In brief, 10 μg of HBV plasmid DNA was incubated with 20 μl
211
Lipofectamine 2000 (Life Technologies, Carlsbad, CA) for 20 min at room
212
temperature to form the DNA–Lipofectamine 2000 complex. Then, the complexes
213
were injected into the tail veins of mice in a volume of PBS equivalent to 8% of the
214
mouse body weight within 5 seconds.
215 216
Detection of serum HBV DNA and intrahepatic core-associated HBV DNA.
11
Cao et al., HBV quasispecies and viral pathogenesis 217
Purification
of
218
quantification of HBV DNA were performed as described previously (23, 26). The
219
real-time
220
5’-AAATCTCCAGTCACTCACCAACC-3’ (nt321-343); P6, 5’-CATAGCAGCAGG
221
ATGCAGAGG-3’ (nt423-403); TaqMan probe, 5’-FAM-TCCTCCAATTTGTCCT
222
GGTTATCGCT-MGB-3’ (nt349-374). The plasmid pHBV-B was used in 10-fold
223
serial dilutions ranging from 101 to 109 copies per reaction as a standard curve.
PCR
core-associated
primers
P5/P6
HBV
and
the
DNA
TaqMan
in
liver
probe
tissue
are
used:
and
P5,
224 225
Immunohistochemistry (IHC). Liver tissues were collected from mice
226
sacrificed at 5 days post-hydrodynamic injection (dpi) and subjected to IHC staining
227
as described previously (20). Intrahepatic HBcAg and HBsAg were detected by IHC
228
staining of formalin-fixed, paraffin-embedded liver tissue sections using anti-HBc
229
antibody (Dako, Carpinteria, CA) and anti-HBs antibody (Thermo Scientific Pierce，
230
Rockford, IL) with an appropriate HRP-conjugated secondary antibody and visualized
231
by the Envision System.
232 233
Intracellular cytokines staining and flow cytometry. Splenocytes and
234
intrahepatic lymphocytes were isolated from mice at 14 and 28 days and subjected to
235
flow cytometry analysis (27). Lymphocytes were supplemented with CD28 (1:1000,
236
ebioscience，San Diego, CA) and Brefeldin A (1:1000, ebioscience，San Diego, CA),
237
stimulated with or without peptide derived from HBcAg and HBsAg corresponding to
238
S protein CD8+ T cells epitope (Kb/S190–197, VWLSVIWM), core protein CD8+ T
12
Cao et al., HBV quasispecies and viral pathogenesis 239
cells epitope (Kb/C93–100, VWLSVIWM), S protein CD4+ T cells epitope (S182-196,
240
FFLLTFULTIFQSLD)
241
TPPAYRPPNAPIL), respectively, and then stained for surface marker CD4 and CD8
242
(BD Biosciences, San Jose, CA). Lymphocytes were fixed and permeabilized in
243
Cytofix/Cytoperm solution (Cytofix/CytopermTM kit, BD Biosciences, San Jose, CA)
244
followed by intracellular staining for IFN-γ (BD Biosciences, San Jose, CA). Dead
245
cells were excluded by staining with 7-aminoactinomycin D (7AAD, Biolegend, San
246
Diego, CA).
and
core
protein
CD4+
T
cells
epitope
(C128-140,
247 248
Statistical
analysis.
The
statistical
analysis
was
carried
out
using
249
GraphPadPrism 5.0 software (Graphpad Software). Two tailed t-test was used to
250
determine the differences in multiple comparisons. A P value < 0.05 was considered
251
statistically significant. Results were presented as mean ± standard deviation (SD).
252
13
Cao et al., HBV quasispecies and viral pathogenesis RESULTS
253 254
Sequence analysis of HBV genomes. First, HBV DNA was isolated from the
255
serum of the patient and subjected to sequence analysis. A comparison of the cloned
256
HBV DNA sequences with published HBV sequences in Genbank indicated that the
257
isolates belong to the genotype B and serotype adw2 and harbor the A1762T/G1764A
258
double mutation in the BCP region and the G1896A mutation in the preC region (Fig.
259
2A). The A1762T/G1764A double mutation has been reported to reduce the
260
production of HBeAg and to enhance the HBV replication level in vitro (28, 29),
261
whereas the G1896A stop codon mutation abolishes HBeAg expression (30). In 8 of
262
10 sequenced HBV genomes, 2 deletions in the preS1 region (nt 2976-3102) and in
263
the preS2 promoter and preS2 open reading frame (ORF) region (nt 3203-3215, nt
264
1-31) were found (Fig. 2A), resulting in a stop codon mutation in the ORF of large
265
HBsAg (LHBsAg) and the deletion of the start codon in the ORF of middle HBsAg
266
(MHBsAg), respectively. In addition, two typical point mutations, N146S and P120S,
267
were present. The mutation N146S prevents the glycosylation of HBV envelope
268
proteins (31). The mutation P120S may be associated with immune escape (Fig. 2A)
269
(32). Thus, two major HBV variants coexisted in this patient; the one with complete
270
HBsAg was designated as SH, and the other one with deletion mutations in HBsAg
271
ORFs was designated as SH-DPS. The ratio of SH to SH-DPS was 1 to 4 based on the
272
numbers of the sequenced clones.
273 274
Phenotypic characterization of SH and SH-DPS in vitro. The plasmids pSH
14
Cao et al., HBV quasispecies and viral pathogenesis 275
and pSH-DPS containing the respective 1.3-fold over-length HBV genomes were
276
transfected into Huh7 cells. PSH produced much less intracellular and supernatant
277
HBsAg (52% and 30%, respectively) than PC (32) (Fig. 2B). PSH-DPS did not
278
produce detectable HBsAg in supernatant but produced a low level of intracellular
279
HBsAg. These results suggested that HBsAg with deletions and mutations encoded by
280
SH-DPS was secretion deficient. No HBeAg was detected in culture supernatants,
281
consistent with the presence of the G1896A mutation in the preC region that abolishes
282
HBeAg expression (Fig. 2C) (1, 2). WB with anti-HBs antibodies indicated that
283
glycosylated and un-glycosylated bands corresponding to L, M and SHBsAg were
284
detected for both SH and PC as reported previously (33), while only the
285
non-glycosylated form of SHBsAg could be detected for SH-DPS, consistent with the
286
sequence information that included the termination of L and MHBsAg and the loss of
287
the glycosylation site by N146S substitution (Fig. 2D). Based on immunofluorescence
288
(IF) staining with anti-HBs antibodies, HBsAg was evenly distributed in the
289
cytoplasm of cells transfected with pSH or PC (Fig. 2E) (23). In contrast, a dot-like
290
distribution of HBsAg was observed in the cytoplasm of cells transfected with
291
pSH-DPS (Fig. 1E). Thus, the mutations in HBsAg expressed from pSH-DPS caused
292
abnormal localization of HBsAg and prevented its secretion.
293 294
The coexistence of SH and SH-DPS at a ratio of 1 to 4 increased HBV
295
replication. We co-transfected pSH and pSH-DPS at different ratios and analyzed
296
HBV gene expression and replication by enzyme linked immunosorbent assay
15
Cao et al., HBV quasispecies and viral pathogenesis 297
(ELISA), Western blot, Southern blot, and IF staining (Fig. 3). Increasing amounts of
298
pSH-DPS led to a decrease of HBsAg production in supernatant (Fig. 3A), as well as
299
a decrease of HBcAg expression level in cell lysates (Fig.3B). Both the pSH and
300
pSH-DPS clones were replication competent. However, pSH-DPS produced much
301
more core-associated DNA than pSH (Fig. 3B and C). Interestingly, when pSH and
302
pSH-DPS were co-transfected at different ratios, the highest amount of
303
core-associated HBV replication intermediates was detected at a ratio of 1 to 4, which
304
yielded a 15% increase compared to pSH-DPS alone (Fig. 3B and C). This
305
demonstrated a synergistic effect of HBV quasispecies on genomic replication.
306 307
The coexistence of SH and SH-DPS led to a predominant nuclear
308
localization of HBcAg. In Fig. 3D, HBcAg evenly distributed in both the cytoplasm
309
and nucleus in cells transfected with pSH-DPS but only in the cytoplasm in cells
310
transfected with pSH and PC. When the ratio of pSH and pSH-DPS was 4 to 1, both
311
HBsAg and HBcAg were evenly distributed in the cytoplasm, similar to pSH
312
transfection. At a ratio of 1 to 4, HBsAg aggregated in the cytoplasm and HBcAg was
313
located both in the cytoplasm and nucleus similar to the distribution with pSH-DPS
314
transfection.
315 316
Phenotypic characterization of SH and SH-DPS in vivo. We further
317
characterized these two HBV variants using an HBV HI mouse model (23, 25).
318
pAAV-SH and pAAV-SH-DPS were generated by cloning the 1.3-fold over-length
16
Cao et al., HBV quasispecies and viral pathogenesis 319
HBV genomes into the pAAV plasmid. After HI of HBV constructs into C57BL/6
320
mice, serum HBsAg levels were monitored from day 1 on and up to day 63 post-HI. A
321
high serum HBsAg level was detected in all mice receiving pAAV-PC and pAAV-SH
322
at 1 dpi to 7 dpi, then declined to an undetectable level at 28 dpi (Fig. 4A). HBsAg
323
was not detected in mice receiving pAAV-SH-DPS, consistent with the secretion
324
deficiency of mutated HBsAg (Fig. 4A). For the pAAV-SH and pAAV-SH-DPS
325
groups co-injected at a ratio of 4 to 1, HBsAg was detected at 1 dpi and
326
seroconversion occurred at 21 dpi (Fig. 4A). However, when pAAV-SH and
327
pAAV-SH-DPS were co-injected at a ratio of 1 to 4, no HBsAg was detected (Fig.
328
4A).
329 330
Further, we detected serum and hepatic HBV DNA in mice by real-time PCR at
331
the indicated time points after HI. In pAAV-PC-injected mice, the serum HBV DNA
332
level was 1.4×106 copies/ml at 3 dpi and increased to 2.4×107 copies/ml at 5 dpi (Fig.
333
4B). HI with pAAV-SH in mice resulted in significantly lower serum HBV DNA
334
levels, at approximately 6% of the levels in the control with pAAV-HBV-B. The
335
serum HBV DNA levels were below the detection limit in other mice receiving
336
pAAV-SH-DPS
337
intrahepatic HBV DNA levels at 5 dpi were positive in all mice injected with
338
pAAV-SH, pAAV-SH-DPS, and pAAV-SH/pAAV-SH-DPS but were less than 10% of
339
that of the PC group (Fig. 4C), indicating that both SH and SH-DPS were replication
340
competent in vivo but at a lower level. However, there were no significant differences
and
pAAV-SH/pAAV-SH-DPS
co-injection
(Fig.
4B).
The
17
Cao et al., HBV quasispecies and viral pathogenesis 341
among these groups. Southern blot hybridization detected HBV replication
342
intermediates from the PC group but significantly lower levels of intermediates from
343
the liver samples from the other groups (Fig. 4C).
344 345
Immunohistochemistry (IHC) staining for HBsAg and HBcAg with liver tissue
346
sections collected at 5 dpi showed a cytoplasmic distribution of HBsAg in mice
347
injected
348
pAAV-SH-DPS-injected mice (Fig. 5). The staining of HBcAg showed both
349
cytoplasmic and nuclear distribution (Fig. 5B). When SH and SH-DPS were
350
co-injected, both diffused distribution and dot-like distribution of HBsAg in the
351
cytoplasm were observed (Fig. 5A). Additionally, the percentage of HBsAg- or
352
HBcAg-positive hepatocytes was calculated and positively related to the intrahepatic
353
HBV DNA levels of each group (Figs. 5A and B).
with
pAAV-PC
and
pAAV-SH
and
a
dot-like
distribution
in
354 355
HBV-specific antibody- and cell-mediated immune responses. The levels of
356
HBsAg in the sera of all mice receiving pAAV-PC and pAAV-SH declined to an
357
undetectable level at 28 dpi. Inversely, anti-HBs antibodies were initially undetectable
358
but became positive at 21 dpi and then were maintained at a high level through the
359
experiment period in mice of both groups (Fig. 6A). However, like HBsAg, anti-HBs
360
antibodies were not detected in mice receiving pAAV-SH-DPS, consistent with the
361
secretion deficiency of mutated HBsAg (Fig. 4A). Strikingly, when pAAV-SH and
362
pAAV-SH-DPS were co-injected at a ratio of 1 to 4, a significantly higher anti-HBs
18
Cao et al., HBV quasispecies and viral pathogenesis 363
antibody response was induced in the mice of this group, although we could not detect
364
HBsAg previously (Figs. 4A, 6A and B). When mice were co-injected with pAAV-SH
365
and pAAV-SH-DPS at a ratio of 4 to 1, the HBsAg levels decreased before 21 dpi and
366
anti-HBs antibody levels increased after 21 dpi (Figs. 4A and 6A). Therefore, the
367
coexistence of SH and SH-DPS variants enhanced the HBsAg-specific immune
368
responses.
369 370
The IgG1/IgG2a antibody responses could serve as an indication for the Th bias
371
of specific immune responses. Thus, we determined the subtypes of HBsAg-specific
372
IgGs. The IgG1 response to HBsAg was comparable in all mice receiving SH,
373
SH/SH-DPS at the ratio of 1 to 4 and PC. Interestingly, HBsAg-specific IgG2a
374
antibodies were only detected in mice receiving SH/SH-DPS (Figs. 6C and D),
375
suggesting that the coexistence of SH and SH-DPS induced a significantly stronger
376
Th1 response.
377 378
Next, we further examined the induction of cell-mediated, specific immune
379
responses to HBsAg and HBcAg. Splenocytes and intrahepatic lymphocytes were
380
isolated at 14 and 28 dpi, respectively, and stimulated with two H-2kb-restricted CTL
381
epitope peptides derived from HBsAg and HBcAg, as well as a CD4+ T cell epitope
382
peptide. Flow cytometric analysis was performed for the detection of CD4+ and
383
CD8+ IFN-γ-producing cells. CD8+ IFN-γ-producing cell populations specific to
384
HBsAg were detected in mice from all groups except the mock group at 14 dpi and 28
19
Cao et al., HBV quasispecies and viral pathogenesis 385
dpi. Notably, at 28 dpi, the frequencies of CD8+ IFN-γ-producing cells were the
386
highest if pAAV-SH and pAAV-SH-DPS were co-injected at a ratio of 1:4 (Figs. 7A
387
and B), consistent with the production of anti-HBs antibody. However, the HBsAg-
388
and HBcAg-specific CD4+ T cell response and the HBcAg-specific CD8+ T cell
389
response in all mice were not detected in both splenocytes and intrahepatic
390
lymphocytes (Figs. 7C, D, E, F, G and H).
391 392
DISCUSSION
393
HBV mutants are thought to play a role in the progression of HBV-associated
394
liver diseases (1, 6-8). The HBV population in the host exists in the form of
395
quasispecies and contains a great spectrum of mutants (5). The role of HBV
396
quasispecies in the pathogenesis of HBV infection is currently not understood. In the
397
present study, we found that the coexistence of two naturally occurring HBV variants
398
at a ratio of 1 to 4 increased HBV replication and led to a predominant nuclear
399
localization of HBcAg. More importantly, significantly stronger intrahepatic CTL
400
responses and antibody responses specific to HBsAg were induced in mice by these
401
HBV variants when co-applied by HI. These findings revealed the potential role of
402
HBV quasispecies and their mutations in the pathogenesis of HBV infection.
403 404
The cell-mediated immune response is considered to contribute to both viral
405
clearance and liver injury in HBV infection (34). In mice receiving HI with HBV
406
genomes, a higher frequency of HBsAg-specific CD8+ T cells was detected in the
407
liver than in the spleen. Strikingly, the coexistence of SH and SH-DPS at a ratio of 1
20
Cao et al., HBV quasispecies and viral pathogenesis 408
to 4 induced a stronger CTL response than each HBV vector alone (Figs. 7A and B).
409
At the same time, the magnitude and the IgG subtype distribution of anti-HBs
410
antibody responses were significantly changed by the coexistence of the HBV
411
quasispecies (Figs. 6B, C and D). Both higher CTL frequencies and the appearance of
412
the IgG2a subtype of anti-HBs antibodies indicated a shift of HBV-specific immune
413
responses toward the Th1 type. The enhancement of HBV-specific immune response
414
may be due to secretion defect of HBsAg. In addition, increased HBV replication
415
activity when both HBV clones were co-injected and changed subcellular localization
416
of HBsAg may also contribute to the induction of stronger specific immune responses.
417 418
Though our study does not prove a direct link between the HBV quasispecies and
419
HBV-associated severe liver diseases, it hints at a possible explanation for the nearly
420
obligatory presence of HBV variants in patients suffering from acute LF. It also gives
421
an idea for why the characterization of HBV variants in cell culture systems seldom
422
delivers a clear answer about their pathogenic potential. We could hypothesize that
423
only the combination of different WT and mutant proteins effectively trigger specific
424
host immune responses and leads to observed immunopathology in LF patients. This
425
hypothesis implies that HBV variants are not innocent bystanders selected by
426
decreased liver functions but are the major causes for LF, and thus may need attention
427
in clinical monitoring.
428 429
Previously, the A1762T/G1764A and G1896A mutations in BCP/preC were
21
Cao et al., HBV quasispecies and viral pathogenesis 430
found to affect HBV replication and to abrogate expression of HBeAg. It was
431
suspected that these mutations may contribute to the development of LF (1, 6-8).
432
However, most of these results were based on in vitro transfection of single HBV
433
replication-competent plasmids with such mutations. Here, co-transfection of two
434
HBV replication-competent plasmids increased viral replication in the ratio of 1 to 4,
435
suggesting that the HBV quasispecies should be paid more attention in clinical
436
monitoring.
437 438
HBcAg tended to localize in the cytoplasm in the presence of the
439
A1762T/G1764A double mutation (35, 36). However, the localization of HBcAg from
440
a fulminant HBV strain was changed to the nucleus when both G1862T and G1896A
441
were also present (3). Early studies on clinical samples indicated that the subcellular
442
localization of HBcAg was closely correlated with hepatitis activity in
443
HBeAg-positive patients (37, 38). However, the relationship between intracellular
444
distribution of HBcAg and hepatitis activity is unknown in HBeAg-negative or
445
anti-HBe-positive patients. Here, we observed a predominant nuclear localization of
446
HBcAg when SH DNA and SH-DPS DNA were transfected into cells at a ratio of 1 to
447
4 (Fig. 3C), suggesting that the coexistence of SH and SH-DPS changed the
448
preferential localization of HBcAg from the cytoplasmic to nuclear compartment. To
449
clarify the mechanism, we performed Western blot and found that increasing amounts
450
of pSH-DPS led to a decrease of HBcAg expression level (Fig.3B). Low level of
451
HBcAg could not drive capsid assembly and virion formation in cytoplasm for
22
Cao et al., HBV quasispecies and viral pathogenesis 452
budding (39) but could freely enter the nucleus by nuclear pore complex (40).
453
Moreover, mature nucleocapsids could be targeted back to the nucleus and amplify the
454
pool of cccDNA, or be targeted to the Endoplasmic reticulum to bud and exit the cell.
455
Envelope proteins play a regulatory role in the process (41). However, SH-DPS
456
produced a low level of intracellular HBsAg (Fig.2). Therefore the difference in core
457
protein localization between SH, SH-DPS and the co-existence of SH and SH-DPS
458
was observed. However, the mechanism determining the shuttle of HBcAg between
459
the nucleus and cytoplasm is not completely understood. Nuclear localization/export
460
signals and some host factors may play a role in this process (42). The presence of
461
different HBV proteins including mutated HBsAg may change the expression or
462
function of such viral and host factors. Future investigation is required to identify the
463
factors involved in the regulation of HBcAg localization.
464 465
To our knowledge, the present study demonstrated for the first time that the
466
interaction of HBV quasispecies may enhance HBV replication and induce stronger
467
host immune responses, thereby potentially contributing to acute exacerbation of liver
468
diseases and the development of liver failure.
469 470 471 472
ACKNOWLEDGMENTS We are grateful to Bin Yan, Xuefang An, Yuan Zhou, and Xue Hu for excellent technical support. We also thank Pei-Jer Chen for helpful suggestions.
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This work was supported by the National Basic Research Priorities Program of
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China (2012CB519001) and the National Nature Science Foundation of China 23
Cao et al., HBV quasispecies and viral pathogenesis 475
(31200699).
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REFERENCES
477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515
1.
Ren X, Xu Z, Liu Y, Li X, Bai S, Ding N, Zhong Y, Wang L, Mao P, Zoulim F, Xu D. 2010. Hepatitis B virus genotype and basal core promoter/precore mutations are associated with hepatitis B-related acute-on-chronic liver failure without pre-existing liver cirrhosis. J Viral Hepat 17:887-895.
2.
Kay A, Zoulim F. 2007. Hepatitis B virus genetic variability and evolution. Virus Res
3.
Inoue J, Ueno Y, Nagasaki F, Wakui Y, Kondo Y, Fukushima K, Niitsuma H,
127:164-176. Shimosegawa T. 2009. Enhanced intracellular retention of a hepatitis B virus strain associated with fulminant hepatitis. Virology 395:202-209. 4.
Bernal W, Auzinger G, Dhawan A, Wendon J. 2010. Acute liver failure. Lancet
5.
Pawlotsky JM. 2005. The concept of hepatitis B virus mutant escape. J Clin Virol 34 Suppl
6.
Chen L, Zheng CX, Lin MH, Huang ZX, Chen RH, Li QG, Li Q, Chen P. 2013. Distinct
376:190-201. 1:S125-129. quasispecies characteristics and positive selection within precore/core gene in hepatitis B virus HBV associated acute-on-chronic liver failure. J Gastroenterol Hepatol 28:1040-1046. 7.
Baumert TF, Rogers SA, Hasegawa K, Liang TJ. 1996. Two core promotor mutations identified in a hepatitis B virus strain associated with fulminant hepatitis result in enhanced viral replication. J Clin Invest 98:2268-2276.
8.
Tong S, Kim KH, Chante C, Wands J, Li J. 2005. Hepatitis B Virus e Antigen Variants. Int J
9.
Sterneck M, Gunther S, Santantonio T, Fischer L, Broelsch CE, Greten H, Will H. 1996.
Med Sci 2:2-7. Hepatitis B virus genomes of patients with fulminant hepatitis do not share a specific mutation. Hepatology 24:300-306. 10.
Chun YK, Kim JY, Woo HJ, Oh SM, Kang I, Ha J, Kim SS. 2000. No significant correlation exists between core promoter mutations, viral replication, and liver damage in chronic hepatitis B infection. Hepatology 32:1154-1162.
11.
Bock CT, Tillmann HL, Maschek HJ, Manns MP, Trautwein C. 1997. A preS mutation isolated from a patient with chronic hepatitis B infection leads to virus retention and misassembly. Gastroenterology 113:1976-1982.
12.
Chisari FV, Filippi P, Buras J, McLachlan A, Popper H, Pinkert CA, Palmiter RD, Brinster RL. 1987. Structural and pathological effects of synthesis of hepatitis B virus large envelope polypeptide in transgenic mice. Proc Natl Acad Sci U S A 84:6909-6913.
13.
Fan YF, Lu CC, Chang YC, Chang TT, Lin PW, Lei HY, Su IJ. 2000. Identification of a pre-S2 mutant in hepatocytes expressing a novel marginal pattern of surface antigen in advanced diseases of chronic hepatitis B virus infection. J Gastroenterol Hepatol 15:519-528.
14.
Gilles PN, Guerrette DL, Ulevitch RJ, Schreiber RD, Chisari FV. 1992. HBsAg retention sensitizes the hepatocyte to injury by physiological concentrations of interferon-gamma.
24
Cao et al., HBV quasispecies and viral pathogenesis 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559
Hepatology 16:655-663. 15.
Wang HC, Wu HC, Chen CF, Fausto N, Lei HY, Su IJ. 2003. Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress. Am J Pathol 163:2441-2449.
16.
Gunther S, Li BC, Miska S, Kruger DH, Meisel H, Will H. 1995. A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients. J Virol 69:5437-5444.
17.
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 U S A 103:17862-17867.
18.
Alcantara FF, Tang H, McLachlan A. 2002. Functional characterization of the interferon regulatory element in the enhancer 1 region of the hepatitis B virus genome. Nucleic Acids Res 30:2068-2075.
19.
Li L, Shen H, Li A, Zhang Z, Wang B, Wang J, Zheng X, Wu J, Yang D, Lu M, Song J. 2013. Inhibition of hepatitis B virus (HBV) gene expression and replication by HBx gene silencing in a hydrodynamic injection mouse model with a new clone of HBV genotype B. Virol J 10:214.
20.
Qin B, Budeus B, Cao L, Wu C, Wang Y, Zhang X, Rayner S, Hoffmann D, Lu M, Chen X. 2013. The amino acid substitutions rtP177G and rtF249A in the reverse transcriptase domain of hepatitis B virus polymerase reduce the susceptibility to tenofovir. Antiviral Res 97:93-100.
21.
Xu S, Pei R, Guo M, Han Q, Lai J, Wang Y, Wu C, Zhou Y, Lu M, Chen X. 2012. Cytosolic phospholipase A2 gamma is involved in hepatitis C virus replication and assembly. J Virol 86:13025-13037.
22.
Pei R, Zhang X, Xu S, Meng Z, Roggendorf M, Lu M, Chen X. 2012. Regulation of hepatitis C virus replication and gene expression by the MAPK-ERK pathway. Virol Sin 27:278-285.
23.
Wu C, Deng W, Deng L, Cao L, Qin B, Li S, Wang Y, Pei R, Yang D, Lu M, Chen X. 2012. Amino acid substitutions at positions 122 and 145 of hepatitis B virus surface antigen (HBsAg) determine the antigenicity and immunogenicity of HBsAg and influence in vivo HBsAg clearance. J Virol 86:4658-4669.
24.
Wu C, Zhang X, Tian Y, Song J, Yang D, Roggendorf M, Lu M, Chen X. 2010. Biological significance of amino acid substitutions in hepatitis B surface antigen (HBsAg) for glycosylation, secretion, antigenicity and immunogenicity of HBsAg and hepatitis B virus replication. J Gen Virol 91:483-492.
25.
Yin Y, Wu C, Song J, Wang J, Zhang E, Liu H, Yang D, Chen X, Lu M, Xu Y. 2011. DNA immunization with fusion of CTLA-4 to hepatitis B virus (HBV) core protein enhanced Th2 type responses and cleared HBV with an accelerated kinetic. PLoS One 6:e22524.
26.
Weinberger KM, Wiedenmann E, Bohm S, Jilg W. 2000. Sensitive and accurate quantitation of hepatitis B virus DNA using a kinetic fluorescence detection system (TaqMan PCR). J Virol Methods 85:75-82.
27.
Qu D, Lanier G, Yuan ZH, Wen YM, Howard CR, Ahmed R. 2008. Localization of CD8+ cells specific for hepatitis B virus surface protein in the liver of immunized mice. J Med Virol 80:225-232.
25
Cao et al., HBV quasispecies and viral pathogenesis 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603
28.
Buckwold VE, Xu Z, Chen M, Yen TS, Ou JH. 1996. Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication. J Virol 70:5845-5851.
29.
Moriyama K, Okamoto H, Tsuda F, Mayumi M. 1996. Reduced precore transcription and enhanced core-pregenome transcription of hepatitis B virus DNA after replacement of the precore-core promoter with sequences associated with e antigen-seronegative persistent infections. Virology 226:269-280.
30.
Carman WF, Jacyna MR, Hadziyannis S, Karayiannis P, McGarvey MJ, Makris A, Thomas HC. 1989. Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet 2:588-591.
31.
Ito K, Qin Y, Guarnieri M, Garcia T, Kwei K, Mizokami M, Zhang J, Li J, Wands JR, Tong S. 2010. Impairment of hepatitis B virus virion secretion by single-amino-acid substitutions in the small envelope protein and rescue by a novel glycosylation site. J Virol 84:12850-12861.
32.
Shin D, Kim SI, Kim M, Park M. 2006. Efficient inhibition of hepatitis B virus replication
33.
Lepere-Douard C, Trotard M, Le Seyec J, Gripon P. 2009. The first transmembrane
by small interfering RNAs targeted to the viral X gene in mice. Virus Res 119:146-153. domain of the hepatitis B virus large envelope protein is crucial for infectivity. J Virol 83:11819-11829. 34.
Chisari FV. 2000. Rous-Whipple Award Lecture. Viruses, immunity, and cancer: lessons from hepatitis B. Am J Pathol 156:1117-1132.
35.
Kawai K, Horiike N, Michitaka K, Onji M. 2003. The effects of hepatitis B virus core promoter mutations on hepatitis B core antigen distribution in hepatocytes as detected by laser-assisted microdissection. J Hepatol 38:635-641.
36.
Liu CJ, Jeng YM, Chen CL, Cheng HR, Chen PJ, Chen TC, Liu CH, Lai MY, Chen DS, Kao JH. 2009. Hepatitis B virus basal core promoter mutation and DNA load correlate with expression of hepatitis B core antigen in patients with chronic hepatitis B. J Infect Dis 199:742-749.
37.
Chu CM, Liaw YF. 1987. Intrahepatic distribution of hepatitis B surface and core antigens in chronic hepatitis B virus infection. Hepatocyte with cytoplasmic/membranous hepatitis B core antigen as a possible target for immune hepatocytolysis. Gastroenterology 92:220-225.
38.
Chu CM, Yeh CT, Sheen IS, Liaw YF. 1995. Subcellular localization of hepatitis B core antigen in relation to hepatocyte regeneration in chronic hepatitis B. Gastroenterology 109:1926-1932.
39.
Seifer M, Standring DN. 1995. Assembly and antigenicity of hepatitis B virus core particles.
40.
Rabe B, Delaleau M, Bischof A, Foss M, Sominskaya I, Pumpens P, Cazenave C,
Intervirology 38:47-62. Castroviejo M, Kann M. 2009. Nuclear entry of hepatitis B virus capsids involves disintegration to protein dimers followed by nuclear reassociation to capsids. PLoS Pathog 5:e1000563. 41.
Lentz TB, Loeb DD. 2011. Roles of the envelope proteins in the amplification of covalently closed circular DNA and completion of synthesis of the plus-strand DNA in hepatitis B virus. J Virol 85:11916-11927.
42.
Li HC, Huang EY, Su PY, Wu SY, Yang CC, Lin YS, Chang WC, Shih C. 2010. Nuclear
26
Cao et al., HBV quasispecies and viral pathogenesis 604 605 606
export and import of human hepatitis B virus capsid protein and particles. PLoS Pathog 6:e1001162.
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FIGURE LEGENDS
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Fig. 1. Clinical course of the patient with HB-LF. (A) The level of serum
610
transaminase (ALT and AST), total bilirubin (TBIL) and direct bilirubin (DBIL) level.
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(B) The level of PT (prothrombin time), PTA (prothrombin time activity percentage),
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APTT (activated partial thromboplastin time) and FIB (fibriogen).
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Fig. 2. Expression, secretion and subcellular localization of viral proteins. (A)
615
Schematic representation of SH and SH-DPS. Stop codon in the preS1 ORF is
616
indicated by an asterisk. The mutations and deletions are marked by dark arrows. (B,
617
C, D and E) Huh7 cells were transfected with PC, pSH or pSH-DPS. At 72 hours (h)
618
post transfection, HBsAg (B) and HBeAg (C) in culture supernatants and cell lysates
619
were detected by ELISA. Surface proteins (D) were detected by Western blot. (E)
620
Cells were fixed 48 h after transfection and stained for HBsAg. The nuclei were
621
stained with Hoechst 33258. Higher-magnification images of the selected area are
622
also shown. PC, Huh7 cells transfected with a HBV replication-competent plasmid
623
pHBV-B originated from genotype B as a positive control. Mock, Huh7 cells
624
transfected with a mock plasmid.
625 626
Fig. 3. SH and SH-DPS synergistically enhance viral replication and change
627
subcellular localization of the core protein. Huh7 cells were transfected with pSH, 27
Cao et al., HBV quasispecies and viral pathogenesis 628
pSH-DPS or pSH and pSH-DPS at the indicated ratios. (A) HBsAg in culture
629
supernatants was detected by ELISA. (B) HBV replication intermediates were
630
detected by Southern blot. The positions of relaxed circular (RC), double stranded
631
linear (DL), and single stranded (SS) DNA are indicated (upper panel). HBcAg were
632
detected by Western blot. The last lane was mock (Middle panel). The levels of
633
β-actin served as a loading control (lower panel). Relative band intensities for HBV
634
replication intermediates or HBcAg were quantified using NIH ImageJ software and
635
presented as the percentage of DNA or protein in positive control (PC). (C) Viral
636
DNA levels of three independent Southern blot experiments were quantified and
637
plotted as relative level (mean±SD) of PC samples. (D) Cells were fixed and stained
638
for HBsAg and HBcAg. The nuclei were stained with Hoechst 33258.
639
Higher-magnification images of the selected area are also shown. PC, Huh7 cells
640
transfected with a HBV replication-competent plasmid pHBV-B originated from
641
genotype B as a positive control. NC, Huh7 cells transfected with a HBV
642
replication-deficient plasmid pHBV1.3-rtG244Y as a negative control. Mock, Huh7
643
cells transfected with a mock plasmid.
644 645
Fig. 4. Phenotypic characterization of SH and SH-DPS in vivo. Hydrodynamic
646
injection (HI) was performed with HBV constructs at the indicated ratios in C57BL/6
647
mice (n=5). (A) The levels of HBsAg in the serum were detected by ELISA. Results
648
were presented as the OD 450 value. HBV DNA expression in mouse sera (B) or liver
649
samples (C) collected at 5 days post HI (dpi) was determined by real-time PCR and
28
Cao et al., HBV quasispecies and viral pathogenesis 650
Southern blot. PC, mice HI with a HBV replication-competent plasmid pHBV-B
651
originated from genotype B as a positive control. Mock, mice HI with PBS as a
652
negative control. Two tailed t-test was used to determine the differences in multiple
653
comparisons (*, P
