1
Characterization of the Nucleosome Positioning in Hepadnaviral cccDNA 1
Minichromosomes 2
Liping Shi1, Shaohua Li2, Fang Shen1, Haodong Li2, Shuiming Qian1, Daniel H.S. Lee1, 3
Jim. Z Wu1, and Wengang Yang1* 4
1: Roche Pharma Research and Early Development China, Shanghai 201203; 2: WuXi 5
AppTec Co., Ltd. Shanghai 201131 6
7
*Corresponding author 8
Tel: 1-(203) 889-8783 9
E-mail: [email protected] 10
Running title: Nucleosome positioning in hepadnaviral cccDNA minichromosomes 11
Number of tables: 0 12
Number of figures: 6 13
Word count of abstract: 214 14
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Virol. doi:10.1128/JVI.00535-12 JVI Accepts, published online ahead of print on 11 July 2012
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Abstract 21
Hepadnaviral covalently closed circular DNA (cccDNA) exists as an episomal 22
minichromosome in the nucleus of virus infected hepatocytes and serves as the 23
transcriptional template for the synthesis of viral mRNAs. To obtain insight on the 24
structure of hepadnaviral cccDNA minichromosomes, we utilized ducks infected with 25
the duck hepatitis B virus (DHBV) as a model and determined in vivo nucleosome 26
distribution pattern on viral cccDNA by the micrococcal nuclease (MNase) mapping 27
and genome-wide PCR amplification of isolated mononucleosomal DHBV DNA. 28
Several nucleosome-protected sites in a region of DHBV genome (nt. 2000-2700), 29
known to harbor various cis-transcription regulatory elements, were consistently 30
identified in all DHBV-positive liver samples. In addition, we observed other 31
nucleosome protection sites in DHBV minichromosomes that may vary among 32
individual ducks, but the pattern of MNase mapping in those regions is transmittable 33
from the adult ducks to the newly infected ducklings. These results thus imply that the 34
nucleosomes along viral cccDNA in the minichromosomes are not randomly but 35
sequence-specifically positioned. Furthermore, we showed in ducklings a significant 36
portion of cccDNA possesses a few negative superhelical turns, suggesting the presence 37
of intermediates of viral minichromosomes assembly in the livers where dynamic 38
hepatocytes growth and cccDNA formation occur. This study supplies initial 39
framework for the understanding of the overall complete structure of hepadnaviral 40
cccDNA minichromosomes. 41
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Introduction 42
43
Currently, about 350 million individuals worldwide are chronically infected with the 44
hepatitis B virus (HBV). 15-40% of the infected people will develop severe sequelae in 45
their life time, most notably liver cirrhosis and hepatocellular carcinoma (18). The 46
treatment of chronic hepatitis B has been improved dramatically in the past 10 years, 47
mainly due to successful development and application of nucleoside(tide) drugs 48
targeting HBV polymerase and interferon (9, 24). These treatment options, although 49
significantly delaying disease progress by inhibiting viral replication and modulating 50
host immune functions in certain populations of HBV patients, fail to cure the majority 51
of HBV patients. A predominant reason for this failure is attributed to the persistence of 52
viral covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes 53
during the treatment with nucleoside(tide) analogs (8, 20, 38). Without interfering the 54
cccDNA maintenance within the infected hepatocytes, nucleoside(tides) only have a 55
limited effect on HBV DNA replication and disease progression. 56
57
Hepadnaviruses are small DNA-containing viruses that replicate their DNA genomes 58
through reverse transcription of an RNA intermediate called pregenomic RNA (32). 59
The template of the pregenomic RNA is a pool of cccDNA located in the hepatocyte 60
nuclei (34, 41). cccDNA is converted from a relaxed circular double stranded DNA 61
(RC DNA) that is transported into the nucleus from the cytoplasm where viral DNA 62
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replication occurs within naked capsid particles (29). A small percentage of the 63
cccDNA is converted from double stranded linear DNA through nonhomologous 64
recombination that generates sequence variations around the joint region (39). In the 65
nucleus, cccDNA exists as an individual minichromosome with a “beads-on-a-string” 66
structure revealed by electron microscope (2, 25). Histones as well as non-histone 67
proteins are either binding directly to the cccDNA or are indirectly recruited to viral 68
minichromosomes through protein-protein interactions (2, 20, 25, 26, 36). Using 69
cccDNA chromatin IP with anti-acetylated H3/H4 antibodies, it was shown that the 70
acetylation status of H3/H4 in cccDNA minichromosomes plays an important role in 71
HBV RNA transcription (26). Besides host proteins that, as components of 72
minichromosomes, involve in cccDNA functions, virally encoded proteins core and 73
HBx have also been shown to bind to this structure and result in either a reduction of 74
the nucleosomal spacing in HBV minichromosomes or an overall enhancement of HBV 75
replication, respectively (1, 3, 43). In contrast to viral RNA transcription and its 76
regulatory factors, we know little about the structure of viral minichromosomes and the 77
maintenance mechanism of cccDNA in the nucleus of hepatocytes. 78
79
Here, we used ducks congenitally infected with the duck hepatitis B virus (DHBV) to 80
study in vivo structures of viral cccDNA minichromosomes, especially the nucleosome 81
positioning on cccDNA. We found a unique distribution pattern of nucleosomes of 82
DHBV minichromosomes through micrococcal nuclease (MNase) mapping and PCR 83
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amplification of mononucleosomal viral DNA. By comparing the mapping results 84
among DHBV-positive ducks, we showed that nucleosome binding patterns are more 85
conserved in a region of nt. 2000-2700 where various cis-elements and the binding sites 86
of trans-elements of RNA transcription exist (4, 6, 14, 21-23). MNase mapping in other 87
regions of DHBV genomes is more or less variable in different individuals and the 88
variations were passed to the newly infected ducklings. In addition, cccDNA with a few 89
supercoiled turns or bound nucleosomes were consistently found in the livers of 90
ducklings infected either congenitally or horizontally with DHBV, where liver growth 91
and virus spreading are active compared to that of adults. These results benefit our 92
understandings toward the formation and structure of hepadnaviral cccDNA 93
minichromosomes and may facilitate the identification of novel targets for curing HBV 94
infections. 95
96
Materials and Methods 97
98
Isolation of duck hepatocyte nuclei. 99
All animal studies were approved by the Ethics Committee for Animal Experiments of 100
WuXi AppTec. Inc. These studies were conducted in accordance with the current 101
facility’s Standard Operating Procedures (SOPs) and in compliance with the Animal 102
Welfare Act. Researchers of Roche Pharma Research and Early Development 103
monitored all activities in animal studies including the IACUC approval, performance, 104
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and compliance. Isolation of hepatocyte nuclei from domestic ducks that were 105
congenitally infected with DHBV was performed as previously described (25) with 106
modifications. Briefly, 100-300 mg of liver tissue were rinsed in solution H (0.25 M 107
sucrose, 3 mM MgCl2, 10 mM NaH2PO4, pH 6.5) and then disrupted in a loose-fitting 108
Dounce homogenizer. The homogenate was strained through four layers of cheesecloth 109
and centrifuged at 2,500 rpm for 20 min. The pellet was suspended in 7 to 10 volumes 110
of solution H' (1.8 M sucrose, 3 mM MgCl2, 10 mM NaH2PO4, pH 6.5). The suspension 111
was subjected to a centrifugation in a Beckman type T40i rotor at 22,000 rpm at 4°C for 112
1 h. The supernatant was decanted. The nuclear pellet was washed twice with solution 113
H' and the nuclei were counted under microscope after stained with ethidium bromide. 114
115
Nuclease treatment of isolated nuclei. 116
Nuclei (2×108 nuclei/ml) were treated with 0.5 or 2 U/ml micrococcal nuclease (MNase, 117
Takara) or a concentration mentioned specifically in the text in Buffer A (10 mM 118
Tris-HCI, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.3 M sucrose, 10 mM CaC12) at 37°C 119
for 20 min (35). The reaction was stopped by mixing with an equal volume of 2 x stop 120
buffer (100 mM Tris-HCI, pH 7.5, 200 mM NaCl, 2 mM EDTA, 1% SDS). The 121
mixture was then treated with 150 μg/ml of DNase-free RNaseA at 37°C for 1.5 h. 122
Genomic DNA was extracted with phenol and precipitated with ethanol. Total DNA 123
was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and OD260 nm was 124
measured before further analysis. 125
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126
Isolation of cccDNA from duck livers. 127
cccDNA was extracted with Hirt method (39). Briefly, DHBV-positive duck liver was 128
homogenized in a 2-ml Dounce homogenizer. Homogenates were mixed with an equal 129
volume of 4% SDS. Then, majority of cellular components including nucleic acid and 130
proteins were precipitated by mixing with 1/4 volume of 2.5 M KCl. After a 131
centrifugation at 4°C for 10 min, supernatant was transferred to a different tube and 132
extracted with phenol. After ethanol precipitation, nucleic acids including cccDNA 133
were dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). 134
135
Indirect end labeling. 136
Five probes, named as A, B, C, D, and E, were used in this study. Each probe was 137
synthesized by using a PCR DIG Probe Synthesis Kit (Roche Applied Science) with 138
dNTP mix containing Digoxigenin (DIG)-11-dUTP. DHBV16 plasmid was used as the 139
template of PCR amplifications. As shown in Fig.1 and 4, probe A spans at nt. 42~385 140
(close to EcoR I site, 344 bp in length); probe B spans at nt. 399~747 (close to Bgl II 141
site, 348 bp in length); probe C spans at nt. 49~385 (close to Bgl II site, 337 bp in 142
length); probe D spans at nt. 1298~1623 (close to Kpn I site, 353 bp in length); probe E 143
spans at nt. 1003~1284 (close to Kpn I site, 282 bp in length). DNA (~50 μg) extracted 144
from MNase-treated nuclei was digested with a restriction enzyme that has a unique site 145
on DHBV genome. DNA was extracted again with phenol followed by ethanol 146
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precipitation. The pellet was dissolved in 50 μl TE and subjected to an electrophoresis 147
of 2% agarose gel in 1 x TAE buffer overnight. After denaturation and neutralization, 148
DNA was blotted onto a Hybond-N+ membrane (GE Healthcare) in 20×SSC and 149
hybridized with a DIG-labeled DHBV DNA probe targeting a specific region of DHBV 150
DNA. After incubating blots with an alkaline-phosphatase-conjugated anti-DIG 151
antibody, hybridization signals were detected in a standard chemiluminescence 152
reaction. 153
154
Real-time PCR analysis. 155
Isolated hepatocyte nuclei from DHBV-positive ducks were digested with 16 U/ml 156
micrococcal nuclease in Buffer A described above at 37°C for 20 min. Total DNA was 157
then extracted with phenol and subjected to a 1.5% agarose gel electrophoresis. The 158
band corresponding to mononucleosomal DNA was cut and DNA was extracted with a 159
gel extraction kit (Qiagen). cccDNA from the same duck(s) was extracted as described 160
above and used as reference DNA of MNase mapping and PCR amplification. PCR was 161
performed on a LightCycler® 480 II (Roche Diagnostics) in a final volume of 20 μl in 162
which 1 μl of purified mononucleosomal DNA or cccDNA was included as PCR 163
templates. Amplification was done as follows: denaturation program (95°C for 10 min), 164
amplification and quantification program with 45 times repeating (95°C for 10 s, 55°C 165
for 30 s, 72°C for 30 s with a single fluorescence measurement), melting curve program 166
(60 to 95°C with a heating rate of 0.1°C per second and a continuous fluorescence 167
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measurement) and finally a cooling step to 40°C. The amplification efficiency of 168
mononuclesomal DNA was normalized by comparing Ct value of mononucleosomal 169
DNA to that of cccDNA (ratiomononucleosomal DHBV DNA/cccDNA= 2 -∆Ct) in each reactions. 170
Thirty-six overlapping regions on DHBV16 genome were chosen for amplification (Fig. 171
3B and Fig. 4C). The starting and ending nucleotides (nt.) of each region are listed as 172
following: region 1 (1656-1857); region 2 (1726-1929); region 3 (1798-1978); region 4 173
(1876-2082); region 5 (1949-2133); region 6 (1973-2201); region 7 (2062-2303); 174
region 8 (2183-2380); region 9 (2228-2436); region 10 (2256-2489); region 11 175
(2291-2524); region 12 (2404-2607); region 13 (2493-2704); region 14 (2822-3006); 176
region 15 (2859-38); region 16 (2922-98); region 17 (3003-211); region 18 (70-298); 177
region 19 (164-390); region 20 (238-446); region 21 (333-539); region 22 (359-612); 178
region 23 (458-693); region 24 (497-753); region 25 (627-850); region 26 (689-916); 179
region 27 (765-998); region 28 (863-1053); region 29 (863-1112); region 30 180
(958-1154); region 31 (969-1227); region 32 (1123-1327); region 33 (1233-1454); 181
region 34 (1368-1542); region 35 (1458-1677); region 36 (1529-1711). 182
183
Comparison of levels of DHBV RNA and core protein in duck and duckling livers. 184
100 mg DHBV-positive duck or duckling liver samples were homogenized in 1.5 ml TE 185
buffer (50 mM Tris-HCl and 1 mM EDTA, pH 8.0) in a 2-ml Dounce homogenizer. The 186
homogenate was aliquoted into four parts to extract viral cccDNA, replicative 187
intermediate DNA, total RNA, and to prepare protein samples for the measurement of 188
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DHBV core protein. Total liver RNAs in the homogenate were extracted with RNeasy 189
Mini kit (Qiagen) by following the manufacturer’s instructions. Contaminated DNA was 190
eliminated by an on-column DNase digestion step. After quantitation and normalization 191
of RNA samples, RNA was reverse-transcribed with iScriptTM cDNA synthesis kit 192
(Bio-Rad) in which a mixture of oligo (dT) and random primers were used. Then, 193
DHBV cDNA was measured by real-time PCR and normalized with the cDNA of 194
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using LightCycler® 480 (Roche 195
Diagnostics). Primers for DHBV real-time PCR amplification are 196
5’-TTTGGATAGGGCTAGGAGATTG-3’ (sense, nt.42-63) and 197
5’-AGGCGAGGGAGATCTATGGTG-3’ (antisense, nt.385-405). This set of primers is 198
to measure of the levels of PreC/C viral RNAs due to the position of amplification. 199
Primers for duck GAPDH real-time PCR amplification are 200
5’-CATCGTGCACCACCAACTG-3’ (sense) and 5’-CGCTGGGATGATGTTCTGG-3’ 201
(antisense). To measure DHBV core protein, homogenate was treated briefly with 202
CA-630 at a final concentration of 2%. Nuclei and other cellular debris were removed 203
by a short centrifugation. Protein concentration of the supernatant was determined by the 204
Bradford method. Equal amounts of total liver proteins were loaded on a 4-12% 205
SDS-PAGE gel and level of DHBV core protein was evaluated in a western blot analysis. 206
Beta-actin was used as an internal control. 207
208
Infection of ducklings. 209
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DHBV-negative Cherry Valley ducklings (3 days old) were inoculated intravenously 210
with DHBV-positive sera at a volume of 0.2 ml per ducklings (40). Nine days after 211
inoculation, the infected ducklings were sacrificed and the livers were removed and 212
stored at -80°C until use. 213
214
Results 215
216
MNase mapping of hepadnaviral cccDNA minichromosomes. 217
Although previous studies showed that hepadnaviral cccDNA exists in the nucleus of 218
hepatocytes as individual minichromosomes, details about the distribution of host 219
nucleosomes in these "beads-in-a-string" structures are largely unknown (25). In this 220
study, we used ducks congenitally infected with DHBV to map the pattern of 221
nucleosome distribution on DHBV cccDNA minichromosomes in vivo. 222
223
As a general scheme, DHBV-positive liver homogenates were subjected to a 224
centrifugation in which nuclei passed through a sucrose cushion. The isolated nuclear 225
fractions that contain both cellular chromatin and viral minichromosomes were partially 226
digested with MNase (33, 35). As a starting point of MNase mapping, total DNA after 227
treated with MNase at different concentrations was extracted and subjected to an 228
agarose gel electrophoresis and Southern blot hybridization using a full-length DHBV 229
DNA probe. As reported previously (25) and shown in Fig. 1B, a typical pattern of 230
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mono-, di-, tri-nucleosomes that were generated from DHBV cccDNA 231
minichromosomes as a result of MNase digestion was detected. To locate the positions 232
of MNase cleavage on viral cccDNA, EcoRI, possessing a unique site in DHBV 233
genome, was chosen to cut DNA fragments generated by MNase. Viral cccDNA 234
fragments containing one end derived from MNase cleavage and the other end from 235
EcoRI digestion were detected in a Southern hybridization with a short probe (probe A, 236
nt. 42-385 of DHBV16 genome) that is close to EcoRI site (Fig. 1A). As shown in Fig. 237
1C, following MNase digestions which covered MNase concentrations from 0.1 to 8 238
U/ml, nuclear fractions showed a consistent pattern with distinctive bands in a Southern 239
blot. Based on the sizes of these bands, positions of MNase cleavage on DHBV genome 240
were inferred. As more MNase was added in the reactions, signals of most bands, 241
especially large ones, were gradually reduced and finally disappeared. In contrast, 242
naked cccDNA, which was purified from the same liver sample, displayed different 243
hybridization patterns (Fig. 1C). After a 5-minute MNase treatment at a concentration 244
of 8 U/ml, no hybridization signal was detected in naked cccDNA while a weak but 245
clear pattern was still observable in the minichromosome fractions, even the duration of 246
treatment was longer (20 min) for the latter, suggesting a higher accessibility/sensitivity 247
of naked cccDNA to MNase. At a lower concentration of 2 U/ml, MNase digestion of 248
naked cccDNA generated individual bands that were buried in high backgrounds. 249
Unlike purified cccDNA molecules, specific regions of DHBV minichromosomes in 250
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the nuclei fraction were protected from MNase disgestion, raising a possibility that 251
these regions are bound with nucleosomes or other cellular/viral structures. 252
253
Unique pattern of MNase mapping is shared among DHBV infected ducks. 254
In order to see if a similar pattern of MNase mapping described above could be 255
observed among individuals where viral DNA sequences and host general status might 256
be different, four liver samples of DHBV-positive ducks were harvested and MNase 257
mapping of nuclear fractions of hepatocytes was performed and compared in a 258
side-by-side manner. As shown in Fig.2, besides the presence or absence of several 259
MNase cleavage bands (marked on the right side of the blot), overall MNase mapping 260
showed similar MNase-less-accessible regions (marked with a series of brackets) 261
among all four ducks (including the previous one shown in Fig.1, that is 2# in Fig. 2). 262
Patterns in a region of approximate nt. 2000 - 2700 in cccDNA were highly 263
reproducible among four samples. The presence of distinctive and consistent patterns in 264
terms of MNase accessible sites/resistant regions suggested that nucleosomes are not 265
randomly distributed on the hepadnaviral minichromosomes, at least in some regions of 266
cccDNA. 267
268
Determination of nucleosome binding positions on cccDNA minichromosomes by 269
real-time PCR. 270
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To supply more evidences to the claim that the protected regions of DHBV cccDNA in 271
the viral minichromosomes are associated with nucleosomes, mononucleosomal DNAs 272
generated by MNase treatment were prepared. We assumed that these short DNA 273
fragments, according to their sizes, should bind one nucleosome. DHBV-specific PCR 274
amplifications on isolated mononucleosomal DNA were performed to determine the 275
size and the boundary of the protected regions. In general, mononucleosomal DNA was 276
gel-purified after an extensive MNase digestion of nuclear fractions that broke down 277
most cccDNA minichromosomes to mononucleosomes, as verified by a Southern 278
hybridization (data not shown). Within a part of DHBV genome (nt. 1650 to 2700) in 279
which a reproducible MNase mapping was observed in viral minichromosomes of the 280
four DHBV-positive ducks (Fig. 2), thirteen consecutive, overlapping PCR 281
amplification regions were allocated (region 1 or R1 to region 13 or R13, shown at the 282
bottom of Fig. 3B). Each amplification region contained two sets of individual PCR 283
reactions, for example, region one (R1) has set 1 and set 2 and region two has set 3 and 284
set 4, and so on (Fig. 3A). In each set there were four individual PCR amplifications. 285
One primer from one side of amplification was fixed and primers on the other side were 286
different, generating PCR products of 75 – 120 bp in length (Fig. 3A). In order to 287
normalize the efficiencies of mononucleosomal amplifications at various positions of 288
DHBV genome, isolated mononucleosomal DNA and naked cccDNA were used as 289
templates and amplified in parallel for all PCR amplifications. Ct values of individual 290
mononucleosomal amplifications were aligned with that of the corresponding cccDNA 291
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template to obtain a ratio of mononucleosomal DHBV DNA vs. cccDNA. As shown in 292
Fig. 3B, products amplified from mononucleosomal DNA were detected in five out of 293
thirteen amplification regions (region 1 or R1, R7, R9, R11, and R13). In each of these 294
regions, some PCRs in the inner part produced amplification signals that were 50% or 295
more when compared to that of naked cccDNA. As the distance between the two 296
primers became bigger, amplification signals were attenuated. This result was 297
consistent with MNase mapping where all five regions were clearly less accessible to 298
MNase digestion (gel image on the top of Fig. 3B). In addition, several amplifications 299
in the region of around nt. 1800-1950 (Fig. 3B, region 3) showed a moderate ratio of 300
mononucleosomal DNA/cccDNA Ct values, suggesting presence of a weak protection 301
site that was probably due to a nucleosome binding in this region. On the contrary, 302
mononucleosomal amplifications in the rest seven regions (R2, R4, R5, R6, R8, R10, 303
and R12) were relatively inefficient as reflected by the lower Ct values that were ~10 304
folds less than that of cccDNA. When aligned with the MNase mapping, it was found 305
that some of these poorly amplified regions straddle two MNase insensitive areas with 306
MNase cleavage sites in the middle and the others are located in the MNase sensitive 307
regions where strong signals derived from multiple MNase cleavages were observed. 308
We named the major MNase cleavage bands shown in the top panel of Fig. 3B and 309
aligned them on DHBV genome according to the estimated sizes of those bands. For 310
the details of designation of MNase cleavage sites that scattered throughout whole 311
DHBV genome, please refer to descriptions in the result section and the figure legend 312
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of Fig. 4 (below). Taken together, in a region of nt. 1650-2700 of DHBV genome, the 313
location of nucleosome-protected viral DNA revealed by real-time PCR of isolated 314
mononucleosomal DNA correlates well with the pattern of nucleosome binding mapped 315
by a partial MNase digestion (Fig. 1 and 2). 316
317
Genome-wide mapping of nucleosome binding of DHBV cccDNA 318
minichromosomes. 319
In order to obtain a genome-wide mapping of nucleosome binding of DHBV cccDNA 320
minichromosomes, we prepared nuclear fractions of hepatocytes from one 321
DHBV-positive duck and employed the two approaches described above: Southern blot 322
hybridization to show MNase cleavage patterns of cccDNA minchromosomes and PCR 323
amplifications of isolated mononucleosomal DHBV DNA that cover the rest 2/3 of 324
DHBV genome. For the first approach, we chose three restriction enzymes, EcoRI, 325
BglII, and KpnI, each of them has a unique site in DHBV genome, to do MNase 326
mapping. Southern blot hybridization with five different probes, A, B, C, D, and E from 327
two orientations (determined by relative positions between a probe and a restriction site) 328
are shown in Fig. 4A and 4B. Probes A, B, and D share a clockwise orientation. 329
Hybridization with these three probes (first, second, and fourth panels in Fig. 4B) 330
produced similar and overlapping MNase mapping patterns. Specific regions of viral 331
genome presented in individual blots, when combined together, cover the whole DHBV 332
cccDNA. Probes C and E share a counter-clockwise orientation. Hybridization with the 333
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two probes (third and fifth panels in Fig. 4B) resulted in MNase mapping that was 334
arranged in a way opposite to the mapping of probes A, B, and D. By aligning 335
individual bands shown in each blots with a standard length curve of DNA markers, 336
sizes and positions of MNase bands on DHBV genome were toughly determined. Based 337
on the estimated sizes and patterns of individual bands shown in different blots, 24 338
major MNase-cleavage bands were named in an alphabetical way and marked on the 339
right side of each blot in Fig. 4B as well as on a linearized DHBV genome in Fig. 4C. 340
Because of different probes and restriction enzymes used in individual blots, an 341
individual band or a group of bands with the same designations was located at different 342
positions or was arranged at opposite orientations in different blots. While the majority 343
of these bands were consistently observed, some new bands were detected when 344
different restriction enzymes and probes were employed which might reflect a 345
resolution change. For example, a group of bands condensed on the top of a gel were 346
separated well around the bottom of a gel when a different restriction enzyme was used. 347
For the second approach, mononucleosomal DNA generated from MNase digestion was 348
gel-purified and subjected to random PCR amplifications, similar to the amplifications 349
described in Fig. 3. As shown in Fig. 4C, 23 regions that cover most of the rest 2/3 350
DHBV genome were amplified through 46 sets. Combining MNase cleavage sites and 351
the ratios of mononucleosomal DHBV DNA vs. cccDNA in different amplification 352
regions, we identified several other nucleosome binding positions, especially, positions 353
between L3 – M (R26), N3 – O (R20), O-P (R18), P-A (R16). It is worth to mention 354
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that in the area ranging from nt. ~900 to nt. ~1200 we failed to obtain an appreciable 355
ratio of mononucleosomal DHBV DNA vs. cccDNA with five overlapping 356
amplification regions (R27-R31) which are upstream of small S coding region. The 357
detailed positions of all amplification regions in DHBV genome were shown in Fig. 4C 358
as well as in the Materials and Methods section. The relation between bound 359
nucleosomes and DHBV cis-acting elements (4, 6, 14, 21-23, 37) was shown in Fig. 4C 360
(bottom). 361
362
Patterns of MNase mapping of cccDNA minichromosomes passed from adult 363
ducks to horizontally infected ducklings. 364
As shown in Fig. 2, although the patterns of MNase mapping of cccDNA 365
minichromosomes were very similar among the four ducks, a closer inspection revealed 366
that several distinct DNA fragments did not appear in all the ducks examined. While it 367
is possible that the observed differences in nucleosome positioning in the certain 368
regions of cccDNA minichromosomes among the different ducks are due to differences 369
of cccDNA sequences, viral RNA transcription status, and other unknown host or viral 370
factors, it is more interesting to know whether the traits are inheritable features of these 371
viruses. To address this question, two MNase mappings with distinguishable bands (Fig. 372
5A) were chosen and the corresponding serum samples were used to inoculate newly 373
hatched DHBV-negative ducklings (two ducklings in each group). Nine days later, liver 374
and serum samples were harvested for MNase mapping and viral DNA sequencing. As 375
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shown in Fig. 5B, characteristic bands in two regions of minichromosomes in adult 376
ducks were visualized in MNase mapping of all infected ducklings, correspondingly. 377
DNA sequence changes in dominant viral species between adults and infected 378
ducklings were not detected. These results indicate that although differences such as 379
kinetics of virus replication, virus spreading, and liver microenvironments exist 380
between adults and ducklings, overall minichromosome structures reflected by the 381
patterns of MNase mapping are inheritable. 382
383
In addition, we aligned the two original DHBV DNA sequences that were obtained 384
from duck 5# and 6# for a possible relationship between sequence variations and a 385
specific MNase pattern. We found both variable areas marked in Fig. 5A associated 386
with a higher nucleotide substitution rate when compared to the surrounding regions in 387
DHBV genome. DNA covered by the bracket in Fig. 5A encodes part of PreS and the 388
overlapping spacer region of DHBV polymerase and was highly variable between the 389
two sequences (there are 37 base-pair substitutions scattering in the region of ~260 bp 390
in length). Another region marked by an arrow in Fig. 5A encodes a part of viral 391
polymerase and also showed a high substitution rate between the two sequences (21 392
substitutions in the region of ~320 bp in length). Although we had data of viral DNA 393
sequences and MNase mapping for the two DHBV-positive ducks, we, however, were 394
unable to pinpoint or correlate a sequence variation with a specific MNase pattern. We 395
thought this might be due to, at least partially, the fact that MNase mapping here has a 396
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lower resolution when compared to DNA sequencing and the fact that multiple 397
substitutions scattered in the regions. Moreover, it is possible that a sequence variation 398
might result in some structural changes on viral minichromosomes that are away from 399
the original position of the sequence variation through protein-DNA and protein-protein 400
interactions. 401
A portion of cccDNA in duckling livers has only a few supercoiled turns. 402
During the comparison of MNase mappings of cccDNA minichromosomes between 403
adult ducks and ducklings, we found that it was always the case that more isolated 404
nuclear fractions of ducklings were required to generate signals equivalent to that of 405
adult ducks in Southern hybridization after a partial MNase digestion though other 406
conditions were the same, indicating a possibility that duckling hepatocytes might 407
contain more partially assembled cccDNA minichromosomes that are easily accessible 408
to MNase digestion. Initial confirmation by Southern blot hybridization, however, 409
failed to show a significant difference between adult and duckling cccDNA samples. 410
We then performed a more careful electrophoresis in which a small sample volume (~ 411
10 µl) was loaded to a well and cccDNA was separated through a 25 cm-long, 0.9% 412
agarose gel overnight. It was repeatedly observed in duckling samples that besides a 413
predominant, fast moving cccDNA band, a portion of cccDNA molecules migrated 414
slowly to form several discrete bands. These slowly moving bands were marked with 415
arrows in Fig. 6A and represented cccDNA molecules with less superhelical turns that 416
differ by one turn in neighboring bands. Between cccDNA prepared from DHBV 417
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congenitally or horizontally infected ducklings (4-14 days old) there was no apparent 418
difference in terms of patterns and positions of these topoisomer bands (Fig. 6A, 419
compare lanes 5, 6, 7, 8 to lanes 9 and 10). In contrast, less slowly moving topoisomers 420
of cccDNA were detected in cccDNA preparations of adult ducks (Fig. 6A, lanes 1, 2, 3, 421
and 4). To reinforce the claim that the slowly moving bands detected in duckling livers 422
are cccDNA with less supercoiled turns, duckling cccDNA was further analyzed with 423
different treatments. First, mobilities of cccDNA and the slowly moving bands were the 424
same after a heat denaturation (Fig. 6B, lane 2). This treatment converted nicked 425
double-stranded circular and double-stranded linear DNA into single-stranded DNA. 426
Supercoiled DNAs, however, were renatured and migrated in electrophoresis at the 427
same mobility as their native forms after heat treatment. Secondly, treatment with 428
E.coli. topoisomerase I that efficiently relaxes cccDNA with nagetively superhelical 429
turns converted most of cccDNA including those slowly moving bands into relaxed 430
circular DNA and much less supercoiled cccDNA (Fig.6B, lane 4). These results 431
support the notion that the slowly moving species extracted from DHBV-positive 432
duckling livers are cccDNA with less negatively superhelical turns. Since the number 433
of superhelical turns in a cccDNA is equal to the number of bound nucleosomes on this 434
molecule (25), detection of a significant portion of cccDNA with a few negatively 435
supercoiled turns in duckling hepatocytes suggest that cccDNA molecules with a few 436
bound nucleosomes are more prevalent in young ducks where liver growth and 437
cccDNA formation are drastically occurring. 438
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439
In order to study if the superhelicity of cccDNA affects viral RNA levels, we chose four 440
DHBV congenitally infected birds (two ducks and two ducklings) to extract cccDNA, 441
viral replicative intermediate DNA, total RNA, and to prepare protein samples. 442
cccDNA with less superhelical turns was detected in two duckling samples (Fig. 6C) as 443
shown in Fig. 6A. Equal amounts of total RNA were used for measuring the levels of 444
DHBV RNA and RNA of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by 445
real-time RT PCR. DHBV RNA in the two ducklings was 10-25 folds higher than that 446
of ducks (Fig. 6C). As a control, GAPDH RNA in ducklings was 4-8 folds higher than 447
that in ducks (Fig. 6C). Therefore, the ratio of DHBV PreC/C RNA vs. GAPDH RNA 448
in ducklings was 2-3 folds higher than that of ducks. We inferred from these results that 449
cccDNA in ducklings are associated with a higher level of viral RNAs but could not 450
rule out a possibility that in ducklings those RNAs have a longer half-life. Different 451
from viral RNA, DHBV core protein and replicative intermediates were nearly equally 452
detected in ducks and ducklings with some variations. 453
454
Discussion 455
456
It has been reported that the genome of several DNA viruses exists as an individual 457
minichromosome in the nucleus of infected host cells. For long-term episomal 458
maintenance of this viral structure from a parental cell to two daughter cells, viral DNA 459
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segregation must occur. This process involves “chromosome tethering” in many DNA 460
viruses in which a viral protein binds to both a specific viral sequence, such as ori, and 461
a host chromosome (15, 16, 19, 30). Such association enables the acentric viral 462
minichromosome to utilize the chromosomal centromere in trans, thereby achieving 463
efficient transmission of viral DNA genomes during cell division. The mechanism of 464
the maintenance of hepadnaviral cccDNA minichromosomes in the nucleus of 465
hepatocytes, however, is unclear. Given that hepatocytes are long-living cells, pressures 466
on viral DNA segregation might be less or attenuated in the case of hepadnaviruses. For 467
persistent hepadnaviral infection, stably maintaining cccDNA in the nucleus is crucial 468
since the replenishment of viral RC DNA, the precursor of cccDNA, might be 469
inefficient in the presence of viral large envelope protein, the negative regulator of RC 470
DNA nuclear translocation (10, 34). As the first steps of understanding of hepadnaviral 471
cccDNA maintenance in the nucleus, it is informative to clarify the overall structure of 472
cccDNA minichromosomes, especially nucleosome positioning on cccDNA. 473
474
In this study, we used MNase mapping and PCR amplification of isolated 475
mononucleosomal DNA to study nucleosomes positioning of hepadnaviral cccDNA 476
minichromosomes. Decision of choosing DHBV congenitally infected ducks as a 477
surrogated model for this purpose was based on the following considerations: First, 478
using DHBV-positive ducks we could study viral minichromosome structures in 479
normally differentiated hepatocytes. Secondly, it is experimentally practical to transmit 480
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virus into individuals of various physiological conditions, for example, ducks or 481
ducklings that supplies opportunities to study viral minichromosomes under different 482
liver microenvironments (28). 483
484
In eukaryotic genomes including human DNA, positioning of nucleosome occupancy 485
and depletion along DNA strands is determined by both trans-elements such as 486
transcription factors, chromatin remodelers, and RNA polymerase and cis-elements 487
represented by nucleosome sequence preferences (5, 17, 27, 42). The latter are specific 488
patterns of DNA sequence that affect DNA local bendability around a small histone 489
octamer core (17, 42). In hepadnaviruses, considering the fact that viral RNA 490
transcription is regulated by many cis- and trans- elements (6, 7, 13, 23) and only a few 491
copies of cccDNA minichromosomes, the transcription template, exist in each 492
hepatocytes (41, 44), viral minichromosomes are expected to have a specific structure 493
in terms of nucleosome positioning and other features to fulfill the complexity and 494
efficiency of its functions. Besides the viral sequence that could be one of determinants 495
of nucleosome binding through nucleosome sequence preferences as described in other 496
species, part of uniqueness of cccDNA minichromosomes structure might be derived 497
from the asymmetry nature of the precursor of cccDNA, RC DNA. The asymmetries of 498
RC DNA include a short RNA oligomer at the 5’end of the plus stranded DNA, a 499
redundant sequence at the two ends of the minus stranded DNA, a cohesive region 500
between the 5’ ends of plus and minus DNA strands, and a single-stranded region in 501
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RC DNA due to uncompleted elongation of plus-strand DNA (11, 12). Once 502
translocated into nucleus, RC is the target of host DNA repair machinery that fixes 503
those asymmetries and converts RC to cccDNA. The specific locations of the 504
asymmetries would cause an uneven distribution of cellular DNA repair machinery on 505
RC DNA (31). These “hot” spots enriched with host proteins might act as the first 506
players in the formation of cccDNA minichromosomes by triggering preferential 507
nucleosome binding on viral genome or expelling through steric hindrance the binding 508
of proteins from this region that perform a negative role on the assembly of cccDNA 509
minchromosomes. It is worthwhile to notify that a highly reproducible pattern of 510
nucleosome binding shown in several ducks as well as ducklings (Figs. 2 and 5) 511
coincides with the region of nt. 2000-2600 of DHBV genome where most of the 512
asymmetries of RC DNA locate. 513
514
Another clue revealed in this study that might be useful for addressing the formation 515
and structure of cccDNA minichromosomes is that a portion of viral cccDNA in the 516
livers of ducklings carries a few nucleosomes reflected by a low number of superhelical 517
turns (Fig. 6). This is apparently different from cccDNA detected in adult ducks where 518
much less cccDNA with < 8 supercoiled turns was observed in a Southern blot. It has 519
been claimed that there are two populations of cccDNA, dependent upon the number of 520
bound nucleosomes (25), an inactive or less active form of cccDNA in terms of viral 521
RNA transcription where nucleosomes are fully loaded on cccDNA; an active form of 522
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cccDNA for transcription in which only part of cccDNA is bound with nucleosomes. 523
The results described in this study might raise another possibility for the reasons of 524
existence of cccDNA that is partially loaded with nucleosomes: They may be 525
intermediates of a process in which nucleosomes are gradually bound to cccDNA. 526
Given that the total number of hepatocytes is rapidly increased in ducklings, total 527
amounts of cccDNA have to be proportionally increased by means of de novo infection 528
and/or intracellular cccDNA amplification (34), dependent on the mechanism 529
underlying virus spreading to match the liver growth. Therefore, it is not surprising to 530
detect cccDNA with a few negative supercoils during a time when a large amount of 531
cccDNA is converted from RC and other forms of precursors. If these cccDNAs were 532
really initial intermediates of fully nucleosome-loaded cccDNA minichromosomes, it is 533
worth to separate these minichromosomes and detect protected virus sequences to see 534
whether or not some specific regions of cccDNA are preferentially loaded with 535
nucleosomes. Careful analysis of these special populations of viral minichromosomes 536
and comparison between liver samples of different ages and physiological status might 537
disclose the kinetics and sequence of nucleosomes loading on hepadnaviral cccDNA 538
minichromosomes. An alternative scenario that could be involved in the presence of the 539
less-supercoiled cccDNA in the duckling livers may be related to the rapid liver growth 540
in these young ducks where host factors like histones are used for the formation of both 541
cellular chromosomes and viral minichromosomes. A possible competition for these 542
key chromosome components might slow down the assembly process of viral 543
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minichromosomes, resulting in cccDNA with less negatively supercoiled turns in the 544
duckling livers. 545
546
In order to fully understand the functionality including the maintenance mechanism of 547
cccDNA, it is important to obtain information of protein components bound at specific 548
sites of viral minichromosomes. Though various host proteins as well as HBV core and 549
X antigen have been reported to be associated with minichromosomes (1-3, 20, 25, 26), 550
exact binding positions and mechanisms of action of these components are not clear. 551
Complete elucidation of hepadnaviral minichromosome structures is a challenge despite 552
extensive efforts have been made. A main hurdle was a heavy contamination of host 553
counterpartners during the process of isolation of viral minichromosomes, though 554
different purification methods were employed in a tandem manner such as sucrose 555
gradient centrifugation, gel filtration, and immunoprecipitation with different 556
antibodies. The other difficulty was the overall instability of minichromosomes during 557
the isolation: dissociation of some components with a lower binding affinity from 558
minichromosomes might cause inconsistence when comparing protein ID of different 559
purification preparations. Nevertheless, the results of nucleosome mapping on viral 560
minichromosomes reported here will supply initial framework information for the 561
understanding of overall complete structure of this key viral component that may 562
provide a basis of new therapeutic interventions for the elimination of cccDNA from 563
HBV infected livers. 564
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565
566
567
Reference 568
569
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40. Zhang, Y. Y., D. P. Theele, and J. Summers. 2005. Age-related differences in 680 amplification of covalently closed circular DNA at early times after duck 681 hepatitis B virus infection of ducks. J Virol 79:9896-903. 682
41. Zhang, Y. Y., B. H. Zhang, D. Theele, S. Litwin, E. Toll, and J. Summers. 683 2003. Single-cell analysis of covalently closed circular DNA copy numbers in a 684 hepadnavirus-infected liver. Proc Natl Acad Sci U S A 100:12372-7. 685
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696 697 Figure Legend 698 699
Fig. 1. Mapping of nucleosome binding on DHBV cccDNA minichromosomes. 700
(A) Strategy of MNase mapping of viral minichromosomes with a probe (Probe A) that 701
hybridizes a small region close to EcoRI site, a unique position on DHBV genome. 702
Curves with an arrow represent fragments of cccDNA generated from MNase and 703
EcoRI cleavage. (B) DHBV DNA fragments detected by Southern blot hybridization 704
following MNase digestion. Nuclei fractions of hepatocytes were prepared from liver 705
samples of a DHBV-infected duck (see the section of Materials and Methods) and were 706
treated with MNase at different concentrations. A probe of full-length DHBV DNA 707
genome (DHBV 16) was used in hybridization. Mono, Di, and Tri represent 708
mononucleosomal, dinucleosomal, and trinucleosomal DHBV DNA, respectively. (C) 709
MNase cleavage on purified cccDNA and viral minichromosomes in isolated nuclei. 710
MNase concentrations (U/ml) and treatment duration (in minutes) employed in this 711
experiment are labeled on the top of the blots. Probe A (nt. 42-385) was used in 712
hybridization. A DNA ladder with known sizes (bp) is shown on the left of the blots. 713
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714
Fig. 2. Comparison of MNase mapping among four DHBV-positive adult ducks. 715
The experimental conditions were the same as that used in Fig. 1C. Specifically, each 716
reaction contained MNase at a concentration of 8 U/mL and the vials were incubated at 717
37˚C for 20 minutes. Variations of MNase cleavages on viral minichromosomes of 718
individual ducks are marked with arrow heads and MNase-less-accessible regions were 719
marked with brackets shown on the right side of the blot. A DNA ladder with known 720
sizes (bp) is shown on the left of the blot. 721
722
Fig. 3. Nucleosome-protected regions on viral minichromosomes revealed by 723
real-time PCR. (A) A diagram of the relation between nucleosome binding and 724
positions of different amplification sets. Sets 1, 2, 5, and 6 locate at nucleosome 725
protected regions (R1 and R3). Sets 3 and 4 cross a linker region (R2). In each set, four 726
PCR amplifications were performed. One primer from one side of amplification is at a 727
fixed position (S1 in set 1, 3, and 5; AS1 in set 2, 4 and 6). Primers on the other side of 728
each amplification set are at different positions. (B) Alignment of MNase mapping with 729
the data of real-time PCR that covered one-third of viral genome (nt. 1650 – 2700). 730
Mononucleosomal DNA was isolated after an extensive MNase cleavage (16 U/mL for 731
20 minutes) and used for real-time PCR as described in the section of Materials and 732
Methods. Ct values of individual monochromosomal PCR were normalized with that of 733
amplification of naked cccDNA extracted from the same bird. Each line in the figure 734
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represents one specific PCR amplification. It contains information of the two primer 735
positions (dots at the two ends of each line) relative to DHBV genome that is shown in 736
numbers (nt) in X-axis dimension as well as information of the ratio of 737
minichromosomal DHBV DNA vs. naked cccDNA as the height of each line (Y-axis). 738
Lines in red color represent PCR amplification sets that are located in putative 739
nucleosome protected regions according to MNase mapping shown on the top of Fig. 740
3B. Lines in blue color are PCR amplifications that straddle two nucleosome protected 741
regions or in regions where multiple MNase cleavages were observed in MNase 742
mapping. MNase mapping was done as described in Fig. 1 and 2 in which MNase 743
partially-digested nuclear DNA was completely digested with EcoRI and followed by 744
Southern blot hybridization with probe A. Major MNase cleavage sites were named in 745
an alphabetical manner and marked on the blot as well as on a linearized DHBV 746
genome in Fig. 3B. Positions of thirteen amplification regions (R1 to R13) are shown at 747
the bottom of Fig. 3B. 748
749
Fig. 4. Overall mapping of nucleosome binding of DHBV cccDNA 750
minichromosomes. (A) A diagram of positions of probes and unique restriction sites 751
on DHBV genome. Arrow of each probe points an orientation of the detected 752
MNase-cleavage fragments: starting from a specific restriction site and circling around 753
viral genome either clockwise (probes A, B, and D) or counter-clockwise (probes C and 754
E). (B) MNase mapping with individual probes following a partial MNase digestion 755
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and a complete restriction enzyme digestion and Southern hybridization. Sizes of a 756
series of DNA markers are shown on the left. MNase cleavage bands were aligned with 757
DNA markers and labeled in an alphabetic manner based on sizes calculated in 758
different blots. Restriction enzyme, probe, and concentrations of MNase used in each 759
blot are shown on the top. (C) Alignment of MNase mapping and real-time PCR in the 760
rest two-third of viral genome (nt. 2800 – 1700 shown in X-axis). The ratios of 761
minichromosomal DHBV DNA vs. naked cccDNA detected in PCR are shown as the 762
height of each line (Y-axis). Lines in red color represent PCR amplification sets with 763
relatively high ratios that are located in putative nucleosome protected regions 764
according to MNase mapping shown in Fig. 4A. Lines in blue color are PCR 765
amplifications that straddle two nucleosome protected regions or in regions with 766
relatively low amplification signals of mononucleosomal DHBV DNA.Positions of 767
twenty-three amplification regions (R14 to R36) and the rough positions of MNase 768
cleavage bands marked in Fig. 4B are shown beneath amplification data..Schematic 769
view of nucleosome binding on linearlized DHBV genome and their relations to 770
cis-acting elements of viral RNA transcription are shown at the bottom. Positions of the 771
initiation sites of viral transcripts, PreS, S, and PreC/C, polyadenylation site, promoters, 772
enhancer, pet (positive effector of transcription), and binding sites of several host 773
factors that play roles in viral RNA transcription initiation are marked by blue arrows 774
and bars with different colors, respectively (4, 6, 14, 21-23, 37). 775
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Fig. 5. Patterns of MNase mapping passed through adult ducks to newly infected 777
ducklings. Three-day old DHBV-negative ducklings were inoculated intravenously 778
with the serum of adult duck 5# or 6# (0.2 ml per duckling), respectively. Nine days 779
after infection, liver samples of two ducklings of each group were collected for MNase 780
mapping. (A) MNase mapping patterns of DHBV minichromosomes of the two adult 781
ducks. (B) MNase mapping patterns in ducklings. Two noticeable variations in MNase 782
mapping that passed to viral minichromosomes of ducklings are labeled with an arrow 783
and a bracket, respectively, on the left of the gels. 784
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Fig. 6. Topoisomers of cccDNA detected in adult ducks and ducklings. (A) 786
cccDNA was extracted simultaneously from the livers of DHBV-positive adult ducks as 787
well as DHBV horizontally- and vertically-infected ducklings. DNA was separated in a 788
gel (25 cm long) slowly (1 volt/cm) before blotting and hybridization. Negative 789
supercoiled turns of cccDNA are marked on the left of the gels. (B) Superhelicity 790
changes of duckling DHBV cccDNA following different treatments. cccDNA untreated 791
control (lane 1 and 3); Heat denaturation (100゜C, 2 min, lane 2); E.coli topoisomerase I 792
treatment (1 unit enzyme /reaction, 37゜C for 1 hour, lane 4). (C) Comparison of levels 793
of DHBV RNA and other viral components in duck and duckling livers. For each bird, 794
after RNA extraction and concentration normalization, triplicated RNA samples were 795
reversed-transcribed and PCR amplified. The experiment was repeated once for all birds 796
used. Numbers under each blot were relative signal intensities among different samples. 797
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For RI, all DNA bands were included for the comparison. RC: relaxed circular DNA; 798
DL: double-stranded linear DNA; CCC: cccDNA; RI: replicative intermediate DNA; 799
DHBc: DHBV core protein. 800
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