Characterization of the Nucleosome Positioning in Hepadnaviral ...

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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

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*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

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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


region 15 (2859-38); region 16 (2922-98); region 17 (3003-211); region 18 (70-298); 177



region 27 (765-998); region 28 (863-1053); region 29 (863-1112); region 30 180


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

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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

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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

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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

1. Belloni, L., T. Pollicino, F. De Nicola, F. Guerrieri, G. Raffa, M. Fanciulli, 570 G. Raimondo, and M. Levrero. 2009. Nuclear HBx binds the HBV 571 minichromosome and modifies the epigenetic regulation of cccDNA function. 572 Proc Natl Acad Sci U S A 106:19975-9. 573

2. Bock, C. T., P. Schranz, C. H. Schroder, and H. Zentgraf. 1994. Hepatitis B 574 virus genome is organized into nucleosomes in the nucleus of the infected cell. 575 Virus Genes 8:215-29. 576

3. Bock, C. T., S. Schwinn, S. Locarnini, J. Fyfe, M. P. Manns, C. Trautwein, 577 and H. Zentgraf. 2001. Structural organization of the hepatitis B virus 578 minichromosome. J Mol Biol 307:183-96. 579

4. Büscher., M, W. Reiser, H. Will, and H. Schaller.1985. Transcripts and the 580 putative RNA pregenome of duck hepatitis B virus: implications for reverse 581 transcription. Cell 40:717-24 582

5. Cairns, B. R. 2005. Chromatin remodeling complexes: strength in diversity, 583 precision through specialization. Curr Opin Genet Dev 15:185-90. 584

6. Crescenzo-Chaigne, B., J. Pillot, and A. Lilienbaum. 1995. Interplay between 585 a new HNF3 and the HNF1 transcriptional factors in the duck hepatitis B virus 586 enhancer. Virology 213:231-40. 587

7. Crescenzo-Chaigne, B., J. Pillot, A. Lilienbaum, M. Levrero, and E. Elfassi. 588 1991. Identification of a strong enhancer element upstream from the pregenomic 589 RNA start site of the duck hepatitis B virus genome. J Virol 65:3882-6. 590

8. Dandri, M., and J. Petersen. 2005. Hepatitis B virus cccDNA clearance: 591 killing for curing? Hepatology 42:1453-5. 592

9. Dienstag, J. L. 2008. Hepatitis B virus infection. N Engl J Med 359:1486-500. 593 10. Funk, A., M. Mhamdi, H. Will, and H. Sirma. 2007. Avian hepatitis B 594

viruses: molecular and cellular biology, phylogenesis, and host tropism. World J 595 Gastroenterol 13:91-103. 596

11. Gao, W., and J. Hu. 2007. Formation of hepatitis B virus covalently closed 597 circular DNA: removal of genome-linked protein. J Virol 81:6164-74. 598

12. Guo, H., D. Jiang, T. Zhou, A. Cuconati, T. M. Block, and J. T. Guo. 2007. 599 Characterization of the intracellular deproteinized relaxed circular DNA of 600 hepatitis B virus: an intermediate of covalently closed circular DNA formation. 601 J Virol 81:12472-84. 602


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

29

13. Hu, J., D. Flores, D. Toft, X. Wang, and D. Nguyen. 2004. Requirement of 603 heat shock protein 90 for human hepatitis B virus reverse transcriptase function. 604 J Virol 78:13122-31. 605

14. Huang, M., and J. Summers. 1994. pet, a small sequence distal to the 606 pregenome cap site, is required for expression of the duck hepatitis B virus 607 pregenome. J Virol 68:1564-72. 608

15. Ilves, I., S. Kivi, and M. Ustav. 1999. Long-term episomal maintenance of 609 bovine papillomavirus type 1 plasmids is determined by attachment to host 610 chromosomes, which Is mediated by the viral E2 protein and its binding sites. J 611 Virol 73:4404-12. 612

16. Kanda, T., and G. M. Wahl. 2000. The dynamics of acentric chromosomes in 613 cancer cells revealed by GFP-based chromosome labeling strategies. J Cell 614 Biochem Suppl Suppl 35:107-14. 615

17. Kaplan, N., I. K. Moore, Y. Fondufe-Mittendorf, A. J. Gossett, D. Tillo, Y. 616 Field, E. M. LeProust, T. R. Hughes, J. D. Lieb, J. Widom, and E. Segal. 617 2009. The DNA-encoded nucleosome organization of a eukaryotic genome. 618 Nature 458:362-6. 619

18. Lavanchy, D. 2004. Hepatitis B virus epidemiology, disease burden, treatment, 620 and current and emerging prevention and control measures. J Viral Hepat 621 11:97-107. 622

19. Lehman, C. W., and M. R. Botchan. 1998. Segregation of viral plasmids 623 depends on tethering to chromosomes and is regulated by phosphorylation. Proc 624 Natl Acad Sci U S A 95:4338-43. 625

20. Levrero, M., T. Pollicino, J. Petersen, L. Belloni, G. Raimondo, and M. 626 Dandri. 2009. Control of cccDNA function in hepatitis B virus infection. J 627 Hepatol 51:581-92. 628

21. Lilienbaum., A, B. Crescenzo-Chaigne, A. Sall, J. Pillot, and E. Elfassi. 1993. 629 Binding of nuclear factors to functional domains of the duck hepatitis B virus 630 enhancer. J Virol. 67:6192-200. 631

22. Liu., C, L.Condreay, J. Burch, and W. Mason. 1991. Characterization of the 632 core promoter and enhancer of duck hepatitis B virus. Virology 184:242-52. 633

23. Liu, C., W. S. Mason, and J. B. Burch. 1994. Identification of factor-binding 634 sites in the duck hepatitis B virus enhancer and in vivo effects of enhancer 635 mutations. J Virol 68:2286-96. 636

24. Lok, A. S., and B. J. McMahon. 2007. Chronic hepatitis B. Hepatology 637 45:507-39. 638

25. Newbold, J. E., H. Xin, M. Tencza, G. Sherman, J. Dean, S. Bowden, and S. 639 Locarnini. 1995. The covalently closed duplex form of the hepadnavirus 640 genome exists in situ as a heterogeneous population of viral minichromosomes. 641 J Virol 69:3350-7. 642

26. Pollicino, T., L. Belloni, G. Raffa, N. Pediconi, G. Squadrito, G. Raimondo, 643 and M. Levrero. 2006. Hepatitis B virus replication is regulated by the 644


http://jvi.asm.org/

Dow

nloaded from

http://jvi.asm.org/

30

acetylation status of hepatitis B virus cccDNA-bound H3 and H4 histones. 645 Gastroenterology 130:823-37. 646

27. Radman-Livaja, M., and O. J. Rando. Nucleosome positioning: how is it 647 established, and why does it matter? Dev Biol 339:258-66. 648

28. Schultz, U., E. Grgacic, and M. Nassal. 2004. Duck hepatitis B virus: an 649 invaluable model system for HBV infection. Adv Virus Res 63:1-70. 650

29. Seeger, C., and W. S. Mason. 2000. Hepatitis B virus biology. Microbiol Mol 651 Biol Rev 64:51-68. 652

30. Skiadopoulos, M. H., and A. A. McBride. 1998. Bovine papillomavirus type 1 653 genomes and the E2 transactivator protein are closely associated with mitotic 654 chromatin. J Virol 72:2079-88. 655

31. Sohn, J. A., S. Litwin, and C. Seeger. 2009. Mechanism for CCC DNA 656 synthesis in hepadnaviruses. PLoS One 4:e8093. 657

32. Summers, J., and W. Mason. 1982. Replication of the genome of a hepatitis 658 B--like virus by reverse transcription of an RNA intermediate. Cell 29:403-415. 659

33. Thoma, F. 1996. Mapping of nucleosome positions. Methods Enzymol 660 274:197-214. 661

34. Tuttleman, J. S., C. Pourcel, and J. Summers. 1986. Formation of the pool of 662 covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 663 47:451-60. 664

35. Verdin, E., P. Paras, Jr., and C. Van Lint. 1993. Chromatin disruption in the 665 promoter of human immunodeficiency virus type 1 during transcriptional 666 activation. EMBO J 12:3249-59. 667

36. Vivekanandan, P., D. Thomas, and M. Torbenson. 2008. Hepatitis B viral 668 DNA is methylated in liver tissues. J Viral Hepat 15:103-7. 669

37. Welsheimer, T., and J. Newbold. 1996. A functional hepatocyte nuclear factor 670 3 binding site is a critical component of the duck hepatitis B virus major surface 671 antigen promoter. J Virol 70:8813-20. 672

38. Werle-Lapostolle, B., S. Bowden, S. Locarnini, K. Wursthorn, J. Petersen, 673 G. Lau, C. Trepo, P. Marcellin, Z. Goodman, W. E. t. Delaney, S. Xiong, C. 674 L. Brosgart, S. S. Chen, C. S. Gibbs, and F. Zoulim. 2004. Persistence of 675 cccDNA during the natural history of chronic hepatitis B and decline during 676 adefovir dipivoxil therapy. Gastroenterology 126:1750-8. 677

39. Yang, W., and J. Summers. 1995. Illegitimate replication of linear 678 hepadnavirus DNA through nonhomologous recombination. J Virol 69:4029-36. 679

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


http://jvi.asm.org/

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http://jvi.asm.org/

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42. Zhang, Y., Z. Moqtaderi., B.P. Rattner., G. Euskirchen., M. Snyder., J.T. 686 Kadonaga., X.S. Liu and K. Struhl. 2009. Intrinsic histone-DNA interactions 687 are not the major determinant of nucleosome positions in vivo. Nat Struct Mol 688 Biol. 16: 847. 689

43. Zimmerman, K. A., K. P. Fischer, M. A. Joyce, and D. L. Tyrrell. 2008. 690 Zinc finger proteins designed to specifically target duck hepatitis B virus 691 covalently closed circular DNA inhibit viral transcription in tissue culture. J 692 Virol 82:8013-21. 693

44. Zoulim, F. 2005. New insight on hepatitis B virus persistence from the study of 694 intrahepatic viral cccDNA. J Hepatol 42:302-8. 695

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

785

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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Characterization of the Nucleosome Positioning in Hepadnaviral ...

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