Characterization of Nucleosome Positioning in HepadnaviralCovalently Closed Circular DNA Minichromosomes
Liping Shi,a Shaohua Li,b Fang Shen,a Haodong Li,b Shuiming Qian,a Daniel H. S. Lee,a Jim Z. Wu,a and Wengang Yanga
Roche Pharma Research and Early Development China, Shanghai, China,a and WuXi AppTec Co., Ltd., Shanghai, Chinab
Hepadnaviral covalently closed circular DNA (cccDNA) exists as an episomal minichromosome in the nucleus of virus-infectedhepatocytes, and serves as the transcriptional template for the synthesis of viral mRNAs. To obtain insight on the structure ofhepadnaviral cccDNA minichromosomes, we utilized ducks infected with the duck hepatitis B virus (DHBV) as a model and de-termined the in vivo nucleosome distribution pattern on viral cccDNA by the micrococcal nuclease (MNase) mapping and ge-nome-wide PCR amplification of isolated mononucleosomal DHBV DNA. Several nucleosome-protected sites in a region of theDHBV genome [nucleotides (nt) 2000 to 2700], known to harbor various cis transcription regulatory elements, were consistentlyidentified in all DHBV-positive liver samples. In addition, we observed other nucleosome protection sites in DHBV minichro-mosomes that may vary among individual ducks, but the pattern of MNase mapping in those regions is transmittable from theadult ducks to the newly infected ducklings. These results imply that the nucleosomes along viral cccDNA in the minichromo-somes are not random but sequence-specifically positioned. Furthermore, we showed in ducklings that a significant portion ofcccDNA possesses a few negative superhelical turns, suggesting the presence of intermediates of viral minichromosomes assem-bled in the liver, where dynamic hepatocyte growth and cccDNA formation occur. This study supplies the initial framework forthe understanding of the overall complete structure of hepadnaviral cccDNA minichromosomes.
Currently, about 350 million individuals worldwide are chron-ically infected with the hepatitis B virus (HBV). Of the in-
fected people, 15 to 40% will develop severe sequelae in their life-time, most notably liver cirrhosis and hepatocellular carcinoma(18). The treatment of chronic hepatitis B has been improveddramatically in the past 10 years, mainly due to the successfuldevelopment and application of nucleoside(tide) drugs targetingHBV polymerase and interferon (9, 24). These treatment optionsdelay disease progress by inhibiting viral replication and modulat-ing host immune functions in certain populations of HBV pa-tients, but fail to cure the majority of HBV patients. A predomi-nant reason for this failure is attributed to the persistence of viralcovalently closed circular DNA (cccDNA) in the nuclei of infectedhepatocytes during the treatment with nucleoside(tide) analogs(8, 20, 38). Without interfering with cccDNA maintenance withinthe infected hepatocytes, nucleosides(tides) only have a limitedeffect on HBV DNA replication and disease progression.
Hepadnaviruses are small DNA-containing viruses that repli-cate their DNA genomes through reverse transcription of an RNAintermediate called pregenomic RNA (32). The template of thepregenomic RNA is a pool of cccDNA located in the hepatocytenuclei (34, 41). The cccDNA is converted from a relaxed circulardouble-stranded DNA (RC DNA) that is transported into the nu-cleus from the cytoplasm, where viral DNA replication occurswithin naked capsid particles (29). A small percentage of thecccDNA is converted from double-stranded linear DNA througha nonhomologous recombination that generates sequence varia-tions around the joint region (39). In the nucleus, cccDNA existsas an individual minichromosome with a “beads-on-a-string”structure, which is revealed by electron microscopy (2, 25). His-tones as well as nonhistone proteins either bind directly to thecccDNA or are indirectly recruited to viral minichromosomesthrough protein-protein interactions (2, 20, 25, 26, 36). UsingcccDNA chromatin IP with antiacetylated H3/H4 antibodies, itwas shown that the acetylation status of H3/H4 in cccDNA
minichromosomes plays an important role in HBV RNA tran-scription (26). Besides host proteins that, as components ofminichromosomes, are involved in cccDNA functions, the virallyencoded proteins core and HBx have also been shown to bind tothis structure and result in either a reduction of the nucleosomalspacing in HBV minichromosomes or an overall enhancement ofHBV replication, respectively (1, 3, 43). In contrast to viral RNAtranscription and its regulatory factors, we know little about thestructure of viral minichromosomes and the maintenance mech-anism of cccDNA in the nucleus of hepatocytes.
Ducks congenitally infected with the duck hepatitis B virus(DHBV) were used to study in vivo structures of viral cccDNAminichromosomes, especially the nucleosome positioning oncccDNA. We found a unique distribution pattern of nucleosomesof DHBV minichromosomes through micrococcal nuclease(MNase) mapping and PCR amplification of mononucleosomalviral DNA. By comparing the mapping results among DHBV-positive ducks, we showed that nucleosome binding patterns aremore conserved in a region of nucleotides (nt) 2000 to 2700,where various cis elements and the binding sites of trans elementsof RNA transcription exist (4, 6, 14, 21–23). MNase mapping inother regions of DHBV genomes is more or less variable in differ-ent individuals and the variations were passed to the newly in-fected ducklings. In addition, cccDNA with a few supercoiledturns or bound nucleosomes were consistently found in the liversof ducklings infected either congenitally or horizontally withDHBV, where liver growth and virus spreading are active com-
pared to those of adults. These results benefit our understand-ings of the formation and structure of hepadnaviral cccDNAminichromosomes and may facilitate the identification ofnovel targets for curing HBV infections.
MATERIALS AND METHODSIsolation of duck hepatocyte nuclei. All animal studies were approved bythe Ethics Committee for Animal Experiments of WuXi AppTec Co. Ltd.These studies were conducted in accordance with the current facility’sstandard operating procedures (SOPs) and in compliance with the Ani-mal Welfare Act. Researchers of Roche Pharma Research and Early De-velopment monitored all activities in animal studies, including theIACUC approval, performance, and compliance. Isolation of hepatocytenuclei from domestic ducks that were congenitally infected with DHBVwas performed as previously described (25), with modifications. Briefly,100 to 300 mg of liver tissue were rinsed in solution H (0.25 M sucrose, 3mM MgCl2, 10 mM NaH2PO4, pH 6.5) and then disrupted in a loose-fitting Dounce homogenizer. The homogenate was strained through fourlayers of cheesecloth and centrifuged at 2,500 rpm for 20 min. The pelletwas suspended in 7 to 10 volumes of solution H= (1.8 M sucrose, 3 mMMgCl2, 10 mM NaH2PO4, pH 6.5). The suspension was subjected to cen-trifugation in a Beckman type T40i rotor at 22,000 rpm at 4°C for 1 h. Thesupernatant was decanted. The nuclear pellet was washed twice with so-lution H= and the nuclei were counted under a microscope after beingstained with ethidium bromide.
Nuclease treatment of isolated nuclei. Nuclei (2 � 108 nuclei/ml)were treated with 0.5 or 2 U/ml micrococcal nuclease (MNase, TaKaRa)or a concentration mentioned specifically in the text in Buffer A (10 mMTris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.3 M sucrose, 10 mMCaCl2) at 37°C for 20 min (35). The reaction was stopped by adding anequal volume of 2� stop buffer (100 mM Tris-HCl, pH 7.5, 200 mMNaCl, 2 mM EDTA, 1% SDS) to the mixture. The mixture was thentreated with 150 �g/ml of DNase-free RNase A at 37°C for 1.5 h. Thegenomic DNA was extracted with phenol and precipitated with ethanol.Total DNA was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA,pH 8.0) and optical density at 260 nm (OD260) was measured beforefurther analysis.
Isolation of cccDNA from duck livers. cccDNA was extracted by theHirt method (39). Briefly, DHBV-positive duck liver was homogenized ina 2-ml Dounce homogenizer. Homogenates were mixed with an equalvolume of 4% SDS. Then, the majority of cellular components, includingnucleic acid and proteins, were precipitated by mixing them with a one-fourth volume of 2.5 M KCl. After a centrifugation at 4°C for 10 min, thesupernatant was transferred to a different tube and extracted with phenol.After ethanol precipitation, nucleic acids, including cccDNA, were dis-solved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
Indirect end labeling. Five probes, named as A, B, C, D, and E, wereused in this study. Each probe was synthesized by using a PCR DIG ProbeSynthesis Kit (Roche Applied Science) with deoxynucleoside triphosphate(dNTP) mix containing digoxigenin (DIG)-11-dUTP. DHBV16 plasmidwas used as the template of PCR amplifications. As shown in Fig. 1 and 4,probe A spans from approximately nucleotides (nt) 42 to 385 (close toEcoRI site, 344 bp in length); probe B spans from approximately nt 399 to747 (close to BglII site, 348 bp in length); probe C spans from approxi-mately nt 49 to 385 (close to BglII site, 337 bp in length); and probe Dspans from approximately nt 1298 to 1623 (close to KpnI site, 353 bp inlength); and probe E spans from approximately nt 1003 to 1284 (close toKpnI site, 282 bp in length). DNA (�50 �g) extracted from MNase-treated nuclei was digested with a restriction enzyme that has a unique siteon the DHBV genome. DNA was extracted again with phenol, followed byethanol precipitation. The pellet was dissolved in 50 �l TE and subjectedto an electrophoresis of 2% agarose gel in 1� TAE buffer overnight. Afterdenaturation and neutralization, DNA was blotted onto a Hybond-N�
membrane (GE Healthcare) in 20� SSC and hybridized with a DIG-labeled DHBV DNA probe targeting a specific region of DHBV DNA.
After incubating blots with an alkaline-phosphatase-conjugated anti-DIGantibody, hybridization signals were detected in a standard chemilumi-nescence reaction.
Real-time PCR analysis. Isolated hepatocyte nuclei from DHBV-pos-itive ducks were digested with 16 U/ml micrococcal nuclease in Buffer Adescribed above at 37°C for 20 min. Total DNA was then extracted withphenol and subjected to a 1.5% agarose gel electrophoresis. The bandcorresponding to mononucleosomal DNA was cut and DNA was ex-tracted with a gel extraction kit (Qiagen). cccDNA from the same duck(s)was extracted as described above and used as reference DNA of MNasemapping and PCR amplification. PCR was performed on a LightCycler480 II (Roche Diagnostics) in a final volume of 20 �l in which 1 �l ofpurified mononucleosomal DNA or cccDNA was included as PCR tem-plates. Amplification was done as follows: the denaturation program was95°C for 10 min, the amplification and quantification program (95°C for10 s, 55°C for 30 s, and 72°C for 30 s with a single fluorescence measure-ment) was repeated 45 times, the melting curve program was 60 to 95°Cwith a heating rate of 0.1°C per s and a continuous fluorescence mea-surement, and finally, the cooling step was to 40°C. The amplificationefficiency of mononucleosomal DNA was normalized by comparingthe cycle threshold (CT) value of mononucleosomal DNA to that ofcccDNA (ratiomononucleosomal DHBV DNA/cccDNA � 2��Ct) in each reac-tion. Thirty-six overlapping regions on the DHBV16 genome werechosen for amplification (Fig. 3B and 4C). The starting and endingnucleotides (nt) of each region are listed as follows: region 1 (1656 to1857); region 2 (1726 to 1929); region 3 (1798 to 1978); region 4 (1876to 2082); region 5 (1949 to 2133); region 6 (1973 to 2201); region 7(2062 to 2303); region 8 (2183 to 2380); region 9 (2228 to 2436); region10 (2256 to 2489); region 11 (2291 to 2524); region 12 (2404 to 2607);region 13 (2493 to 2704); region 14 (2822 to 3006); region 15 (2859to 38); region 16 (2922 to 98); region 17 (3003 to 211); region 18 (70 to298); region 19 (164 to 390); region 20 (238 to 446); region 21 (333 to539); region 22 (359 to 612); region 23 (458 to 693); region 24 (497to 753); region 25 (627 to 850); region 26 (689 to 916); region 27 (765to 998); region 28 (863 to 1053); region 29 (863 to 1112); region 30(958 to 1154); region 31 (969 to 1227); region 32 (1123 to 1327); region33 (1233 to 1454); region 34 (1368 to 1542); region 35 (1458 to 1677);and region 36 (1529 to 1711).
Comparison of levels of DHBV RNA and core protein in duck andduckling livers. A 100-mg DHBV-positive duck or duckling liver samplewas homogenized in 1.5 ml TE buffer (50 mM Tris-HCl and 1 mM EDTA,pH 8.0) and in a 2-ml Dounce homogenizer. The homogenate was ali-quoted into four parts to extract viral cccDNA, replicative intermediateDNA, and total RNA and to prepare protein samples for the measurementof DHBV core protein. Total liver RNAs in the homogenate were ex-tracted with an RNeasy Minikit (Qiagen) by following the manufacturer’sinstructions. Contaminated DNA was eliminated by an on-columnDNase digestion step. After quantitation and normalization of RNA sam-ples, RNA was reverse transcribed with an iScript cDNA synthesis kit(Bio-Rad) in which a mixture of oligonucleotide (dT) and random prim-ers was used. Then, DHBV cDNA was measured by real-time PCR andnormalized with the cDNA of glyceraldehyde-3-phosphate dehydroge-nase (GAPDH) using a LightCycler 480 (Roche Diagnostics). Primers forDHBV real-time PCR amplification are 5=-TTTGGATAGGGCTAGGAGATTG-3= (sense, nt 42 to 63) and 5=-AGGCGAGGGAGATCTATGGTG-3= (antisense, nt 385 to 405). This set of primers measures the levels ofPreC/C viral RNAs due to the position of amplification. Primers for duckGAPDH real-time PCR amplification are 5=-CATCGTGCACCACCAACTG-3= (sense) and 5=-CGCTGGGATGATGTTCTGG-3= (antisense). Tomeasure DHBV core protein, homogenate was treated briefly with CA-630 at a final concentration of 2%. Nuclei and other cellular debris wereremoved by a short centrifugation. Protein concentration of the superna-tant was determined by the Bradford method. Equal amounts of total liverproteins were loaded on a 4 to 12% SDS-PAGE gel and the level of DHBV
core protein was evaluated in a Western blot analysis. Beta-actin was usedas an internal control.
Infection of ducklings. DHBV-negative Cherry Valley ducklings (3days old) were inoculated intravenously with DHBV-positive sera at avolume of 0.2 ml per duckling (40). Nine days after inoculation, the in-fected ducklings were sacrificed and the livers were removed and stored at�80°C until use.
RESULTSMNase mapping of hepadnaviral cccDNA minichromosomes.Although previous studies showed that hepadnaviral cccDNA ex-ists in the nucleus of hepatocytes as individual minichromosomes,details about the distribution of host nucleosomes in these “beads-in-a-string” structures are largely unknown (25). In this study,ducks congenitally infected with DHBV were used to map thepattern of nucleosome distribution on DHBV cccDNA minichro-mosomes in vivo.
As a general scheme, DHBV-positive liver homogenates weresubjected to a centrifugation in which nuclei passed through asucrose cushion. The isolated nuclear fractions that contain bothcellular chromatin and viral minichromosomes were partially di-gested with MNase (33, 35). As a starting point of MNase map-
ping, total DNA after being treated with MNase at different con-centrations was extracted and subjected to an agarose gelelectrophoresis and Southern blot hybridization using a full-length DHBV DNA probe. As reported previously (25) and shownin Fig. 1B, a typical pattern of mono-, di-, and trinucleosomes thatwere generated from DHBV cccDNA minichromosomes as a re-sult of MNase digestion was detected. To locate the positions ofMNase cleavage on viral cccDNA, EcoRI, possessing a unique sitein the DHBV genome, was chosen to cut DNA fragments gener-ated by MNase. Viral cccDNA fragments containing one end de-rived from MNase cleavage and the other end from EcoRI diges-tion were detected in a Southern hybridization with a short probe(probe A, nt 42 to 385 of the DHBV16 genome) that is close to theEcoRI site (Fig. 1A). As shown in Fig. 1C, following MNase diges-tions which covered MNase concentrations from 0.1 to 8 U/ml,nuclear fractions showed a consistent pattern with distinctivebands in a Southern blot. Based on the sizes of these bands, posi-tions of MNase cleavage in the DHBV genome were inferred. Asmore MNase was added in the reaction mixtures, signals of mostbands, especially large ones, were gradually reduced and finallydisappeared. In contrast, naked cccDNA, which was purified from
FIG 1 Mapping of nucleosome binding on DHBV cccDNA minichromosomes. (A) Strategy of MNase mapping of viral minichromosomes with a probe (probeA) that hybridizes a small region close to the EcoRI site, a unique position on the DHBV genome. Curves with an arrow represent fragments of cccDNA generatedfrom MNase and EcoRI cleavage. (B) DHBV DNA fragments detected by Southern blot hybridization following MNase digestion. Nuclear fractions of hepato-cytes were prepared from liver samples of a DHBV-infected duck (see Materials and Methods) and were treated with MNase at different concentrations. A probeof the full-length DHBV DNA genome (DHBV 16) was used in hybridization. Mono, Di, and Tri represent mononucleosomal, dinucleosomal, and trinucleo-somal DHBV DNA, respectively. (C) MNase cleavage on purified cccDNA and viral minichromosomes in isolated nuclei. MNase concentrations (U/ml) andtreatment duration (in minutes) employed in this experiment are labeled on the top of the blots. Probe A (nt 42 to 385) was used in hybridization. A DNA ladderwith known sizes (bp) is shown on the left of the blots.
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the same liver sample, displayed different hybridization patterns(Fig. 1C). After a 5-min MNase treatment at a concentration of 8U/ml, no hybridization signal was detected in naked cccDNAwhile a weak but clear pattern was still observable in the minichro-mosome fractions, and even the duration of treatment was longer(20 min) for the latter, suggesting a higher accessibility/sensitivityof naked cccDNA to MNase. At a lower concentration of 2 U/ml,MNase digestion of naked cccDNA generated individual bandsthat were buried in high backgrounds. Unlike purified cccDNAmolecules, specific regions of DHBV minichromosomes in thenuclear fraction were protected from MNase digestion, raising apossibility that these regions are bound with nucleosomes or othercellular/viral structures.
A unique pattern of MNase mapping is shared amongDHBV-infected ducks. In order to see if the similar patterns ofMNase mapping described above could be observed among indi-viduals where viral DNA sequences and host general status mightbe different, four liver samples of DHBV-positive ducks were har-vested, MNase mapping of nuclear fractions of hepatocytes wasperformed, and mapping patterns were compared in a side-by-side manner. As shown in Fig. 2, besides the presence or absence ofseveral MNase cleavage bands (marked with arrowheads on theright side of the blot), overall MNase mapping showed similar regionswhere MNase was less accessible (marked with a series of brackets)among all four ducks (including the previous one shown in Fig. 1,which is 2 in Fig. 2). Patterns in a region from approximately nt 2000to 2700 in cccDNA were highly reproducible among four samples.The presence of distinctive and consistent patterns in terms of
MNase-accessible sites and -resistant regions suggested that nucleo-somes are not randomly distributed on the hepadnaviral minichro-mosomes, at least in some regions of cccDNA.
Determination of nucleosome binding positions on cccDNAminichromosomes by real-time PCR. To supply more evidencesto the claim that the protected regions of DHBV cccDNA in theviral minichromosomes are associated with nucleosomes, mono-nucleosomal DNAs generated by MNase treatment were pre-pared. We assumed that these short DNA fragments, according totheir sizes, should bind one nucleosome. DHBV-specific PCR am-plifications on isolated mononucleosomal DNA were performedto determine the size and the boundary of the protected regions.In general, mononucleosomal DNA was gel purified after an ex-tensive MNase digestion of nuclear fractions that broke downmost cccDNA minichromosomes to mononucleosomes, as veri-fied by a Southern hybridization (data not shown). Within a partof the DHBV genome (nt 1650 to 2700) in which a reproducibleMNase mapping pattern was observed in viral minichromosomesof the four DHBV-positive ducks (Fig. 2), 13 consecutive, over-lapping PCR amplification regions were allocated (region 1 or R1to region 13 or R13, shown at the bottom of Fig. 3B). Each ampli-fication region contained two sets of individual PCRs; for exam-ple, region one (R1) has set 1 and set 2 and region two has set 3 andset 4, and so on (Fig. 3A). In each set there were four individual PCRamplifications. One primer from one side of amplification was fixedand primers on the other side were different, generating PCR prod-ucts of 75 to 120 bp in length (Fig. 3A). In order to normalize theefficiencies of mononucleosomal amplifications at various positionsof the DHBV genome, isolated mononucleosomal DNA and nakedcccDNA were used as templates and amplified in parallel for all PCRamplifications. CT values of individual mononucleosomal amplifica-tions were aligned with that of the corresponding cccDNA templateto obtain a ratio of mononucleosomal DHBV DNA to cccDNA. Asshown in Fig. 3B, products amplified from mononucleosomal DNAwere detected in five out of 13 amplification regions (region 1 or R1,R7, R9, R11, and R13). In each of these regions, some PCRs in theinner part produced amplification signals that were 50% or morecompared to that of naked cccDNA. As the distance between the twoprimers became bigger, amplification signals were attenuated. Thisresult was consistent with MNase mapping where all five regions wereclearly less accessible to MNase digestion (gel image on the top of Fig.3B). In addition, several amplifications in the region of around nt1800 to 1950 (Fig. 3B, region 3) showed a moderate ratio of mono-nucleosomal DNA to cccDNA CT values, suggesting the presence of aweak protection site that was probably due to a nucleosome bindingin this region. On the contrary, mononucleosomal amplifications inthe other seven regions (R2, R4, R5, R6, R8, R10, and R12) wererelatively inefficient as reflected by CT values that were lower than thatof cccDNA. When aligned by MNase mapping, some of these poorlyamplified regions were found to straddle two MNase-insensitive ar-eas, with MNase cleavage sites in the middle, and the others werefound in the MNase-sensitive regions, where strong signals derivedfrom multiple MNase cleavages were observed. We named the majorMNase cleavage bands shown in the top panel of Fig. 3B and alignedthem in the DHBV genome according to the estimated sizes of thosebands. For the details of designation of MNase cleavage sites that arescattered throughout the DHBV genome, please refer to descriptionsin Results and the figure legend of Fig. 4 (below). Taken together, in aregion of nt 1650 to 2700 of the DHBV genome, the location ofnucleosome-protected viral DNA revealed by real-time PCR of
FIG 2 Comparison of MNase mapping patterns of four DHBV-positive adultducks. The experimental conditions were the same as that used in Fig. 1C.Specifically, each reaction mixture contained MNase at a concentration of 8U/ml and the vials were incubated at 37°C for 20 min. Variations of MNasecleavages on viral minichromosomes of individual ducks are marked witharrowheads and MNase-less-accessible regions are marked with bracketsshown on the right side of the blot. A DNA ladder with known sizes (bp) isshown on the left of the blot.
isolated mononucleosomal DNA correlates well with the pat-tern of nucleosome binding mapped by a partial MNase diges-tion (Fig. 1 and 2).
Genome-wide mapping of nucleosome binding of DHBVcccDNA minichromosomes. In order to obtain a genome-widemap of nucleosome binding of DHBV cccDNA minichromo-somes, nuclear fractions of hepatocytes were prepared from oneDHBV-positive duck and then the two approaches describedabove were employed: Southern blot hybridization to showMNase cleavage patterns of cccDNA minichromosomes and PCR
amplifications of isolated mononucleosomal DHBV DNA thatcover the remaining two-thirds of the DHBV genome. For the firstapproach, we chose three restriction enzymes, EcoRI, BglII, andKpnI, each of which has a unique site in the DHBV genome, to doMNase mapping. Southern blot hybridization with five differentprobes, A, B, C, D, and E, from two orientations (determined bytheir relative positions between a probe and a restriction site) areshown in Fig. 4A and B. Probes A, B, and D share a clockwiseorientation. Hybridization with these three probes (first, second,and fourth panels in Fig. 4B) produced similar and overlapping
FIG 3 Nucleosome-protected regions on viral minichromosomes revealed by real-time PCR. (A) A diagram of the relation between nucleosome binding andpositions of different amplification sets. Sets 1, 2, 5, and 6 are located in nucleosome-protected regions (R1 and R3). Sets 3 and 4 cross a linker region (R2). In eachset, four PCR amplifications were performed. One primer from one side of the amplification is at a fixed position (S1 in sets 1, 3, and 5; AS1 in sets 2, 4, and 6).Primers on the other side of each amplification set are at different positions. (B) Alignment of MNase mapping with the data of real-time PCR that coveredone-third of the viral genome (nt 1650 to 2700). Mononucleosomal DNA was isolated after an extensive MNase cleavage (16 U/ml for 20 min) and used forreal-time PCR as described in Materials and Methods. CT values of an individual monochromosomal PCR were normalized with that of amplification of nakedcccDNA extracted from the same bird. Each line in the figure represents one specific PCR amplification. It contains information about the two primer positions(dots at the two ends of each line) relative to the DHBV genome, which is shown in numbers (nt) in the x axis dimension, as well as information about the ratioof minichromosomal DHBV DNA to naked cccDNA as the height of each line (y axis). Lines in red represent PCR amplification sets that are located in putativenucleosome protected regions according to the MNase mapping shown above panel B. Lines in blue are PCR amplifications that straddle two nucleosomeprotected regions or in regions where multiple MNase cleavages were observed in MNase mapping. MNase mapping was done as described in Fig. 1 and 2, inwhich nuclear DNA partially digestd with MNase was completely digested with EcoRI and then subjected to Southern blot hybridization with probe A. MajorMNase cleavage sites were named in an alphabetical manner and marked on the blot as well as on a linearized DHBV genome in panel B. Positions of 13amplification regions (R1 to R13) are shown at the bottom of panel B.
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FIG 4 Overall mapping of nucleosome binding of DHBV cccDNA minichromosomes. (A) A diagram of positions of probes and unique restriction sites on the DHBVgenome. The arrow of each probe indicates the orientation of the detected MNase cleavage fragments, starting from a specific restriction site and circling around the viralgenome either clockwise (probes A, B, and D) or counter-clockwise (probes C and E). (B) MNase mapping with individual probes following a partial MNase digestionand a complete restriction enzyme digestion and Southern hybridization. The sizes of a series of DNA markers are shown on the left. MNase cleavage bands were alignedwith DNA markers and labeled in an alphabetic manner based on sizes calculated in different blots. The restriction enzyme, probe, and concentrations of MNase usedin each blot are shown on the top. (C) Alignment of MNase maps and results of real-time PCR in the remaining two-thirds of the viral genome (nt 1700 to 2800 shownon the x axis). The ratios of minichromosomal DHBV DNA to naked cccDNA detected by PCR are shown as the height of each line (y axis). Lines in red represent PCRamplification sets with relatively high ratios that are located in putative nucleosome protected regions according to the MNase mapping shown in panel A. Lines in blueare PCR amplifications that straddle two nucleosome protected regions or regions with relatively low amplification signals of mononucleosomal DHBV DNA. Positionsof 23 amplification regions (R14 to R36) and the rough positions of MNase cleavage bands marked in panel B are shown beneath amplification data. A schematic viewof nucleosome binding on the linearized DHBV genome and the relationship of the nucleosomes with cis-acting elements of viral RNA transcription are shown at thebottom. Positions of the initiation sites of the viral transcripts, PreS, S, and PreC/C mRNAs, polyadenylation site, promoters, enhancer, pet (positive effector oftranscription), and binding sites of several host factors that play roles in viral RNA transcription initiation are marked by blue arrows and bars with different colors (4,6, 14, 21–23, 37).
MNase mapping patterns. Specific regions of viral genome pre-sented in individual blots, when combined together, cover thewhole DHBV cccDNA. Probes C and E share a counterclockwiseorientation. Hybridization with the two probes (third and fifthpanels in Fig. 4B) resulted in MNase mapping that was arranged ina way opposite to the mapping of probes A, B, and D. By aligningindividual bands shown in each blot with a standard-length curveof DNA markers, sizes and positions of MNase bands on DHBVgenome were roughly determined. Based on the estimated sizesand patterns of individual bands shown in different blots, 24 ma-jor MNase cleavage bands were named in an alphabetical way andmarked on the right side of each blot in Fig. 4B as well as on alinearized DHBV genome in Fig. 4C. Because of different probesand restriction enzymes used in individual blots, individual bandsor groups of bands with the same designations were located atdifferent positions or were arranged in opposite orientations indifferent blots. While the majority of these bands were consis-tently observed, some new bands were detected when differentrestriction enzymes and probes were employed, which might re-flect a resolution change. For example, a group of bands con-densed on the top of a gel were separated well around the bottomof a gel when a different restriction enzyme was used. For thesecond approach, mononucleosomal DNA generated fromMNase digestion was gel purified and subjected to random PCRamplifications, as with the amplifications described in Fig. 3. Asshown in Fig. 4C, 23 regions that cover most of the remainingtwo-thirds of the DHBV genome were amplified through 46 sets.
Combining MNase cleavage sites and the ratios of mononucleo-somal DHBV DNA to cccDNA in different amplification regions,we identified several other nucleosome binding positions, espe-cially, positions between L3–M (R26), N3–O (R20), O–P (R18),and P–A (R16). It is worth mentioning that in the area rangingfrom approximately nt 900 to nt 1200, we failed to obtain anappreciable ratio of mononucleosomal DHBV DNA to cccDNAwith five overlapping amplification regions (R27 to R31) whichare upstream of a small S coding region. The detailed positions ofall amplification regions in the DHBV genome are shown in Fig.4C as well as in Materials and Methods. The relation betweenbound nucleosomes and DHBV cis-acting elements (4, 6, 14, 21–23, 37) is shown in Fig. 4C (bottom).
Patterns of MNase mapping of cccDNA minichromosomespassed from adult ducks to horizontally infected ducklings. Asshown in Fig. 2, although the patterns of MNase mapping ofcccDNA minichromosomes were similar among the four ducks, acloser inspection revealed that several distinct DNA fragments didnot appear in all the ducks examined. While it is possible that theobserved differences in nucleosome positioning in certain regionsof cccDNA minichromosomes among the different ducks are dueto differences of cccDNA sequences, viral RNA transcription sta-tus, and other unknown host or viral factors, it is more interestingto know whether the traits are inheritable features of these viruses.To address this question, two MNase mapping patterns with dis-tinguishable bands (Fig. 5A) were chosen and the correspondingserum samples were used to inoculate newly hatched DHBV-neg-
FIG 5 Patterns of MNase mapping passed through adult ducks to newly infected ducklings. Three-day-old DHBV-negative ducklings were inoculated intra-venously with the serum of adult duck 5 or 6 (0.2 ml per duckling). Nine days after infection, liver samples of two ducklings of each group were collected forMNase mapping. (A) MNase mapping patterns of DHBV minichromosomes of the two adult ducks. (B) MNase mapping patterns in ducklings. Two noticeablevariations in MNase mapping that passed to viral minichromosomes of ducklings are labeled with an arrowhead and a line with arrowhead on the left of the gels.
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ative ducklings (two ducklings in each group). Nine days later,liver and serum samples were harvested for MNase mapping andviral DNA sequencing. As shown in Fig. 5B, characteristic bands intwo regions of minichromosomes in adult ducks were visualizedby MNase mapping of all infected ducklings, correspondingly.DNA sequence changes in dominant viral species between adultsand infected ducklings were not detected. These results indicatethat although differences such as kinetics of virus replication, vi-rus spreading, and liver microenvironments exist between adultsand ducklings, overall minichromosome structures reflected bythe patterns of MNase mapping are inheritable.
In addition, we aligned the two original DHBV DNA sequencesthat were obtained from duck 5 and 6 for a possible relationshipbetween sequence variations and a specific MNase pattern. Bothvariable areas marked in Fig. 5A were associated with a highernucleotide substitution rate compared to the surrounding regionsin the DHBV genome. The DNA covered by the line with anarrowhead in Fig. 5A encodes part of PreS and the overlappingspacer region of DHBV polymerase, and the two sequences werehighly varied (there are 37 bp substitutions scattered in the region[�260 bp in length]). Another region marked by an arrowhead inFig. 5A encodes a part of viral polymerase and also showed a highsubstitution rate between the two sequences (21 substitutions inthe region, which is �320 bp in length). Although we had data ofviral DNA sequences and MNase mapping for the two DHBV-positive ducks, we were unable to pinpoint or correlate a sequencevariation with a specific MNase pattern. We thought that thismight be due, at least partially, to the fact that MNase mappinghere has a lower resolution compared to DNA sequencing and thefact that multiple substitutions are scattered in the regions. More-over, it is possible that a sequence variation might result in somestructural changes on viral minichromosomes that are away fromthe original position of the sequence variation through protein-DNA and protein-protein interactions.
A portion of cccDNA in duckling livers has only a few super-coiled turns. During the comparison of MNase mappings ofcccDNA minichromosomes between adult ducks and ducklings,we found that it was always the case that more isolated nuclearfractions of ducklings were required to generate signals equivalentto that of adult ducks in Southern hybridization after a partialMNase digestion. Since other conditions were the same, indicat-ing a possibility that duckling hepatocytes might contain morepartially assembled cccDNA minichromosomes that are easily ac-cessible to MNase digestion. Initial confirmation by Southern blothybridization failed to show a significant difference between adultand duckling cccDNA samples. We conducted a careful electro-phoresis in which a small sample volume (�10 �l) was loaded intoa well and cccDNA was separated through a 25-cm-long, 0.9%agarose gel overnight. It was repeatedly observed in duckling sam-ples that besides a predominant, fast moving cccDNA band, aportion of cccDNA molecules migrated slowly to form severaldiscrete bands. These slowly moving bands are marked witharrowheads in Fig. 6A and represent cccDNA molecules with onefewer superhelical turn than in neighboring bands. BetweencccDNA prepared from DHBV congenitally and horizontally in-fected ducklings (4 to 14 days old) there was no apparent differ-ence in terms of the patterns and positions of these topoisomerbands (Fig. 6A, compare lanes 5, 6, 7, and 8 to lanes 9 and 10). Incontrast, fewer slowly moving topoisomers of cccDNA were de-tected in cccDNA preparations of adult ducks (Fig. 6A, lanes 1, 2,
3, and 4). To reinforce the claim that the slowly moving bandsdetected in duckling livers are cccDNA with fewer supercoiledturns, duckling cccDNA was further analyzed with different treat-ments. First, mobilities of cccDNA and the slowly moving bandswere the same after a heat denaturation (Fig. 6B, lane 2). Thistreatment converted nicked double-stranded circular and double-stranded linear DNA into single-stranded DNA. SupercoiledDNAs, however, were renatured and migrated in electrophoresisat the same mobility as their native forms after heat treatment.Second, treatment with Escherichia coli. topoisomerase I, whichefficiently relaxes cccDNA with negatively superhelical turns, con-verted most of the cccDNA, including those slowly moving bands,into relaxed circular DNA and much less supercoiled cccDNA(Fig. 6B, lane 4). These results support the notion that the slowlymoving species extracted from DHBV-positive duckling livers arecccDNA with less negatively superhelical turns. Since the numberof superhelical turns in a cccDNA is equal to the number of boundnucleosomes on this molecule (25), detection of a significant por-tion of cccDNA with a few negatively supercoiled turns in duck-ling hepatocytes suggest that cccDNA molecules with a few boundnucleosomes are more prevalent in young ducks, where livergrowth and cccDNA formation are drastically occurring.
In order to study if the superhelicity of cccDNA affects viralRNA levels, four DHBV congenitally infected birds (two ducksand two ducklings) were chosen to extract cccDNA, viral replica-tive intermediate DNA, fewer total RNA and to prepare proteinsamples. cccDNA with fewer superhelical turns was detected intwo duckling samples (Fig. 6C) as shown in Fig. 6A. Equalamounts of total RNA were used for measuring the levels ofDHBV RNA and RNA of glyceraldehyde-3-phosphate dehydro-genase (GAPDH) by real-time RT-PCR. The level of DHBV RNAin the two ducklings was 10- to 25-fold higher than that of ducks(Fig. 6C). As a control, GAPDH RNA in ducklings was 4- to 8-foldhigher than that in ducks (Fig. 6C). Therefore, the ratio of DHBVPreC/C RNA to GAPDH RNA in ducklings was 2- to 3-fold higherthan that of ducks. We inferred from these results that cccDNA inducklings are associated with a higher level of viral RNAs butcould not rule out the possibility that in ducklings those RNAshave a longer half-life. Differently from viral RNA, DHBV coreprotein and replicative intermediates were nearly equally detectedin ducks and ducklings with some variations.
DISCUSSION
It has been reported that the genome of several DNA viruses existsas an individual minichromosome in the nucleus of infected hostcells. For long-term episomal maintenance of this viral structurefrom a parental cell to two daughter cells, viral DNA segregationmust occur. This process involves “chromosome tethering” inmany DNA viruses in which a viral protein binds to both a specificviral sequence, such as ori, and a host chromosome (15, 16, 19,30). Such association enables the acentric viral minichromosometo utilize the chromosomal centromere in trans, thereby achievingefficient transmission of viral DNA genomes during cell division.The mechanism of the maintenance of hepadnaviral cccDNAminichromosomes in the nucleus of hepatocytes, however, is un-clear. Given that hepatocytes are long-living cells, pressures onviral DNA segregation might be less or attenuated in the case ofhepadnaviruses. For persistent hepadnaviral infection, stablymaintaining cccDNA in the nucleus is crucial since the replenish-ment of viral RC DNA, the precursor of cccDNA, might be inef-
ficient in the presence of viral large envelope protein, the negativeregulator of RC DNA nuclear translocation (10, 34). As the firststep of understanding of hepadnaviral cccDNA maintenance inthe nucleus, it is informative to clarify the overall structure ofcccDNA minichromosomes, especially nucleosome positioningon cccDNA.
In this study, we used MNase mapping and PCR amplificationof isolated mononucleosomal DNA to study nucleosome posi-tioning of hepadnaviral cccDNA minichromosomes. The decisionto choose DHBV congenitally infected ducks as a surrogate modelfor this purpose was based on the following consideration. First,by using DHBV-positive ducks, we could study viral minichromo-some structures in normally differentiated hepatocytes. Second, it
is experimentally practical to transmit virus into individuals ofvarious physiological conditions, for example, ducks or ducklings,which supplies opportunities to study viral minichromosomesunder different liver microenvironments (28).
In eukaryotic genomes, including human DNA, positioning ofnucleosome occupancy and depletion along DNA strands is de-termined by both trans elements, such as transcription factors,chromatin remodelers, and RNA polymerase, and cis elementsrepresented by nucleosome sequence preferences (5, 17, 27, 42).The latter are specific patterns of DNA sequence that affect DNAlocal bendability around a small histone octamer core (17, 42). Inhepadnaviruses, considering the facts that viral RNA transcriptionis regulated by many cis and trans elements (6, 7, 13, 23) and that
FIG 6 Topoisomers of cccDNA detected in adult ducks and ducklings. (A) The cccDNA was extracted simultaneously from the livers of DHBV-positive adultducks and DHBV horizontally and vertically infected ducklings. DNA was separated in a gel (25 cm long) slowly (1 V/cm) before blotting and hybridization.Negative supercoiled turns of cccDNA are marked on the left of the gels. (B) Superhelicity changes of duckling DHBV cccDNA following different treatments.cccDNA untreated control (lanes 1 and 3); heat denaturation (100°C, 2 min) (lane 2); E. coli topoisomerase I treatment (1 unit enzyme/reaction, 37°C for 1 h)(lane 4). (C) Comparison of levels of DHBV RNA and other viral components in adult duck and duckling livers. For each bird, after RNA extraction andconcentration normalization, triplicate RNA samples were reversed transcribed and PCR amplified. The experiment was repeated once for all birds used.Numbers under each blot are relative signal intensities among different samples. For RI, all DNA bands were included for the comparison. RC: relaxed circularDNA; DL: double-stranded linear DNA; CCC: cccDNA; RI: replicative intermediate DNA; DHBc: DHBV core protein.
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only a few copies of cccDNA minichromosomes, the transcriptiontemplate, exist in each hepatocyte (41, 44), viral minichromo-somes are expected to have a specific structure in terms of nucleo-some positioning and other features to fulfill the complexity andefficiency of their functions. Besides the viral sequence that couldbe one of determinants of nucleosome binding through nucleo-some sequence preferences as described in other species, part ofthe uniqueness of the cccDNA minichromosome structure mightbe derived from the asymmetrical nature of the precursor ofcccDNA, RC DNA. The asymmetries of RC DNA include a shortRNA oligomer at the 5= end of the plus-stranded DNA, a redun-dant sequence at the two ends of the minus-stranded DNA, acohesive region between the 5= ends of plus and minus DNAstrands, and a single-stranded region in RC DNA due to uncom-pleted elongation of plus-strand DNA (11, 12). Once translocatedinto the nucleus, RC is the target of host DNA repair machinerythat fixes those asymmetries and converts RC to cccDNA. Thespecific locations of the asymmetries would cause an uneven dis-tribution of cellular DNA repair machinery on RC DNA (31).These “hot spots” enriched with host proteins might act as the firstplayers in the formation of cccDNA minichromosomes by eithertriggering preferential nucleosome binding to the viral genome orexpelling through steric hindrance the binding of proteins fromthis region that perform a negative role in the assembly of cccDNAminichromosomes. It is worthwhile to note that a highly repro-ducible pattern of nucleosome binding shown in several ducks aswell as ducklings (Fig. 2 and 5) coincides with the region of nt 2000to 2600 of the DHBV genome where most of the asymmetries ofRC DNA are located.
Another clue revealed in this study that might be useful foraddressing the formation and structure of cccDNA minichromo-somes is that a portion of viral cccDNA in the livers of ducklingscarries a few nucleosomes, reflected by a low number of superhe-lical turns (Fig. 6). This is apparently different from cccDNA de-tected in adult ducks, where much less cccDNA with �8 super-coiled turns was observed in a Southern blot. It has been claimedthat there are two populations of cccDNA, dependent upon thenumber of bound nucleosomes (25): (i) an inactive or less activeform of cccDNA in terms of viral RNA transcription in whichnucleosomes are fully loaded on cccDNA and (ii) an active form ofcccDNA for transcription in which only part of the cccDNA isbound with nucleosomes. The results described in this studymight raise another possibility for the reasons of existence ofcccDNA that is partially loaded with nucleosome. They may beintermediates of a process in which nucleosomes are graduallybound to cccDNA. Given that the total number of hepatocytes israpidly increased in ducklings, total amounts of cccDNA have tobe proportionally increased by means of de novo infection and/orintracellular cccDNA amplification (34), dependent on the mech-anism underlying virus spreading to match the liver growth.Therefore, it is not surprising to detect cccDNA with a fewnegative supercoils during a time when a large amount ofcccDNA is converted from RC and other forms of precursors. Ifthese cccDNAs were really initial intermediates of fully nucleo-some-loaded cccDNA minichromosomes, it is worth separat-ing these minichromosomes and detecting protected virus se-quences to see whether or not some specific regions of cccDNA arepreferentially loaded with nucleosomes. Careful analysis of thesespecial populations of viral minichromosomes and comparisonbetween liver samples of different ages and physiological status
might disclose the kinetics and sequence of nucleosomes loadingon hepadnaviral cccDNA minichromosomes. An alternative sce-nario that could be involved in the presence of the less-supercoiledcccDNA in the duckling livers may be related to the rapid livergrowth in these young ducks, where host factors like histones areused for the formation of both cellular chromosomes and viralminichromosomes. A possible competition for these key chromo-some components might slow down the assembly process of viralminichromosomes, resulting in cccDNA with fewer negatively su-percoiled turns in duckling livers.
In order to fully understand the functionality, including themaintenance mechanism of cccDNA, it is important to obtaininformation of protein components bound at specific sites of viralminichromosomes. Though various host proteins as well as HBVcore and X antigen have been reported to be associated withminichromosomes (1–3, 20, 25, 26), exact binding positions andmechanisms of action of these components are not clear. Com-plete elucidation of hepadnaviral minichromosome structures isstill a challenge despite the fact that extensive efforts have beenmade. A main hurdle was a heavy contamination of host counter-partners during the process of isolation of viral minichromo-somes, though different purification methods were employed in atandem manner, such as sucrose gradient centrifugation, gel fil-tration, and immunoprecipitation with different antibodies. Theother difficulty was the overall instability of minichromosomesduring the isolation: dissociation of some components with alower binding affinity from minichromosomes might be inconsis-tent when comparing protein identifications of different purifica-tion preparations. Nevertheless, the results of nucleosome map-ping on viral minichromosomes reported here will supply theinitial framework information for the understanding of the overallcomplete structure of this key viral component, which may pro-vide a basis for new therapeutic interventions for the eliminationof cccDNA from HBV-infected livers.
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