Proc. Natl. Acad. Sci. USAVol. 76, No. 5, pp. 2222-2226, May 1979Biochemistry

Cloning in Escherichia coli and physical structure of hepatitis Bvirion DNAt

(Dane particle/bacteriophage X vector/restriction mapping/SI endonuclease/exonuclease III)

PATRICK CHARNAY, CHRISTINE POURCEL, ANNE LouISE, ALEXANDRE FRITSCH, AND PIERRE TIOLLAISRecombinaison et expression genetique (Institut National de la Sante et de la Recherche MWdicale U. 163), Unite de Genie G6n4tique, Institut Pasteur,28 rue du Docteur Roux, 75015 Paris, France

Communicated by Andre Lwoff, February 23, 1979

ABSTRACT A restriction map of hepatitis B virion DNAwas established after cloning of the whole viral genome inEscherichia coli. By use of EcoRI, Xho I, Bgl II, Xba I, BamHI,HincII, and Hae III endonucleases, a total of 28 restriction siteswere mapped. The single-stranded region was localized on therestriction map and the 5' end of the short strand was mappedat a fixed position.

Hepatitis B is a frequent and sometimes severe disease. In someareas of Asia and tropical Africa, 10% of the population carrythe viral surface antigen (1). The Dane particle (2), found inthe sera of patients, is considered to be the etiological agent (3).It contains a circular DNA molecule, hepatitis B virion (HBV)DNA, with a nick (or a short gap) and a single-stranded region(4-6). HBV DNA is the smallest known mammalian viral ge-nome. These unique characteristics of the genome, the fre-quency of hepatitis B, and the likely relationship between thisvirus and primary liver carcinoma (7, 8) justify an accurateinvestigation of the structure of the HBV genome.

In this article we present a physical map of the HBV genome.Twenty eight restriction sites are located, and new informationconcerning the physical structure of HBV DNA is also estab-lished. In particular, the single-stranded region is localized onthe restriction map. The position of the nick is discussed.

MATERIALS AND METHODSDNA Preparations and Cloning. HBV were purified as

described by Summers et al. (4) and their DNA was labeledwith the endogenous DNA polymerase (6), with [32P]dTTP (22Ci/mmol, 1 Ci = 3.7 X 1010 becquerels) as the radioactiveprecursor. After purification of the HBV DNA (4), it appearedfrom electron microscope observation that, under the actionof the endogenous DNA polymerase, the single-stranded regionof about 10% of the DNA molecules was totally repaired.The purified HBV DNA was used for cloning. The circular

HBV genome was cleaved by EcoRI endonuclease at its uniquerestriction site (J. Summers, personal communication) and in-serted in vitro into the DNA of bacteriophage Xgt WES. XBas described (9). After transfection of C600 recBC bacteria withthe hybrid DNA, recombinant phage clones were purified threetimes. For each recombinant clone a first phage stock was madeand, in order to avoid any genetic drift, all subsequent stockswere made from this initial sample. The cloned EcoRI DNAfragment will be referred to as Eco HBV DNA.

After digestion of the DNA of a recombinant clone withEcoRI endonuclease, Eco HBV DNA was purified from the twovector arms by zone centrifugation in a 5-30% sucrose gra-dient.

Enzymatic Digestions and Chemicals. Digestions by thefollowing restriction nucleases (New England BioLabs), EcoRI,BamHI, Sma I,Xho I, Pst I, Xba I, Bgl II, Sst I, Sal I, Kpn I,HincII, and Hae III, were performed in the buffers recom-mended by New England BioLabs. Alkaline phosphatase (P-LBiochemicals) was used in 10 mM Tris-HCI, pH 8.5/10 mMMgCl2. The 5' ends were labeled by means of [y-32P]ATP (NewEngland Nuclear, 3000 Ci/mmol) and polynucleotide kinase(P-L Biochemicals) in 50 mM Tris-HCI, pH 9.5/10 mMMgCl2/5 mM dithiothreitol. SI endonuclease (generous giftfrom F. Rougeon) was used at 25°C in 25 mM NaCOOCH3,pH 4.5/1 mM ZnSO4/125 mM NaCl. DNA was labeled by nicktranslation with [a-32P]dATP (New England Nuclear, 300Ci/mmol) and the three other unlabeled dNTPs at a concen-tration of 200,uM and with Escherichia coli DNA polymeraseI (Boehringer Mannheim) in 50 mM Tris-HCl, pH 7.8/5 mMMgCl2/10 mM 2-mercaptoethanol; DNA concentration was10 ,g/ml; DNA was purified from free dNTPs by gel filtrationon Sephadex G100 (10). E. coli exonuclease III (New EnglandBioLabs) digestion was done in 67 mM Tris-HCI, pH 7.3/90mM NaCl/4 mM MgCl2/4 mM dithiothreitol at 25°C for 30min; the DNA concentration was 1 ug/ml.

Other Methods. Polyacrylamide gel electrophoresis of re-striction DNA fragments was carried out by the method ofAdams et al. (11). DNA fragments from polyacrylamide gelswere purified as described by Maxam and Gilbert (12). Phageplate stocks, extraction of DNA, electrophoretic analysis ofrestriction fragments on agarose gels, and electron microscopicanalysis of DNA heteroduplexes were done as described(13).

Biohazards. Biohazards associated with the experimentsdescribed in this article have been examined previously by theFrench National Control Committee. Growth of recombinantbacteriophages was done under L3B11* conditions. L3 isequivalent to P3 physical containment and B1* is intermediatebetween the EK1 and EK2 biological safety levels (14).

RESULTSAnalysis of Various X-HBV Recombinant Clones. The

DNAs from five different clones, in which an EcoRI DNAfragment of about 3200 base pairs had been identified, wereanalyzed. These DNAs were digested by EcoRI plus Xho I,EcoRI plus BamHI, EcoRI plus Bgl II, and EcoRI plus Hpa Iand submitted to electrophoresis on 2% agarose gels. The re-striction patterns were identical for the five clones (results notshown). Heteroduplexes were formed between the DNA fromone of these clones (clone X-HBV1) and HBV DNA. The nu-

Abbreviation: HBV, hepatitis B virion.t In order to conform to the usual terminology, the Dane particle isdesignated as hepatitis B virion.

2222

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Biochemistry: Charnay et al.

:<~~~~~~~~MI..; ..;.i.*-

: .... ..

... ., ..... .. .' .b

Proc. Natl. Acad. Sci. USA 76(1979) 2223

0:B 7.. 05 ; . ;

f,, , _ j, ,t : ;7b

FIG. 1. Heteroduplexes between DNA from the X-HBV1 recombinant and DNA from Dane particles. (A) One DNA strand from X-HBV1recombinant is hybridized with the long strand from Dane particle DNA. The loop (a) is entirely double stranded. A HBV DNA homoduplexis also observed (b). (B) The other DNA strand from the X-HBV1 recombinant is hybridized with the short strand from Dane particle DNA.The heteroduplex loop (a) contains a single-stranded region. A HBV DNA homoduplex is also seen (b). The bar represents 0.5 ,gm.

merous heteroduplexes observed by electron microscopy havethe expected structure (Fig. 1). No apparent base-pairing de-fects were observed. Both experiments suggest that there wasno major heterogeneity in the original HBV DNA preparation.Clone X-HBV1 was arbitrarily chosen for all further experi-ments.

A

1 7 70 -

1170 -1100 -

520 --

450 -

BCxc

xC --

E_-220 - F

GH

1 2 3 4 5 6 B

Restriction Map ofHBV Genome. The restriction map was

established on the Eco HBV fragment from clone X-HBV1.The fragment was labeled by nick translation, except for theexperiments in which 5'-end labeling was necessary. No re-

striction site was observed for Sma I, Pst I, Sst I, Kpn I, andHpa I endonucleases.

1 2 3 4 5 6

A s

A ABCC

D B

A A

B

E-'Affi.bbF

C-.

C -E _

DB *

.---xc

JK

bb -

-bbFIG. 2. Electrophoretic analysis of restriction DNA fragments obtained from Eco HBV DNA. (A) Autoradiogram of a 4% polyacrylamide

gel. Arrows indicate the positions of simian virus 40 DNA fragments obtained from digestion with HindIII endonuclease. Fragment sizes aregiven in base pairs. Eco HBV DNA was digested with the following endonucleases: lane 1, Hae III; lane 2, HincII; lane 3, Bgl II; lane 4, BamHI;lane 5, Xba I; lane 6, Xho I. (B) Autoradiogram of an 8% polyacrylamide gel. Eco HBV DNA was digested with the following endonucleases:lane 1, Hae III; lane 2, Hae III + HincII; lane 3, Hae III + BamHI; lane 4, Hae III + Bgl II; lane 5, Hae III + Xba I; lane 6, Hae III + Xho I.xc and bb indicate the respective positions of the xylene cyanol and bromophenol blue dyes.

wow:

2224 Biochemistry: Charnay et al.

Table 1. Electrophoretic analysis of restriction DNA fragmentsobtained from Eco HBV DNA*

Enzyme Fragment Size, base pairs

Xho I A 2950B 135

BamHI A 1450B 900C 475D 280

Bgl II A 1880B 430C 420D 350

Xba I A 1675B 1030C 245D 155

HincII A 1000B 740C 650D 465E 220

Hae III A 920B 400C 370D 340E 250F 205G 150H 140I 105J 60K 55L 45M 42N 15

Pst I, Hpa I, Kpn I, Sst I, Sal I, and Sma I do not cleave Eco HBVDNA.* See Fig. 2.

The size of the restriction fragments obtained from digestionby Xho I, BamHI, Bgl II, HincII, Xba I, and Hae III endonu-cleases was determined by the method of Maniatis et al. (15).The results (Fig. 2) are presented in Table 1.The position of the Xho I, BamHI, Bgl II, Xba I, and HincIl

restriction sites was determined as follows. Restriction fragmentscorresponding to the extremities of Eco HBV DNA were

identified through 5'-end labeling with 32P and size measure-ment of restriction fragments from mixed digestions with thepreceding enzymes taken in pairs. The end closest to the XhoI restriction site is arbitrarily defined as the right end of the EcoHBV DNA (Fig. 3A).The Hae III restriction sites have been positioned by a

combination of several methods. (i) Identification of the endfragments by 32p labeling of the 5' ends; (ii) mixed hydrolysisof Eco HBV DNA with Hae III and each of the above-men-tioned endonucleases (Fig. 2B); (iii) hydrolysis by Hae III ofthe purified HincII A, B, C, D, and E restriction fragments; and(iv) partial hydrolysis of Eco HBV DNA with Hae III and pu-rification of the corresponding restriction fragments on a 4%polyacrylamide gel, followed by complete hydrolysis of thesefragments with Hae III and their analysis on an 8% polyacryl-amide gel. The evidence for the location at the left end of EcoHBV DNA of the 15-base-pair Hae III N fragment (observed

on an 8% gel) is the following: after 5'-end labeling of Eco HBVDNA, only one fragment was observed, Hae III M, whichcorresponds to the right end of Eco HBV DNA. On the otherhand, partial hydrolysis of the Eco HBV DNA by Hae III givesa fragment of about 150 base pairs which, upon complete hy-drolysis with Hae III, leads to the Hae III H fragment of 140base pairs. Finally, from other partial hydrolysis, this Hae IIIH fragment has been placed in the vicinity of the left end of theEco HBV fragment. The Hae III restriction map is presentedin Fig. 3A.The analysis of restriction fragments from mixed digestions

allowed not only the positioning on the physical map of thefragments obtained with a given enzyme, but also the accuratelocation of restriction sites for different enzymes relative to eachother (e.g., the Bgl II and HincHI sites at the extreme right).These results are given in Fig. SB.

Structure of HBV DNA. The model for the structure of HBVDNA as initially proposed by Summers et al. (4) has been testedand some additional properties have been determined. Inparticular, this model implies that the 5' ends of the two DNAstrands overlap over a short stretch.To determine how the circular structure of the HBV DNA

is maintained, we denatured the DNA, renatured it, and ob-served it by electron microscopy. (This experiment was thesame as the one described in Fig. 1. Because there was an excessof Dane particle DNA, many HBV DNA homoduplexes wereobtained.) DNA molecules having the initial circular and par-tially single-stranded structure were observed, but also a few"dimer" molecules with double the initial length and with twosingle-stranded gaps (results not shown). These "dimers" werenot observed in the initial HBV DNA preparation, and theirformation is easily explained by end-to-end polymerizationthrough cohesive ends.HBV DNA was also treated by exonuclease III in order to

digest the 3' ends on each strand. From observation by electronmicroscopy it follows that a great majority of the moleculesremained circular whereas their single-stranded region waslengthened by about 50%. It was also observed that aboutone-third of the circular molecules had a second single-strandedregion apparently located at random relative to the first one andprobably due to an accidental single-stranded nick. Both theseobservations prove that digestion by exonuclease III had actu-ally occurred. Since the majority of the molecules remain cir-c'ular, this experiment and the preceding one prove that thecircular structure is due to base pairing of the 5' ends of eachstrand.To determine the position of the single-stranded region, three

samples of HBV DNA were treated with S1 endonuclease, la-beled by nick translation, and then digested with EcoRI, XhoI, and BamHI endonucleases. The resulting restriction frag-ments were analyzed (Fig. 4). Some major bands were observed:one with EcoRI (corresponding to a 1580-base-pair fragment),one with Xho I (corresponding to 1700 base pairs), and two withBamHI (corresponding to 1310 and 750 base pairs). With thislast enzyme, an additional minor band, corresponding to 900base pairs, was also observed. These results are easily explainedby the model given in Fig. 5: the 5' end of the short strand is ata fixed position located at 1580 base pairs from the EcoRI site,1700 base pairs from the Xho I site, and 1310 base pairs fromthe BamHI A site. This location is strengthened by measure-ments made on heteroduplexes identical to the one of Fig. 1B:the long double-stranded stretch which necessarily has theEcoRI site at one end has a constant length of 1500 ± 150 basepairs. The 750-base-pair fragment obtained after BamHI di-gestion corresponds to the sum of the BamHI C and D frag-ments from the Eco HBV DNA. Finally, the broad band is

Proc. Natl. Acad. Sci. USA 76 (1979)

Proc. Natl. Acad. Sci. USA 76 (1979) 2225

A

1000K, I.. - I I

2000 3000ISlI pp1 I I I 1lii P I PP liIpIi i Il

A B

D1 CI B| A

B IDI A C

D | A B C

A D C I B E

NH J C B A KLG F D E MIII IIl lI l l l l

lilAi aI i I, .~ii

BM E3M t E3=-DB y-=3

cr % -Ea, - oE c D 8I ZII I CD II)()A I

1 1 1 I f I 1)IJ15 140 606565 240 180 240 35 155 300

m EaI E3B ° E3E c

W PIT7I II

II oDxIxxIl I l l

265 185554512525 205 105 165 190 50808542

FIG. 3. Restriction map of Eco HBV DNA. Eco HBV DNA is about 3100 base pairs long. The end closest to the Xho I restriction site isdefined as the right end. (A) Position of Xho I, Bgl II, Xba I, BamHI, HincII, and Hae III restriction fragments. (B) Relative positions of restrictionsites corresponding to the preceding enzymes. Lengths of DNA fragments were estimated from their electrophoretic mobility and are expressedin number of base pairs.

generated by a fragment located in the variable single-strandedregion of HBV DNA and corresponds to the Eco HBV BamHIB fragment. Hence, the single-stranded stretch of HBV DNA

A 1 2 3 4

-,-5200

tff L _-w 3835

--1390

B 1 2

94

I

that is of variable length has one fixed end at 1580 nucleotidesfrom the EcoRI site.

Several minor bands have also been observed. From EcoRIdigestion, at least one band 1320 base pairs long is obtained. XhoI digestion gives two minor bands, 1630 and 1460 base pairslong. Similarly, from BamHI digestion one obtains two minorbands 1220 and 1060 base pairs long. From these results it fol-lows that SI endonuclease partially cuts the HBV DNA at po-

sitions 1500 and 1330.

_-1 770

_ 11170 +1100

520--450

FIG. 4. Electrophoretic analysis of DNA fragments obtainedafter digestion ofHBV DNA by S1 endonuclease and by EcoRI, XhoI, and BamHI restriction enzymes. (A) Autoradiogram of a 1% agarose

gel. HBV DNA was mixed with a 10-fold excess of XWES AB DNA,treated with S1 endonuclease, and nick-translated (lane 4). Twosamples were treated in addition with Xho I (lane 1) and EcoRI (lane2) endonucleases. Lane 3 represents Eco HBV DNA. (B) Autoradio-gram of a 2% agarose gel. HBV DNA was mixed with a 10-fold excessof AWES AB DNA, treated with S1 endonuclease, and then digestedwith BamHI endonuclease (lane 1). Eco HBV DNA was digested withBamHI (lane 2). Arrows indicate the positions of linear simian virus40 DNA and simian virus 40 DNA fragments obtained from digestionwith HindIII or Pst I endonucleases. Fragment sizes are given innumber of base pairs.

FIG. 5. Physical structure of the genome of hepatitis B Daneparticles. The circular structure is maintained through base pairingof the 5' extremities of the two DNA strands. These two ends and thesingle-stranded region (dashed line) are located on the restrictionmap. The EcoRI restriction site is chosen as the origin of the physicalmap, and distances are given in number of base pairs. The 5' end ofthe short strand is located at a position estimated to be at 1580 basepairs.

Nucleotide

Xho

BglI I

Xba

Bam H

Hinc I

Hae III

I

Biochemistry: Charnay et al.

700-- "' I i

+1170 --6-1100

2226 Biochemistry: Charnay et al.

DISCUSSIONThe size of the cloned HBV genome was estimated to be 3200base pairs, in agreement with the results of Landers et al. (6)and J. Summers (personal communication). The cloned DNAwas digested by various restriction enzymes, and the sum of thefragment sizes from each digest was 3100 base pairs. Thenumber of fragments and their sizes obtained upon Hae IIIendonuclease digestion (Fig. 2) are similar to those obtainedfrom identical digestions of HBV DNA by Summers et al. (4)and Landers et al. (6). In agreement with Summers (personalcommunication), we locate the cohesive ends of the two EcoHBV DNA strands in the Hae III A fragment and find theEcoRI restriction site just opposite the cohesive region. Incontrast, the sizes of certain HincIl restriction fragments (Fig.2A) are different from HincII fragments obtained by Landerset al. (6).

Because digestion of HBV DNA by BamHI endonucleasegives a fragment of 750 base pairs, equal to the sum of thelengths of BamHI C and D fragments of the cloned DNA, weconclude that the original HBV DNA does not contain twoEcoRI restriction sites close to each other and, therefore, thatno HBV DNA was lost upon cloning of the EcoRI fragment.The digestion of HBV DNA by exonuclease III and the for-

mation of HBV DNA "dimers" prove that the circular structureis maintained through base pairing of the 5' ends of each DNAstrand, which confirms the structural model of Dane particleDNA proposed by Summers et al. (4). We have now located thesingle-stranded region relative to the restriction map (Fig. 5).Taking the EcoRI restriction site as the origin of the map, the5' end of the short strand is at position 1580 and the single-stranded region is almost completely contained in the BamHIB fragment of HBV DNA.From the analysis of the restriction fragments of HBV DNA

and, particularly, from the existence of the Hae III AA' doublet,Robinson and coworkers (6, 16) suggest that the HBV DNApopulation is heterogeneous. On heteroduplexes and HBV DNAhomoduplexes, as exemplified in Fig. 1, we did not detect anyapparent base-pairing defect, which could mean that theeventual heterogeneity is not localized in a particular regionof HBV DNA. Digestion patterns of this DNA by Si plus XhoI, Si plus EcoRI, and Si plus BamHI nucleases (Fig. 4) provethat, in the great majority of the molecules, the Xho I, EcoRI,and BamHI restriction sites are at fixed positions relative to the5' end of the short HBV DNA strand and that no major amountof heterogeneity exists in the double-stranded region.

After digestion of HBV DNA with SI plus Xho I, Si plusEcoRI, and Si plus BamHI nucleases, minor DNA bands arealso observed (Fig. 4); we propose three possible explanationsfor their origin. (i) Si endonuclease does not cut HBV DNA atthe nick of the long strand; the DNA preparation then contains

two additional minor populations in which the 5' end of theshort strand is shifted to positions 1500 and 1320. (ii) S1 endo-nuclease cuts part of the DNA molecules at the nick and twoDNA populations are present with a different location of thenick. (iii) Each HBV DNA molecule contains two nicks and S1endonuclease cuts only part of them. The first two hypothesesimply that the HBV DNA population is heterogeneous. Uponincrease of the S1 endonuclease concentration, the minor bandscorresponding to position 1500 become weaker relative to theother minor bands. We have no explanation for this observa-tion.A comparison of restriction patterns from different clones

of Eco HBV DNA should give some more insight into thisheterogeneity problem.We thank Dr. P. Maupas for the supply of virion-rich serum, Dr. J.

Summers for his help in the purification of HBV DNA, and Dr. F.Rougeon for helpful discussion. This work was supported by grantsfrom the Institut National de la Sante et de la Recherche Medicale(INSERM), the Centre National de la Recherche Scientifique (CNRS),the Delegation Generale a la Recherche Scientifique et Technique(DGRST), the Universite Paris VII, the North Atlantic Treaty Orga-nization (NATO), and the Fondation pour la Recherche Medicale.

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695-698.3. World Health Organization (1977) Progress in Viral Hepatitis,

WHO Technical Report Series no. 602 (WHO, Geneva, Swit-zerland).

4. Summers, J., O'Connel, A. & Millman, I. (1975) Proc. Nat!. Acad.Sci. USA 72, 4597-4601.

5. Hruska, J. F., Clayton, D. A., Rubenstein, J. L. R. & Robinson,W. S. (1977) J. Virol. 21, 666-682.

6. Landers, T. A., Greenberg, H. B. & Robinson, W. S. (1977) J.Virol. 23, 368-376.

7. Maupas, P., Coursaget, P., Goudeau, A., Drucker, J., Sankale, M.,Linhard, J. & Diebolt, G. (1977) Ann. Microbiol. (Paris) 128,245-253.

8. Summers, J., O'Connel, A., Maupas, P., Goudeau, A., Coursaget,P. & Drucker, J. (1978) J. Med. Virol. 2, 207-214.

9. Fritsch, A., Pourcel, C., Charnay, P. & Tiollais, P. (1978) C. R.Hebd. Seances Acad. Sci., Ser. D 287, 1453-1456.

10. Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448.11. Adams, J. M., Jeppesen, P. G. N., Sanger, F. & Barrel, B. G. (1969)

Nature (London) 223, 1009-1014.12. Maxam, A. M. & Gilbert, W. (1977) Proc. Natl. Acad. Sci. USA

74,560-564.13. Charnay, P., Fritsch, A., Louise, A., Perrin, D. & Tiollais, P. (1979)

Mol. Gen. Genet. 170, 171-178.14. Commission franoaise de recombinaison genetique in vitro:

rapport d'activite (1977) Prog. Sci. 191, 86-94.15. Maniatis, T., Jeffray, A. & Van de Sande, H. (1975) Biochemistry

14,3787-3794.16. Robinson, W. S. (1977) Annu. Rev. Microbiol. 31,357-377.

Proc. Natl. Acad. Sci. USA 76 (1979)