VIROLOGY

183,

555-565

(199 1)

Hepatitis 6 Defective Virus with Rearrangements in the PreS Gene during Chronic HBV Infection GUIDO GERKEN,*+’ DINA KREMSDORF,* FRANCIS CAPEL,+ MARIE ANNE PETIT,+ CHARLES DAUGUET,§ MICHAEL P. MANNS,l- KARL-HERRMANN MEYER ZUM BijSCHENFELDE,t AND CHRISTIAN BRECHOT**” ‘INSERM Unit6 75 C. H. U. Necker, Paris, France; tlnstitut Med. Klinik und Poliklinik, Universitat Mainz, Germany; Paris, France; §lnstitut Pasteur, Unit6 d’Oncologie Virale, Paris, France; and “lnstitut Pasteur, Laboratoire Received

December

28,

1990;

accepted

April

23,

WNSERM Hybridotest,

Unite 13 1 Clamart. Paris, France

199 1

We have found a defective form of HBV’ in a HBsAgand anti-HBe-positive patient with liver cancer. Viral deletions were identified in the preS coding region using PCR. The presence of deleted HBV forms was observed in serum, PBMC, and liver samples. After sequencing 12 clones were analyzed (subtype adr). In 9 out of 12 clones a 183-bp in-frame deletion was recorded in the preS1 region (2995 to 3177). Three out of 9 clones also yielded rearrangements of the preS2 N-terminal part. Four out of 9 showed numerous point mutations in the preS1 and preS2 sequence. In addition, 3 out of 12 clones, which did not show the 183-bp preS1 deletion were found to have small deletions and insertions in the same part of the preS1 gene. Immunological mapping using monoclonal anti-preS antibodies showed loss of preS epitopes located at the 3’-part of preS1 and the 5’-part of preS2. On the other hand, epitopes mapped to the 5’-part of preS1 and 3’ of preS2 were conserved. PBMC were also tested and solely PCR showed the major form of defective HBV with preS1 183-bp deletion. However, viral deletions in the preS gene eliminated the preS2 promotor region and B- and T-cell recognition sites. In contrast to this, the preS1 binding site to hepatocytes was conserved. Therefore. such deletions would potentially lead to an impairment in viral clearance without affecting viral penetration in liver cells, possibly accounting for chronic HBV infection. 0 1991 Academic Press, Inc.

tified in anti-HBe although HBV DNA positive individuals (Carman el al., 1989; Brunetto el a/., 1990). These mutations will result in the lack of HBeAg secretion despite the presence of a persistent viral multiplication. It has been hypothesized that such HBV strains might be involved either in the establishment of the chronic HBV carrier state after acute infection and/or in the development of severe forms of chronic hepatitis. The envelope of HBV consists of three polypeptides which are encoded in one open reading frame (S-ORF) (Tiollais et a/., 1985). Three translation initiation sites are present within this S-ORF, i.e., preS1, preS2, and S allowing the expression of the large protein (LHBs), the middle protein (MHBs), and the small HBs protein (SHBs) (Heermann et al,, 1984; Alberti et a/., 1990). The preS region elicits immune responses in the courses of HBV infection (Budkowska eta/., 1987; Klinkert et a/., 1986). PreS antibodies precede responses to S-protein and appear important for the clearance of HBV. The preS1 region of the HBV envelope contains the binding site to Hep G2 cells liver membrane within residues preS1 (2 1-47) (Neurath era/., 1986; Petit eta/., in press). The PreS2 region has been found to bear the binding site for polymerized human serum albumin, which is believed to be involved in the attachment of HBV to human hepatocyte membrane (Pontisso et al., 1989; Krone et a/., 1990). Chimpanzees immunized

INTRODUCTION The various factors involved in the development of the chronic hepatitis B virus (HBV) carrier state in some infected individuals as well as in the different severity of associated liver diseases are still under discussion (Oldstone, 1989; Hoofnagle, 1987). Although some viral components may have a direct toxic effect, the immune response plays a major role both in liver cell necrosis and viral persistence. Viral envelope and capsid proteins have been shown to be potential targets for humoral and cellular immune responses (Mondelli er al., 1988). Studies on the DNA sequence of various HBV subtypes previously failed to demonstrate differences in the rate of progression of chronic liver diseases and hepatocellular carcinoma (HCC). However, mutations in the preC coding sequence have recently been iden-

’ To whom correspondence and reprint requests should be addressed at INSERM Unite 75 C.H.U. Necker. 156 Rue de Vaugirard, F-1501 5 Paris, France. Fax: 33-l-45 67 53 33. ’ Abbreviations used: DHBV, duck hepatitis B virus; EIA, enzyme immune assay; HBV, hepatitis B virus; HBcAg, hepatitis B core-antigen; HBeAg, hepatitis B e-antigen; HBsAg, hepatitis B surface-antigen; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HIV, human immunodeficiency virus; MAb, monoclonal antibody; PCR, polymerase chain reaction; pHSA, polymerized human serum albumin; RIA, radioimmunoassay. 555

0042-6822191

$3.00

CopyrIght 0 1991 by Academtc Press. Inc. AH rights of reproduction in any form reserved.

556

GERKEN ET AL.

with peptides derived from the N-terminal part of the preS2 sequence (120- 140) were protected against HBV infection (Neurath et a/., 1986; ltoh et a/., 1986). Dominant B- and T-cell epitopes could furthermore be identified to enhance the immune response to S-protein and to overcome nonresponsiveness to S-proteins (Milich et al., 1985, 1986, 1987; Ferrari et a/., 1989). Rearrangements of the preS gene might therefore significantly modify the course of HBV infection. It has recently been shown that C-terminal truncated preS/S proteins may have a transactivating activity both in free viral DNA and in integrated forms (Kekule et al., 1990). Moreover, overproduction and storage of the large envelope protein has been demonstrated to induce liver cell injury and initiation of liver cell transformation in transgenic mice (Chisari et al., 1989). In this study we report on the molecular characterization of a defective hepatitis B virus with deletions and mutations in the preS coding region. This viral DNA form has been identified in serum viral particles as well as PBMC and liver samples of an anti-HBe positive HBsAg carrier with hepatocellular carcinoma. This might therefore contribute to the understanding of the molecular basis of viral persistence in chronic HBV infection. PATIENTS

AND METHODS

Patient A 40-year-old male of South East Asian origin had a 5-year-old history of chronic hepatitis B before the diagnosis of primary hepatocellular carcinoma. Because of rapid progression due to terminal liver failure the patient died within 3 months. All procedures performed were part of the patient’s clinical management after written consent was obtained. Viral serology Blood samples were tested for HBsAg, anti-HBs, anti-HBc, HBeAg, and anti-HBe by commercial kits (Abbott Laboratories, Chicago). HBsAg level was determined by titers of serum dilutions and by immunoelectrophoresis. For routine detection of HBV DNA, a standardized quantitative solution hybridization assay using a 1251-labeled HBV-DNA probe (Abbott Laboratories) was performed (Kuhns et a/., 1989). The detection limit of this assay is 0.15 pg HBV-DNA/ml, or approximately 4 x 1 O4 genomes. Binding sites for pHSA were determined by means of radioimmunoassay (Hansson et a/., 1979). Anti-preS2 antibodies were detected by using a synthetic PreS2 specific peptide (aa 120-l 45) (Coursaget et a/., 1988). The patient was found negative for anti-6 and anti-HIV as tested by standard immu-

noassays (Abbott Laboratories). Anti-HCV was also negative using C 10013 as a recombinant antigen for EIA (ORTHO Diagnostics, Raritan, USA). Detection

of preS encoded

proteins

Detection of preS1 and preS2 proteins was performed by immunoblot and radioimmunoassay technique as described in detail elsewhere (Gerken et a/., 1987, 1989; Petit et al., 1987, 1990). For preS1 domains MAbs MA1817 and F35.25 were applied. MAb F35.25 recognizes the region 21-47 of preS1 amino acid sequence of HBV, thus corresponding to the sequence involved in binding to Hep G2 cell membranes (Petit et al., 1989, and in press). MAb MA1 8/7 binds specifically to a sequential epitope localized between aa 28 and 120 of the preS1 domain (Deepen et al,, 1990). The preS specific MAb F52 recognizes an epitope located between aa residues 94-107 and 117125 (M. A. Petit, unpublished results). For the detection of preS2 domains we applied MAb Ql S/l 0 against the preS2-glycan (Deepen et al., 1990) the MAb F124, which recognizes the sequential preS2 N-terminal region 120 to 126 (Neurath et a/., 1987) and the conformational central region between aa 140 and 150 of the preS2 sequence (M. A. Petit, unpublished results). MAb F376 which recognizes an epitope located between aa 132 and 140/l 45 (Neurath et a/., 1986) was also applied. Total HBsAg activity was determined by using S-specific MAb 39.20, which recognizes a disulfide bond-dependent conformational epitope (Petit et al., 1990). Preparations

of viral particles from serum

Viral particles were purified from the serum by isopycnic sucrose gradient (Petit eta/., 1985). In a polypropylene centrifuge tube 0.2 ml of serum were loaded onto a gradient of 20 to 60% sucrose (w/w) in 10 mM Tris HCI, pH 7.2 containing 140 ml\/l NaCI. After 18 hr of centrifugation at 220.000 g, the fractions were collected and tested using HBs-, preSl-, and preS2-specific radioimmunoassays (Petit et a/., 1990). For PCR assays viral particle preparations were treated with lysis buffer and proteinase K using the classical DNA extraction method. Viral particles were visualized by electron microscopical examination directly after negative staining with 0.5% uranyl acetate using a YEOL 100 CX electron microscope (Jeol Ltd., Japan). Preparation

of DNA samples

Two hundred microliters of serum were incubated at 50” for 2 hr in proteinase K (100 pg/ml) and 2 vol of lysis buffer (400 mnll NaCI, 40 mM EDTA, 3% SDS). DNA

PreS

GENE

REARRANGEMENTS

IN HEPATITIS TABLE

HBV

Gene

B DEFECTIVE

557

1

PRIMERS AND PROBES USED FOR PCR AND HYBRIDIZATION

Localization (subtype awl

region

VIRUS

Sequences Primers

preS

C, preS,

S

S X C

MD MD MD MD MD MD MD MD MD MD MD MD

16 19 17 18 34 11 03 12 24 26 27 31

187-170 2816-2833 2933-2952 3064-3045 2636-2658 437-42 760-743 135-151 1400-1423 1627-1610 1961-1880 2286-2270

1

+ + + + + + -

5’-GTCCTAGGAATCCTGATG-3’ 5’.GGGTCACCATATI-CmGG-3’ 5’~AAATCCAGATTGGGACTKA-3’ 5’~CCCTGAGCCTGAGGGCTCCA-3’ 5’-ATTATGCCTCCCAGGmATCC-3’ 5’.AAGAAGATGAGGCATAG-3 5’-CCCAATATCACATCATCC-3’ 5’GGACTGGGGACCCTGCGC-3 5’-CTGGATCCTGCGCGGGACGTCCTT-3’ 5’-GTTCACGGTGGTCTCCAT-3’ ~‘ACTG~XAAGCCTCCAJIGCT-3’ 5’AGTGCGAATCCACACTC.3’

Probes preS2 preS 1 S X C

MD 37 MD 38 MD 09 MD 29 MD28

58-94 3093-3128 652-696 1428-1471 1884-1922

was extracted with phenol/chloroform, precipitated with ethanol in the presence of dextran T40, and dissolved in 100 ~1 of 10 mM Tris HCI, pH 8, 1 mM EDTA (TE) as described in detail elsewhere (Thiers et al., 1988). PBMC were prepared by centrifugation over Ficoll Hypaque and immediately lysed for nucleic acid isolation in a lysis buffer containing 10 mM Tris HCI, pH 8, 10 mlM EDTA, pH 8, 10 mM NaCI, 0.5% SDS, and 200 pg/ml proteinase K for 12 hr at 37”. After phenol/chloroform extraction the DNA was precipitated with ethanol and redissolved in sterile TE buffer. Sera and PBMC of patients with chronic hepatitis B and healthy blood donors without any HBV markers were used as positive and negative controls, respectively. DNA was also extracted from liver biopsy specimen obtained from our patient using the classical phenol/ chloroform method. DNA extracted from HBsAg-negative as well as HBsAg-positive HCC was studied as negative and positive controls respectively. PCR assay Following a comparison with available nucleotide sequences from hepadnaviruses, different sets of oligonucleotide primers from different HBV gene regions (preS, S, C, X) were synthesized on a 308 A DNA synthesizer by the methoxyphosphoramide method (Applied Biosystems). For the specific mapping of subfrag-

5’-CCTGCTGGTGGCTCCAGTI-CAGGAACAGTAAACCCTG-3 5’CCTCCTGCCTCCACCAATCGGCAGTCAGGAAGGCAGCCT-3 5’-CCTGCTGGTGGCTCCAG-ITCAGGAACAGTAAACCCTG-3’ 5’-TACGTCCCGTCGGCGCTGAATCCTGCGGACGACCCllCTCGGGG-3’ 5’.CClTGGGTGGClTl-GGGGCATGGACATCGACCC-ITATAA-3’

ments of the PreS region additional primer sets were constructed (Table 1). DNA amplification was performed in 50 ~1 reaction mixture containing 10 ~1 of serum DNA preparation, PCR buffer, 10 pmol of each primer, 200 mM of each dNTPs, and 1 unit of Tag polymerase (Perkin-Elmer/ Cetus, Emeryville) as described in detail elsewhere (Thiers et a/., 1988). Using a DNA thermocycler (Perkin-Elmer/Cetus) we carried out 40 cycles of amplification by using a step program (94”, 1 min; 55”, 1 min; 72”, 1 min) followed by a 10-min final extension at 72”. Analysis of PCR products After amplification 25% of the reaction product was subjected to electrophoresis in a 2% agarose gel in Tris borate buffer and transferred to a nylon membrane (Gene Screen Plus, NEN, Boston). The filters were prehybridized and hybridized in 3X SCC (1 X SSC: 150 m/M NaCI, 15 mM sodium citrate), 5X Denhardt’s solution (1 X Denhardt’s: 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 0.5% SDS, 30% formamide, and salmon sperm DNA (100 pg/ml). After 1 hr at 42” hybridization was carried out over night at 42” by the addition of an oligonucleotide probe specific for the amplified fragment labeled at the 5’-end with phosphorus 32 (Table 1). Filters were then washed in 2X SSC, 0.1% SDS for 10 min at 55”. The autoradiograms were exposed for 2, 6, 12, 24, and 48 hr with an

558

GERKEN

ET AL.

quenase kit, U.S. Biochemicals, al., 1977).

*

553

bp

+

553

bp

B MD 19116 -mm-

MD 34111

MD17118

MD 19118

MD17116 I

r 12

FIG. 1. Detection

of serum HBV-DNA by PCR (A) and subsequent PCR mapping of PreS-deleted viral sequences (6). (A) Ethidium bromide-stained agarose gel electrophoresis after PCR using preS primers (MD16/19) followed by Southern blot using preS probe (MD37). Negative control, no reagent control, i.e., PCR-mix without DNA; Positive control, 100 ng DNA extracted from human liver tissue of a HBsAg-positive hepatocellular carcinoma containing one copy HEW-DNA per cell: lanes 1-4, serum DNA from different other HBV-DNA carriers with chronic hepatitis B; lane 5, serum DNA with mutant HEW genomes derived from our patient with chronic hepatitis 6, rapidly evolved to hepatocellular carcinoma. (B) Ethidium bromide-stained gel after PCR analysis of serum DNA from the patient from lane 5 of A(lanes 1) in relation to a viremic HBsAg-carrier (lanes 2) using different primer sets for the amplification of subfragments of the preS region; i.e., MD 16/l 9 (predicted amplimer length: 553 bp); MD34/11 (1083 bp); MD 17/18 (131 bp); MD 19/18 (248 bp); MD 16/l 7 (436 bp).

intensifying screen at -80”. Our PCR assay with subsequent hybridization detected as few as 30 copies of serum viral DNA and 100 ag of cloned HBV-DNA, respectively (Gerken et a/., 199 1). Ml 3-Cloning

and sequencing

of PCR products

The PCR product amplified using the preS-primers MD 16 and MD 19 (Table 1) was purified, digested with Hind III restriction enzyme and inserted into the Hindllll Hincll-digested Ml 3mpl9 vector, using standard cloning procedures (Ml 3 Cloning kit, Amersham, Buckinghamshire, UK). The insert of different clones was sequenced by the dideoxy extention method (Se-

Cleveland) (Sanger et

RESULTS Detection of serum HBV-DNA with PCR Serum-, liver-, and PBMC-derived DNA samples from a chronic anti-HBe positive, HBsAg carrier who developed HCC were analyzed by PCR assay. The HBsAg titer determined by dilution of serum was 1: 1000 positive. Using immunoelectrophoresis the concentration of HBsAg in serum was 0.18 pg/ml, roughly comparable to the level usually observed in nonviremic carriers. Serum HBV-DNA was found negative with a standard hybridization test. However, PCR allowed amplification of HBV-DNA sequences. Serum HBV-DNA amplification of the surface, core, and X coding regions showed the size predicted from the HBV genetic map (Table 1). PCR assay for the amplification of the preS gene by contrast yielded DNA fragments with altered migration, consistent with deletions and mutations in this region (Fig. 1A). Among the several bands identified, one appeared to have the expected size. PCR mapping and nucleotide sequencing of the preS region The expression of the preS coding region of HBVDNA from our patient was further analyzed by using different sets of HBV-primers. PCR experiments using primers MD34 and MD1 1, which are located outside the preS sequence (Table l), mainly showed the presence of a smaller fragment than the expected normal size (Fig. 1 B). This confirmed the deletions of the preS coding region. To further localize the deleted sequence, we performed PCR assay using primers located inside the preS coding region (Table 1, Fig. 1B). After amplification of the 5’-part of preS1 sequence using MD17/MD18 and MD19/MD18 primer pairs, only normal size fragments were obtained. By contrast, fragments with abnormal migration size were shown after PCR analysis with MD1 6/l 9 and MD1 6/l 7 primer pairs, which cover the 3’-part of the preS1 region and the 5’- part of the preS2 region. The deletion could thus be localized at the end of the preS1 sequence and at the beginning of the preS2 coding region. Amplified products obtained using MD16 and MD19 primers were therefore cloned into M 13 bacteriophage and 12 clones were sequenced (Tables 2 and 3). The sequence was most similar to Southeast Asian HBV-DNA isolates which express HBsAg subtype adr (Ono et a/., 1987; Fujiama er a/., 1983). None of these clones corresponded to the HBV wild-type. All clones showed numerous point mutations. In 9 out of 12 clones a 183bp in-frame deletion in the 3’-part of preS1 coding region from nucleotide position 2995 to 3177 was re-

PreS GENE REARRANGEMENTS

559

IN HEPATITIS B DEFECTIVE VIRUS

TABLE 2 REARRANGEMENT

IN THE

PreS GENE OF DEFECTIVE HBV AFTER CLONING AND SEQUENCING

(CLONES

l-9)

ORF P

Whbp-

DELETION

l

2643 A+ C

2919 r*G

157

&lo

299s

3117

pi&zq

002 IQI. (10 bp)

003 r+c

009-019 pz--J

009

023

A*G

C*T

.

t t

Oh9 @

K2A

cT

t

t

t

t

t

t

t

t

Cloaa 1

t

t

2

t

t

3

t

t

4

-

t

3

t

t

6

t

t

7

-

t

9

-

t

9

-

+

0

*

0

t

-

+

t

0

t

-

t

0

t

0

t

0

t

t t t

.

t

t

t

t

-

t

t

-

t

t

+

-

-

+

t

-

t

t

0

t

.

t

l

0

t

s

t

t

0

t

.

-

Note. Top: The position of the genes of wild-type HBV-DNA (subtype adr) are indicated above. ORFP, the P gene coding for DNA polymerase; ORFS, the S gene coding for small hepatitis B surface protein (S-HBs; p24/gp27); PreSl , the preS1 region coding for large surface protein (L-HBs; p39Igp42); PreS2, the preS2 region coding for middle surface protein (M-HBs; gp33Igp36). Initiation codons are indicated by black dots. Numbers give nucleotide positions of point mutations, deletions (Del.), and insertions (Ins.) detected in the preS sequence. Numbering of nucleotide sequence is according to One et a/. (1987).

corded (Fig. 2 and Table 2). In addition, 3 out of these 9 clones showed marked rearrangements in the Y-part of preS2 sequence. This included insertion (10 bp) followed by deletion (10 bp) as well as numerous point mutations (Table 2). In 3 other clones the 183-bp deletion of preS1 could not be identified. In fact, in the 3’-part of preS1 sequence 2 of these 3 clones showed point mutations, small deletions (l-3 bp) and insertions (3 bp). In one of these clones, a 54-bp deletion in the 5’-part of the preS2 region from nucleotide position 4 to 57 was observed (Table 3). Immunological mapping translation products

of preS-encoded

Analysis of serum viral proteins by immunoblot and radioimmunoassay showed loss of expression of epi-

topes recognized by MAb MA1 8/7 in the preS1 region, Ql9/10 and F376 in the 5’preS2 region, and F52 in the 3’ preS 1 and 5’ preS2 regions (Table 4). The analysis of epitopes recognized by MAb F35.25 in the 5’ preS1 and by F124 in the central part of preS2 by contrast yielded positive results, Furthermore, no binding activity to pHSA confirming rearrangement of the N-terminal part of preS2 domain could be detected. In addition, the serum of our patient showed no reactivity in antipreS2 peptide (aa 120-l 45) immunoassay. Analysis samples

of serum viral particles,

PBMC, and liver

Using an isopycnic sucrose gradient centrifugation, HEW-associated particles were isolated (Fig. 3A). As shown in Fig. 3A, HBsAg (F39.20), preS2 (F124)-, and

GERKEN ET AL.

560

TABLE 3 REARRANGEMENT IN THE PreS GENE OF DEFECTIVEHBV AFTERCLONINGAND SEQUENCING(CLONES 1O-l 2)

ORF P PRE-Sl 2995

PRE-S2

t

t

ORFS

1 DELETION OF CLONES NCb 1 TO P’y

.

29.9

MM\\ 2999

y-q

4

3180

157

so00

pq

9094-99

G

;;;,,;,

r;;l;,

;;,-q

2107 c4 t

2120 9129 c+ A A4 0

2127 te c

2190 A+ 0

9191 002 ?a A t*li

999-0s~ 0 Dal. (I4 bp)

CIOM

Note. For legend, see Table 2.

preS1 (F35.25)-specific epitopes were detected in a narrow peak in fractions 8, 9, and 10 banding at 40% sucrose. After HBV-DNA extraction from fractions of sucrose gradient, PCR analysis showed the presence of the preS-deleted HBV-DNA fragments in fraction 9/10 (Fig. 38, lane 2). Electron microscopic examination of fractions 9/l 0 (40% sucrose) where the preS-deleted fragments were

T T C i! \

Insertion (10

C

bp) : c

; C A A G G/ T

found (Fig. 3C) revealed the presence of viral particles of approximately 60 nm, which may correspond to hepatitis B defective virus with rearrangements in the preS/S gene. After amplification of DNA extracted from PBMC with preS specific primers, one single PCR fragment could be visualized (Fig. 4) corresponding to the 183-bp-deleted PCR product sequenced from serum particles. When using a preS1 specific oligonucleotide probe corresponding to a sequence inside the 183-bpdeleted region (MD38, Table 1) hybridization was negative, thus confirming the presence of homogeneous preS1 -deleted molecules in PBMC. PCR assay of liver-derived DNA using preS primers showed the presence of a similar mixture of deleted and undeleted preS fragments to the one previously obtained from the serum and the viral particles in our patient (Fig. 4). DISCUSSION

5’ FIG. 2. Sequence analysis of amplified and cloned HBV-DNA with rearrangements in the preS region (clone 2 corresponding to Table 2, HBV-DNA subtype adr).

The results of this study allowed the identification of an HBV defective form in a patient whose chronic HBV infection rapidly evolved to hepatocellular carcinoma within 5 years. It is important to note that we used PCR as the most sensitive technique available so far for detecting HBV-DNAsequences in an HBsAg-positive individual who was found nonviremic based on the routine hybridization method. Using PCR to investigate the HBV-DNA sequence of different clones obtained from

PreS GENE REARRANGEMENTS

TABLE SEROIMMUNOLOGICAL

ANALYSIS OF

561

IN HEPATITIS B DEFECTIVE VIRUS

PreS- AND S-ENCODED

4 SURFACE PROTEINS

USING MONOCLONAL

ANTIBODIES

Note. Top: Organization and rearrangement of the preS region with predicted amino acid exchange (aa exe) specified by each mutation and with location of predicted aa loss due to preS1 and preS2 nucleotide deletions and insertions detected after cloning and sequencing (see Tables 2 and 3, clones l-l 2).

the amplification product of the preS gene in fact showed that none of them corresponded to the wildtype HBV strain. The nucleotide sequence variation in the preS region of our adr isolate is much more important than that obtained for the adr subtypes so far (Okamoto et a/., 1987; Ono et al., 1987). However, a mixture of different deleted HBV-DNA molecules was observed. The fact that various HBV-DNA forms may coexist in the same individual has been previously shown in other studies (Okamoto ef al., 1987; Kaneko et a/., 1989). A large 183-bp in-frame preS1 deletion, present in 9 of the clones examined, as well as small deletions and insertions, resulted in a deficiency of preS1 and preS2 epitopes. The predicted amino acid sequence from aa 58 to 118 was deleted within the preS1 region. Furthermore, the N-terminal pat-t of preS2 region was markedly rearranged between aa

120 and 129 and aa 124 and 143, respectively (Tables 2 and 3). These findings were confirmed by the application of monoclonal antibodies to test the expression of viral envelope proteins in the serum and on HBV particles isolated from our patient (Table 4). We thus demonstrated the absence of epitopes mapped to the C-terminal part of preS1 and to the N-terminal part of preS2. On the other hand, epitopes mapped to the Nterminal part of preS1 and the C-terminal part of preS2 were conserved. By means of electron microscopic examination of fractions of sucrose gradient, we found the presence of modified morphological forms of viral particles. This leads to the suggestion that HBV-DNA with preS rearrangements might be encapsidated in such particles. The identified 183-bp preS1 deletion eliminates the sequence coding for the overlapping genes of the

562

GERKEN

ET AL

EB

50

MO

1

2

Q

3 to

3 Y I3

to0.6-

4553

bp

Cl330

5

10

PRE Sl ,F.K?j,

I.

b:

I 15 2% Fracl~on number

.I

10

760

Ax) 5

10

15 x) Fraction number

HBS lF3920) 10 0) PRESZlFl24~ (A A)

FIG. 3. Characterization of HBV antigens activities. (B) Subsequent preS2 probe (MD37). EB. ethidium (+), HBV particles isolated from a sucrose gradient (shown in A). Bar

particles. (A) lsopycnic sucrose gradient centrifugation: preS1 (F35.25) preS2 (F124), and PCR analysis of fraction 8 (lane 1) and 9/l 0 (lane 2) of sucrose gradient using preS primers bromide staining; SB, Southern blot. Negative control (-), PCR mix without DNA template; viremic HBsAg positive carrier with normal HBV. (C) Electron microscopic examination of marker represents 50 nm; single arrow indicates HBV-defective forms, i.e.. full (a) and empty(b)

preSZ/S promoter and of the spacer region of the endogenous polymerase (Miller et al., 1989). Recent in vitro studies of DHBV mutants have shown that the P-gene spacer region appears not to be essential for viral replication (Bartenschlager el al., 1988). The preS2 promotor positively directs the synthesis of mRNA for MHBs (GP 33/36) and SHBs (p24/GP27) (Masuda et al., 1990; Raney et al., 1990). A detailed deletion analysis showed that sequences located inside the preS1 deleted fragment contain a regulatory sequence which modulates the preS2/S promoter activity (Antonucci, 1989; Raney et al., 1989). A number of recent investigations has been concerned with the role of preS1 and preS2 antigens. There is considerable evidence indicating that these proteins are of importance in the clearance of and the protection against HBV infection (Heermann et al., 1984; Alberti, 1990). This includes the kinetic of antipreS2 and anti-preS1 antibodies in acute and chronic infection as well as immunization experiments in chimpanzees using synthetic peptides (Neurath et al.,

HBsAg (F39.20) (MD1 6/l 9) and positive control fraction 9/l 0 of viral particles,

1986; ltoh et a/., 1986). B- and T-cell antigenic determinants have furthermore been characterized which elicit bypassing of nonresponsiveness in mice. Particularly, epitopes mapping to the C-terminal PreSl have been suggested as important immunogenes (Milich et al., 1988). PreS encoded proteins are major components of a hepatocyte binding site although the significance of in vitro binding assay of these particles to pHSA is still open to question (Alberti, 1990; Krone ef al., 1990); PreS2 encoded domains are involved in this binding (Machida et a/., 1984). However, corresponding DNA sequences are mutated in this defective virus. Consistent with these findings, there were no circulating antipreS2 antibodies (120-l 45) and no pHSA binding activity in the serum of our patient. On the other hand, the amino acid sequence corresponding to preS1 aa 21 to 47 has been retained in all 12 clones examined. This domain has been shown to mediate attachment of the large envelope protein to Hep G2 cells (Neurath et al., 1986; Petit et a/., in press). Furthermore, the large en-

PreS

Q

PROBE

pf&cj,

0

12

I

-

GENE

REARRANGEMENTS

3

4

5

-

-

I

I,

*

:

L

-

PROBE

FIG. 4. Defective HBV-DNA observed after PCR analysis with preS primers (MD1 6/MD19) and subsequent Southern blot using preS2 (MD37) and preS1 (MD38) probe alternatively. EB, ethidium bromide staining; negative control, PCR mix without template DNA; positive control, 100 ng DNA extracted from HBsAg positive liver tumor tissue containing one copy of HBV-DNA per cell. Lane 1: DNA extracted from serum. Lane 2: DNA extracted from PBMC. Lane 3: liver derived DNA. Lane 4: viral particles (fraction 8). Lane 5: viral particles (fraction 9/l 0).

velope protein has been shown to bind to human hepatocyte membranes (Pontino et al., 1989). Therefore, the modified virus might potentially be infectious. Using in vitro systems, it has recently also been shown that deletions in the preS/S gene of DHBV lead to an accumulation of covalently closed viral DNA forms in the cell (Summers et al., 1990). Previously, data suggested that such viral DNA molecules might be involved in persistence of hepadna viral infection (Wu et al., 1990). It is therefore tempting to speculate that in addition to modification in the immune clearance of the virus, the deletions observed in the preS gene might also modify the pattern of intracellular accumulation of the covalently closed HBV-DNA. One other important finding of our study was that the major PreSl deleted HBV DNA molecule was also detected in PBMC as a homogenous population. The different PCR profile observed in serum and PBMC indicates that this was not merely due to contamination of the cell preparation by the serum particles. PBMC have previously been shown to be a potential reservoir for HBV in humans (Romet-Lemonne et al., 1983; Yoffe et al., 1986; Pasquinelli et a/., 1986; Sureau et a/., 1986)

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and in the woodchuck model (Korba et al., 1986, I 988). After liver transplantation for HBV-related liver disease, they might moreover be implicated in a reinfection of the liver graft (Feray et al., 1990). Our study therefore supports that viral-lymphocyte interaction may play an important role in a selected immunosuppression and result in viral persistence. It is interesting to note that viral variants can be selectively generated in lymphocytes from persistently lymphocytic choriomeningitis virus (LCMV)-infected mice (Ahmed et al., 1988). In conclusion, we used polymerase chain reaction for the highly sensitive and specific detection of HBVDNA sequences present in serum in amounts undetectable in routine hybridization assay. We were thus able to identify a HBV defective variant with marked rearrangements in the preSl/S2 coding sequence. Deletions and insertions could potentially lead to an impairment in the immune clearance of the virus, while retaining a major hepatocyte binding site. It might also interfer with the recycling of covalently closed HBVDNA molecules in the liver cells. Generation of such defective viruses during the course of HBV infection might therefore be an important factor in the persistence of an HBV chronic carrier state with development of HCC as an endstage complication. Finally, the analysis of the epitopes retained or eliminated in such viral forms may provide in viva information complementing that obtained in animal or in vitro models concerning the relative importance of the various viral protein determinants in the control of the viral infection. ACKNOWLEDGMENTS This study was in part supported by grants from INSERM, ARC, LNC, CNAM, MRT, DFG (SFB 31 l), DAAD (PROCOPE 31 l/90), and NATO. G.G. was awarded by the Deutsche Gesellschaft fur Verdauungs und Stoffwechselkrankheiten, sponsored by Asche AG. We thank Prof. Wolfram H. Gerlich (Gl)ttingen. Germany) for providing us with the monoclonal antibodies MA 18/7 and Q 19/10 for quantitation of HBsAg level and for critical evaluation of the manuscript. We are grateful to C. Foster-Schorr for excellent technical assistance.

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