Journal of Hepatology, 1991; 13(Suppl. 4): S93496
s93
0 1991 Elsevier Science Publishers B.V. All rigtits resented. 0148~8278/91/$03.50 HEPAT 01029
G. Gerken’33, ‘INSERT
.
Kremsdorfl,
.A. Petit’, h.4. Manns3,
eyer 2x1
ikxh3nfelde3
and
Unit6 75, CHU Necker, ‘INSERM Unite’136, Ciamart , France, 31. Medioinische Klirrik ~rnl Poliklinik der Johannes Gutenberg Urtiversiriir,Mamz, Federal Republic of Germany and ‘lrrstitutPasiel.r, Paris, France
We have found a defective form of hepatitis B in an antie positive chronic HBsAg carrier with liver can Viral deletions were identified in tk reS coding region u PCR and sequencing. The presence of deleted was observed in seru liver tissue. To co e, rearrangements and deletions in t e preS gene would potentially lead to an impairment in viral clearance without affecting viral penetration in liver cells, possibly accounting for chronic HBV infection.
Chronic HBV infection leads to distinct clinical outcomes varying from asymptomatic sAg carrier state without HBV replication and normal liver function to symptomatic and progressive liver disease with high viremia and poor liver function (1). Epidemiologic evidence supports the close relationship between chronic persistent HBV infection, liver cirrhosis and the development of primary hepatocellular carcinoma ( cently, in vitro amplification of HBV-DNA by means of the polymerase chain reaction has become available as the most sensitive direct assay to monitor HBV replication (3). In this study we report on the discovery and molecular characterization of a preS gene-defective form of HBV in a HBsAg-positive patient with liver cancer.
A 40-year-old male of South-East Asian origin had a 5-year history of chronic hepatitis B before the diagnosis of hepatoceliular carcinoma (HCC). Because of the rapid progression to terminal liver failure, the patient died within 3 months. HBV markers were tested by commercial assays (Abbott Laboratories, Chicago, U.S.A.). HBV-DNA was determined using quantitative solution hybridization assay (Abbott Laboratories, Chicago,
I_J.$.A.). Detection of preS-1 and preS-2 proteins was performed by irnrn~lnob~~t and radioimmunoassay techniques as described in detail elsewhere (4). Viral particles were purified from the serum by isopycmic sucrose gradient and visualized by electronmicroscopical examination. DNA samples were prepared from serum, liver biopsy specimen, viral particles and peripheral blood mononuclear cells (PBMC). Polymerase chain reaction (PCR) assay was performed as described in detail elsewhere (5). The PCR product amplified using the preS primers MD16 and MD19 was purified and inserted into M13MP19 vector using standard cloning procedures (Ml3 cloning kit, Amersham, U.K.). The insert of different clones was sequenced by the didesoxy extension method (Sequenase Kit, USB, Cleveland, U.S.A.).
esdts
The low amount of HBsAg in serum of our patient was roughly comparable to the level usually observed in nonV-DNA was found negviremic carriers. While serum ative with the standard hybridization assay, PCR allowed V-DNA sequences. PC amplification of the amplification of the preS gene yielded DNA-
Correspondence: G. Gerken, M.D., INSERM Unit& 75 CHU Necker, 156 Rue de Vaugirard, F-75730 Paris Cedex 15,
France.
G. GERKEN et al.
s94 3177 (Fig. 2). Three out of nine clones showed additional rearrangements in the 5’ part of the preS-2 sequence. In three other clones, where the 183 bp deletion of preS-1 could not be identified, two clones showed preS-1 point mutations, small deletions and insertions. One clone was deleted in the 5’ part of the preS-2 region from nucleotide position 4 to 57. Analysis of serum viral proteins by immunoblot and radioimmunoassay showed loss of expression of epitopes recognized by preS monoclonal antibodies in the N-terminal part of preS-2 and in the C-terminal part of preS-1 (Fig. 3). After amplification of DNA extracted from P IWC with preS specific primers, one single PCR fragment could be visualized corresponding to the 183 bp deleted PCR product sequenced from serum particles (Fig. 4). Liver-derived DNA showed the presence of a similar mixture of deleted and undeleted preS-PCR fragments to the one previously obtained from serum and viral particles in our patient. Electronmicroscopical examinations revealed the presence of viral particles of approximately 60 nm which may correspond to hepatitis El defective virus with rearrangements in the preS region.
553 bp
LO Oh-
+
553 bp
083 -
Fig. 1, Detection of serum HBV-DNA by PCR using preS specific primers. Ethidium bromide stained agarose gel electrophoresis (EB) followed by Southern blotting (SB) using preS oligonucleotide probe. $, control, i.e., 100 ng DNA from liver tissue of a HBsAgpositive patient with HCC containing one copy HBV-DNA per cell. 8. control, i.e., PCR-mix without DNA. Lanes I-4, different chronic HBV carriers: lane 5, anti-HBe-positive HBsAg carrier with HCC.
fragments with altered migration consistent with deletions and mutations in this region (Fig. 1). PCR mapping and nucleotide sequencing of the preS region showed in 9 out of 12 clones a 183 bp in frame deletion in the 3’ part of the preS-1 gene from nucleotide position 2995 to
SW0
151
FEg. 2. Rearrangements in the preS gene of defective HBV after cloning and sequencing (clone 1-12).
Pre-S DEFBCTIVE
VIRUS IN CHRONIC I-IEPATI’ITS B
S95
F 5 2(94-107
W-125)
0
Deletion t-t&AS: + 1 1000 (Subtype
adr)
/
F 124(140-150)
@
tiBeAg: -
anti -t-t&.: anti-WBe:+ anti-HEc : + anti Pre S2 : HSW-DNA:Clinic’Chronic
Fig. 3.
hepatitis.
HCC
Seroimmunological and molecular characterization of a defective hepatitis B virus with rearrangements in the preS gene sequence.
PRE S2
c PRE St
Fig. 4. Detection of preS-1 defective HBV variant in PBMC after PCR analysis (EB) and subsequent Southern blot using preS-2- (B) and pre-S-l- (C) specific oligonucleotide probe subsequently. Lane 1, DNA sample from asymptomatic HBsAg-carrier; lane 2, PBMC from the HBsAg (@) patient with HCC; lane 3, wild type HBVDNA from HBV-positive serum; lane 4, positive control, i.e., 100 ng DNA from liver tissue of an HBsAg-positive patient with HCC containing one copy HBV-DNA per cell; negative controls, i.e., PCR-mix without DNA and normal DNA extracted from human leucocytes, respectively.
A defective form of V could be identified in a patient whose chronic V infection rapidly evolved into hepatocellular carcinoma within 5 years. After cloning and sequencing a large 183 bp in frame preSldeletion, presently seen in 9 out of 12 clones, could be detected. These findings were confirmed by the application of monoclonal preS-antiiiodies. We thus demonstrated the absence of epitopes mapped to the C-terminal part of preS-l- and the N-terminal part of the preS-2 region. The identified 183 bp preS-l-deletion eliminates the sequence coding for the overlapping genes of the pre§-2 promotor and of the spacer region of the endqenous polymerase (6). However, epitopes mapping to the C-terminal preS-1 region have been suggested as important immunogenetics (7). PreS encoded proteins are major components of a hepatocyte binding site, although the significance of in vitro binding assay of these particles to poly-HSA is still open to encoded proteins are involved in the dire to liver cells. However, corresponding TXA sequences are mutated in this defective virus. On the other hand, the amino acid sequence corresponding to the N-terminal part of preS-1 has been retained in all clones examined. Therefore, the modified virus might potentially be infectious. Another important finding of our studv was that the major preS-l deleted HBV-DNA molecule was also detected in PBMC as a homogenous population. PBMC have previously been shown to be a potential reservoir for HBV in humans (8). Our study therefore supports the proposition that viral lymphocyte detection may play an important role in viral persistence.
G. GERKEN et al.
Acknowledgements This study was in part supported by grants from INSERM, DFG (SFB 311), DAAD (PROCOPE 311-90).
G. Gerken was the recipient of a stipendium from the Deutsche Gesellschaft fiir Verdauungs- und Stoffwechselkrankheiten, sponsored by Asche AG, Hamburg.
References 1 Hoofnagle JH, Shafritz DA, Poppe H. Chronic type B hepatitis in the healthy carrier state. Hepatology 1987; 7: 758-63. 2 Beasly RP, Hawan LY, Linn CC, Yen CS. Hepatocellular carcinoma and HBV. A prospective study of 21707 men in Taiwan. Lancet 1961; ii: 1129-33. 3 Ticrce V, Nakayima E. Kremsdorf B, et al. Hepatitis B seronegative blood can transmit hepatitis B. Polymerase chain reaction evidence of mutant viruses not currently detected. Lancet 1988; ii: 1273-6. 4 Gerken G, Manns M, Gerlich WH, Hess G, Meyer zum BUschenfelde K-H. Immunoblot analysis of viral surface prateins in serum
5
6
7
8
and liver of patients with chronic hepatitis B virus infection. J Med Virol 1989; 29: 261-5. Gerken G, Paterlini P, Manns M, et al. Assay of hepatitis B viru, DNA by polymerase chain reaction and its relationship to pre-S and S encoded viral surface antigens. Hepatoiogy 1991; 13: 15866. Gerken G, Kremsdorf B, Cape1 F, et al. Hepatitis B defective virus with rearrangements in the pre-S gene during chronic HBV infection. Virology 1991; in press. Alberti A. Gerlich W, Heermann KH. Pontisso P. Nature and display of hepatitis B virus envelope proteins and the humoral immune response. Spr Sem Immunpathol 1990; 12: 5-23. Oldstone MBA. Viral persistence. Rev Cell 1989; 56: 517-20.
s93
0 1991 Elsevier Science Publishers B.V. All rigtits resented. 0148~8278/91/$03.50 HEPAT 01029
G. Gerken’33, ‘INSERT
.
Kremsdorfl,
.A. Petit’, h.4. Manns3,
eyer 2x1
ikxh3nfelde3
and
Unit6 75, CHU Necker, ‘INSERM Unite’136, Ciamart , France, 31. Medioinische Klirrik ~rnl Poliklinik der Johannes Gutenberg Urtiversiriir,Mamz, Federal Republic of Germany and ‘lrrstitutPasiel.r, Paris, France
We have found a defective form of hepatitis B in an antie positive chronic HBsAg carrier with liver can Viral deletions were identified in tk reS coding region u PCR and sequencing. The presence of deleted was observed in seru liver tissue. To co e, rearrangements and deletions in t e preS gene would potentially lead to an impairment in viral clearance without affecting viral penetration in liver cells, possibly accounting for chronic HBV infection.
Chronic HBV infection leads to distinct clinical outcomes varying from asymptomatic sAg carrier state without HBV replication and normal liver function to symptomatic and progressive liver disease with high viremia and poor liver function (1). Epidemiologic evidence supports the close relationship between chronic persistent HBV infection, liver cirrhosis and the development of primary hepatocellular carcinoma ( cently, in vitro amplification of HBV-DNA by means of the polymerase chain reaction has become available as the most sensitive direct assay to monitor HBV replication (3). In this study we report on the discovery and molecular characterization of a preS gene-defective form of HBV in a HBsAg-positive patient with liver cancer.
A 40-year-old male of South-East Asian origin had a 5-year history of chronic hepatitis B before the diagnosis of hepatoceliular carcinoma (HCC). Because of the rapid progression to terminal liver failure, the patient died within 3 months. HBV markers were tested by commercial assays (Abbott Laboratories, Chicago, U.S.A.). HBV-DNA was determined using quantitative solution hybridization assay (Abbott Laboratories, Chicago,
I_J.$.A.). Detection of preS-1 and preS-2 proteins was performed by irnrn~lnob~~t and radioimmunoassay techniques as described in detail elsewhere (4). Viral particles were purified from the serum by isopycmic sucrose gradient and visualized by electronmicroscopical examination. DNA samples were prepared from serum, liver biopsy specimen, viral particles and peripheral blood mononuclear cells (PBMC). Polymerase chain reaction (PCR) assay was performed as described in detail elsewhere (5). The PCR product amplified using the preS primers MD16 and MD19 was purified and inserted into M13MP19 vector using standard cloning procedures (Ml3 cloning kit, Amersham, U.K.). The insert of different clones was sequenced by the didesoxy extension method (Sequenase Kit, USB, Cleveland, U.S.A.).
esdts
The low amount of HBsAg in serum of our patient was roughly comparable to the level usually observed in nonV-DNA was found negviremic carriers. While serum ative with the standard hybridization assay, PCR allowed V-DNA sequences. PC amplification of the amplification of the preS gene yielded DNA-
Correspondence: G. Gerken, M.D., INSERM Unit& 75 CHU Necker, 156 Rue de Vaugirard, F-75730 Paris Cedex 15,
France.
G. GERKEN et al.
s94 3177 (Fig. 2). Three out of nine clones showed additional rearrangements in the 5’ part of the preS-2 sequence. In three other clones, where the 183 bp deletion of preS-1 could not be identified, two clones showed preS-1 point mutations, small deletions and insertions. One clone was deleted in the 5’ part of the preS-2 region from nucleotide position 4 to 57. Analysis of serum viral proteins by immunoblot and radioimmunoassay showed loss of expression of epitopes recognized by preS monoclonal antibodies in the N-terminal part of preS-2 and in the C-terminal part of preS-1 (Fig. 3). After amplification of DNA extracted from P IWC with preS specific primers, one single PCR fragment could be visualized corresponding to the 183 bp deleted PCR product sequenced from serum particles (Fig. 4). Liver-derived DNA showed the presence of a similar mixture of deleted and undeleted preS-PCR fragments to the one previously obtained from serum and viral particles in our patient. Electronmicroscopical examinations revealed the presence of viral particles of approximately 60 nm which may correspond to hepatitis El defective virus with rearrangements in the preS region.
553 bp
LO Oh-
+
553 bp
083 -
Fig. 1, Detection of serum HBV-DNA by PCR using preS specific primers. Ethidium bromide stained agarose gel electrophoresis (EB) followed by Southern blotting (SB) using preS oligonucleotide probe. $, control, i.e., 100 ng DNA from liver tissue of a HBsAgpositive patient with HCC containing one copy HBV-DNA per cell. 8. control, i.e., PCR-mix without DNA. Lanes I-4, different chronic HBV carriers: lane 5, anti-HBe-positive HBsAg carrier with HCC.
fragments with altered migration consistent with deletions and mutations in this region (Fig. 1). PCR mapping and nucleotide sequencing of the preS region showed in 9 out of 12 clones a 183 bp in frame deletion in the 3’ part of the preS-1 gene from nucleotide position 2995 to
SW0
151
FEg. 2. Rearrangements in the preS gene of defective HBV after cloning and sequencing (clone 1-12).
Pre-S DEFBCTIVE
VIRUS IN CHRONIC I-IEPATI’ITS B
S95
F 5 2(94-107
W-125)
0
Deletion t-t&AS: + 1 1000 (Subtype
adr)
/
F 124(140-150)
@
tiBeAg: -
anti -t-t&.: anti-WBe:+ anti-HEc : + anti Pre S2 : HSW-DNA:Clinic’Chronic
Fig. 3.
hepatitis.
HCC
Seroimmunological and molecular characterization of a defective hepatitis B virus with rearrangements in the preS gene sequence.
PRE S2
c PRE St
Fig. 4. Detection of preS-1 defective HBV variant in PBMC after PCR analysis (EB) and subsequent Southern blot using preS-2- (B) and pre-S-l- (C) specific oligonucleotide probe subsequently. Lane 1, DNA sample from asymptomatic HBsAg-carrier; lane 2, PBMC from the HBsAg (@) patient with HCC; lane 3, wild type HBVDNA from HBV-positive serum; lane 4, positive control, i.e., 100 ng DNA from liver tissue of an HBsAg-positive patient with HCC containing one copy HBV-DNA per cell; negative controls, i.e., PCR-mix without DNA and normal DNA extracted from human leucocytes, respectively.
A defective form of V could be identified in a patient whose chronic V infection rapidly evolved into hepatocellular carcinoma within 5 years. After cloning and sequencing a large 183 bp in frame preSldeletion, presently seen in 9 out of 12 clones, could be detected. These findings were confirmed by the application of monoclonal preS-antiiiodies. We thus demonstrated the absence of epitopes mapped to the C-terminal part of preS-l- and the N-terminal part of the preS-2 region. The identified 183 bp preS-l-deletion eliminates the sequence coding for the overlapping genes of the pre§-2 promotor and of the spacer region of the endqenous polymerase (6). However, epitopes mapping to the C-terminal preS-1 region have been suggested as important immunogenetics (7). PreS encoded proteins are major components of a hepatocyte binding site, although the significance of in vitro binding assay of these particles to poly-HSA is still open to encoded proteins are involved in the dire to liver cells. However, corresponding TXA sequences are mutated in this defective virus. On the other hand, the amino acid sequence corresponding to the N-terminal part of preS-1 has been retained in all clones examined. Therefore, the modified virus might potentially be infectious. Another important finding of our studv was that the major preS-l deleted HBV-DNA molecule was also detected in PBMC as a homogenous population. PBMC have previously been shown to be a potential reservoir for HBV in humans (8). Our study therefore supports the proposition that viral lymphocyte detection may play an important role in viral persistence.
G. GERKEN et al.
Acknowledgements This study was in part supported by grants from INSERM, DFG (SFB 311), DAAD (PROCOPE 311-90).
G. Gerken was the recipient of a stipendium from the Deutsche Gesellschaft fiir Verdauungs- und Stoffwechselkrankheiten, sponsored by Asche AG, Hamburg.
References 1 Hoofnagle JH, Shafritz DA, Poppe H. Chronic type B hepatitis in the healthy carrier state. Hepatology 1987; 7: 758-63. 2 Beasly RP, Hawan LY, Linn CC, Yen CS. Hepatocellular carcinoma and HBV. A prospective study of 21707 men in Taiwan. Lancet 1961; ii: 1129-33. 3 Ticrce V, Nakayima E. Kremsdorf B, et al. Hepatitis B seronegative blood can transmit hepatitis B. Polymerase chain reaction evidence of mutant viruses not currently detected. Lancet 1988; ii: 1273-6. 4 Gerken G, Manns M, Gerlich WH, Hess G, Meyer zum BUschenfelde K-H. Immunoblot analysis of viral surface prateins in serum
5
6
7
8
and liver of patients with chronic hepatitis B virus infection. J Med Virol 1989; 29: 261-5. Gerken G, Paterlini P, Manns M, et al. Assay of hepatitis B viru, DNA by polymerase chain reaction and its relationship to pre-S and S encoded viral surface antigens. Hepatoiogy 1991; 13: 15866. Gerken G, Kremsdorf B, Cape1 F, et al. Hepatitis B defective virus with rearrangements in the pre-S gene during chronic HBV infection. Virology 1991; in press. Alberti A. Gerlich W, Heermann KH. Pontisso P. Nature and display of hepatitis B virus envelope proteins and the humoral immune response. Spr Sem Immunpathol 1990; 12: 5-23. Oldstone MBA. Viral persistence. Rev Cell 1989; 56: 517-20.
