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Active and inactive replication of hepatitis B virus deoxyribonucleic acid in chronic liver disease
GASTROENTEROLOGY
1985;89:610-6
Active and Inactive Replication of Hepatitis B Virus Deoxyribonucleic Acid in Chronic Liver Disease OSAMU YOKOSUKA, KUNIO OKUDA First Department
MASAO
of Internal Medicine,
OMATA,
Chiba University
Little is known about the replicative forms of hepatitis B virus (HBV) DNA in the liver in chronic liver disease. We therefore analyzed HBV DNA and the changes in DNA signals after endonuclease digestion in liver tissues taken from 64 patients with hepatitis B surface antigen-positive chronic liver disease. The “active” replication pattern, which included various replicative intermediates, was seen in 36 of 38 (95%) hepatitis B e antigen-seropositive patients. This pattern was also found in 5 of 26 (19%) hepatitis B e antigen-seronegative patients who showed the highest mean serum alanine aminotransferase level (403 ? 184 mU/ml]. Most of them had advanced liver disease. Episomal viral DNA of an “inactive” type having only the supercoiled form was found in 3 patients; they showed the lowest mean serum alanine aminotransferase level (27 t 7 mU/ml] and only mild liver disease. As with duck HBV infection, episomal replicative forms of human HBV could be resolved by Southern blot analysis and seem to have clinical implications in human HBV infection.
FUMIO IMAZEKI, School of Medicine,
and
Chiba, Japan
the minus strand of DNA, and synthesis of the plus strand by DNA polymerase reaction. In an experimental transmission study, Mason et al. (3) showed formation of supercoiled viral DNA that preceded the reverse transcription phase of viral DNA, suggesting the importance of this particular species in the initiation of infection. Studies of human liver tissue by Southern blot hybridization have demonstrated integration of HBV DNA into genomic DNA in neoplastic (4-6) and nonneoplastic liver disease (7-9). Information on episomal replicative forms in human liver disease, however, is still limited. We therefore analyzed episomal HBV DNA by the method of Southern blot hybridization and studied changes after endonuclease digestion in liver tissues taken from 64 patients with hepatitis B surface antigen-positive chronic liver disease, and correlated the patterns with clinical features. Materials and Methods Patients
Recently, Summers and Mason proposed a replicative pathway of duck hepatitis B virus (DHBV) DNA, a virus that shares many properties with human HBV (1,2). They demonstrated the presence of a pregenomic RNA intermediate, reverse transcription of
Received November Address
requests
13, 1984. Accepted March 4, 1985. for reprints to: Masao Omata, M.D., First
Department of Medicine, Chiba, Japan 280.
Chiba University
School of Medicine,
This research was supported in part by Japanese Ministry of Education grant (B) 58480215, a grant from the Japan Medical Research Foundation, and a grant from the Japanese Ministry of Health and Welfare. The authors thank Dr. Jesse Summers (Fox Chase, Pennsylvania] for his generous gift of cloned HBV DNA and helpful discussion. 0 1985 by the American Gastroenterological Association
0016-5085/85/$3.30
Percutaneous liver biopsy specimens were taken from 64 patients with chronic liver disease who were seen at the First Department of Medicine, Chiba University. They had been seropositive for hepatitis B surface antigen for at least 6 mo before liver biopsy. None of them had received antiviral treatments or immunosuppressive agents. The ages of patients ranged from 19 to 70 yr (mean 34 yr). Of the 64 patients, 38 were seropositive for hepatitis B e antigen (HBeAg), 7 were negative for both HBeAg and hepatitis B e antibody (anti-HBe), and 19 were positive for anti-HBe. The demographic data on these patients are shown in Table 1. Two needle cores were taken from all patients with a Tru-Cut biopsy needle. One core was processed for routine histologic examination, and the other was immediately frozen and stored at -80°C.
Abbreviation
used in this paper:
DHBV, duck hepatitis
B virus.
September
Table
1985
ACTIVE AND INACTIVE
1. Demographic
Data on 64 Patients
Sex (male/female) Age (mean + SD, yr) Liver histology Chronic persistent hepatitis Chronic active hepatitis Cirrhosis HBeAg status HBeAg (+)/anti-HBe (-) HBeAg (-)/anti-HBe (-) HBeAg (-)/anti-HBe (+) Serum alanine aminotransferase mU/ml: normal 5 40)
Studied 51113 34.2 5 11.1
33 20 11 38 7 19 146 + 204
(mean + SD,
Anti-HBe, hepatitis B e antibody: HBeAg, hepatitis B e antigen: +. positive: -, negative.
Isolation Liver
of Deoxyribonucleic
Acid
From
the
REPLICATION
OF HBV DNA
applied as a sensitivity as well as a size marker. Electrophoresed A-phage DNA digested with Hind III and endlabeled with 32P-dCTP using T,-polymerase was used as another size marker. Autoradiograms were analyzed by a Sakura PDS 15 densitometer (Sakura Co., Tokyo, Japan).
Restriction Endonuclease Deoxyribonucleic Acid
Analysis
of Liver
Ten micrograms of nucleic acid extracted from liver was digested with 10 U of Hind III or Eco RI for 2 h at 37°C with a mixture of 0.02 M Tris-HCl (pH 7.5), 0.05 M NaCl, 0.01% triton, and 1 mM dithiothreitol. To study the kinetics of Eco RI digestion, 20 Fg of liver DNA extracted with a hot phenol-SDS method without prior pronase treatment was digested with 20 U of Eco RI for 15, 30, 45, 60, 90, 120, and 180 min.
A biopsy core (10-25 mg) was homogenized in a mixture of 0.5 ml of 10 mM Tris-HCl (pH 7.4) and 10 mM
ethylenediaminetetraacetic acid (EDTA) by a loose-fitting Dounce homogenizer. To this was added 0.5 ml of 0.2 M NaCl, 0.02 M Tris-HCl (pH 7.4), 2 mM EDTA, 1 mg/ml pronase, and 1% sodium dodecyl sulfate (SDS) and the mixture was incubated for 30 min at 37°C. Extraction of DNA was done twice with 1 ml phenol/chloroform (l:l), and the aqueous phase was then precipitated and washed three times with absolute ethanol chilled to -20°C. The nucleic acid pellets were resuspended in 100 ~1 of a mixture of 5 mM Tris-HCl (pH 7.4) and 1 mM EDTA. The average yield of total nucleic acids was 7.2 t 3.4 pg/mg
Serum Hepatitis B Surface Hepatitis B e Antigen
Antigen
kb
E
uH
’*
-
9.4
-
6.7
-
4.4
4
3.2
-
2.3
-
2.0
??
Analysis of Hepatitis B Virus Deoxyribonucleic Acid Ten micrograms of nucleic acids from liver were electrophoresed on 1.0% or 1.5% agarose gel (Seakam Co., Rockland, Me.) and transferred to nitrocellulose filter (Schleicher & Schuell, Dassel, Federal Republic of Germany) by the Southern blot technique (11). The DNA immobi-
lized on nitrocellulose filter was hybridized with ‘*Plabeled cloned HBV DNA inserted into pBH-20 plasmid (12). The HBV DNA insert was repurified, and the excised HBV DNA segment was used for labeling. The labeling of DNA with “‘P-deoxycytidine triphosphate (dCTP) was done by the nick translation method (13). The specific activity of the HBV DNA probe was 3-9 X lOa cpm/pg DNA. After hybridization, the filter paper was washed twice with 2 x SSC (0.15 M NaCl, 0.015 M sodium citrate)/ 0.1% SDS at room temperature, and washed again with 0.1 X SSCiO.l% SDS at 56°C for 30 min each. After washing, was dried and autoradiographed with (Eastman Kodak Co., Rochester, N.Y.) Du Pont lightning (DuPont Co., Wilscreen at -70°C for l-3 days. Three DNA in 2 pg of calf thymus DNA was
and
Hepatitis B surface antigen was assayed by reversed passive hemagglutination, and HBeAg by radioimmunoassay (Abbott Laboratories, North Chicago, Ill.).
tissue (mean ? SD). In selected samples, a hot phenol-SDS extraction procedure before pronase treatment was used to isolate proteinfree DNA as described by Ruiz-Opazo et al. (10).
nitrocellulose filter Kodak X-Omat film in the presence of mington, Del.) plus picograms of HBV
611
.
Figure
*
1. “Inactive” free hepatitis B virus DNA on 1% agarose gel. Supercoiled (SC) form at 2.0 kb in lane LJ (uncut DNA] shifted to a band at 3.2 kb after Eco RI digestion (Jane E), but remained tion (Jane H).
unchanged
after Hind III diges-
612
Figure
YOKOSUKA
ET AL.
2. Digestion kinetic study of the 2.0-kb form (lane 1) with Eco RI. Liver DNA was extracted with the hot phenol-SDS procedure, and electrophoresed on 1.5% agarose gel. Liver DNA (20 pg) was digested with 20 U of Eco RI for 15 min (lane 2), 30 min (lane 31, 45 min (lane 4). 60 min (lane 51, 90 min (lane 6), 120 min (lane 7), and 180 min (lane 8). After 15 min of digestion, two bands (4.0 kb and 3.2 kb) started to appear. After 180 min, the 2.0-kb form was complete-
GASTROENTEROLOGY
1’2
3
4
56
Vol. 89. No. 3
7
?
RC L SC
ly converted to a 3.2-kb form. AC, relaxed circular; L, linear duplex; SC, supercoiled.
smears from 2.8 kb to 2.0 kb, and below 1.35 kb, and a band at 2.0 kb on 1.0% agarose gel
Results
included
Detection of Episomal Hepatitis B Virus Deoxyribonucleic Acid in Liver Tissue
(Figure 3A). A densitogram of the autoradiogram is shown in Figure 3B. Because the 2.0-kb form was not distinct in the active form (probably due to smears from above), hot phenol-SDS extraction was performed to isolate protein-free DNA. This revealed a distinct band at 2.0 kb with uncut liver DNA, and a 3.2-kb form after Eco RI digestion (Figure 4). With phenol-chloroform extraction, a band appeared at 3.2 kb after Eco RI digestion (Figure 3A). These results indicated that the supercoiled form was present in the active form, but was simply obscured by other replicative intermediate signals. This situation was somewhat similar to that reported with serum (10). As all the radioactive signals were converted after heat treatment to a smear below 1.35 kb, which was resistant to Eco RI (Figure 3A) but was sensitive to Sl nuclease, the smear below 1.35 kb appeared to represent single-stranded DNA. The smear from 2.8 to 2.0 kb was compatible with partially double-stranded DNA (1,2,14). Thus, the active form included various replicative intermediates, and indicated active on-going viral replication.
Two Southern blot hybridization patterns of free viral DNA were recognized on 1.0% agarose gel. One pattern showed a single band at 2.0 kb with uncut liver DNA (Figure 1). After digestion with Hind III, which has no cutting site within HBV DNA, the band remained at 2.0 kb, whereas it moved to 3.2 kb after digestion with Eco RI, which recognizes one site within HBV DNA (Figure 1). A digestion kinetic study of the 2.0-kb form using 20 U of Eco RI and 1.5% agarose gel revealed faint bands at 4.0 kb and 3.2 kb after 15 min of digestion. One band at 4.0 kb became weaker, and one at 3.2 kb became more intense after longer periods of digestion. After digestion for 3 h, only one band was recognized at 3.2 kb (Figure 2). Digestion kinetics of DNA as studied with Sl nuclease showed a similar conversion of the 2.0kb form to a 3.2-kb form. These findings suggested that the 2.0-kb form was supercoiled HBV DNA and the 4.0-kb form was relaxed circular fully doublestranded DNA. A band at 3.2 kb corresponded to a cloned, fully double-stranded linear HBV DNA. These findings in human liver were compatible with previous reports in ducks (2,3) and in chimpanzees (10). Summers and Mason postulated that supercoiled DNA would act as a template for viral replication when infection was initiated in the duck liver (1,3). Because the sole presence of the supercoiled form seems to indicate that no active transcription of viral DNA is occurring, despite the presence of the template, we tentatively designated this Southern blot pattern as “inactive.” In contrast to inactive replication, various radioactive signals were observed in an “active” form that
Patterns of Viral Replication Features
and Clinical
Of the 38 HBeAg-seropositive patients, 36 showed the active pattern with various replicative forms, and none showed the inactive pattern (Table 2). Of the 7 patients negative for HBeAg and anti-HBe, 1 patient showed the active and 1 patient showed the inactive pattern. Of the 19 patients positive for anti-HBe, 4 (21%) and 2 (11%) showed the active and the inactive pattern, respectively (Table 2). (95%)
September
1985
ACTIVE
AND
INACTIVE
A
REPLICATION
OF HBV DNA
613
r-4 (1)
w
(2)
4.4 Figure
3.2
2.32.0
1.35
kb
3. A. “Active” form of hepatitis B virus DNA on 1.0% agarose gel electrophoresis. Various replicative forms including singlestranded (SS), partially double-stranded (PDS), and supercoiled (SC) DNA are present. After Eco RI digestion (lane E). a band at 3.2 kb (relaxed circular or linear duplex, or both) emerged, and the radioactive signal at 2.0 kb became less than that in lane Lr (uncut DNA). The change may be better appreciated on densitograms [Figure 3B). Heating converted all double-stranded DNAs to single-stranded DNAs (lane H). B. Densitograms of uncut (lane U), Eco RI-digested (lane E), and heated (lane H) autoradiograms in A corroborated well the changes observed visually. They showed conversion of a peak at 2.0 kb [w, top (l)], which corresponded to the supercoiled form, to a peak at 3.2 kb [o, middle (2)], which corresponded to relaxed circular or linear duplex. or both, after Eco RI digestion. Heating converted all double-stranded DNAs to single-stranded DNAs [bottom
(311
Of the 38 HBeAg-positive patients, 36 with the active pattern had high serum alanine aminotransferase levels (ALT) (mean 183 mu/ml), whereas 2 patients without viral DNA in liver tissue showed near normal ALT values (mean 55 mu/ml) (Table 3). Of the 26 patients who were either negative for Table
2.
Relationship Between Serum Hepatitis B e Antigen/Hepatitis B e Antibody and Molecular State of Hepatitis B Virus Deoxyribonucleic Acid in the Liver No.
HBeAg (-t )/anti-HBe HBeAg (-)/anti-HBe HBeAg (-)/anti-HBe
(-1 (-) (+)
Total Anti-HBe, hepatitis B e antibody; positive: -, negative.
Active
Inactive
38 7 19
36 1 4
0 1 2
64
41
3
HBeAg,
hepatitis
B e antigen:
HBeAg or positive for anti-HBe, 5 patients with active replication showed the highest mean ALT values (403 mu/ml) and advanced liver disease (chronic active hepatitis or cirrhosis, or both, in 4 patients), whereas 3 patients with the inactive pattern showed normal ALT values (mean 27 mu/ml) and mild liver disease (chronic persistent hepatitis in 3 patients) (Table 3). Eighteen patients without demonstrable viral DNA in the liver showed near normal ALT values (mean 57 mu/ml).
Discussion
f,
Previous Southern blot hybridization analyses in human liver disease put much emphasis on integration of viral DNA into genomic DNA, but little on replicative intermediates of episomal viral DNA. Summers and Mason proposed the following infec-
614
YOKOSUKA
Figure
ET AL.
GASTROENTEROLOGY
4. Southern blot analysis of liver DNA extracted by the hot phenol-SDS procedure without prior pronase treatment revealed a distinct band at 2.0 kb. In this patient, the “active” form with various replicative forms was shown by the ordinary phenol-chloroform extraction method with prior pronase treatment. A band at 2.0 kb (supercoiled) (Jane 1, uncut] moved to 3.2 kb after Eco RI digestion (lane 2).
3.
Demography
and Their
of Patients
Laboratory
With an Active Data
and Inactive
Hepatitis
B Virus Deoxyribonucleic
HBeAg-positive Active
Number Age W SALT (mu/ml) Histology CPH CAH/cirrhosis
36 30 (83%) 31.9 + 10.6" 183 2 236
Acid Replication
Active
_
5 5 (lOO"~,) 38.0 + 9.4 403 2 184"'
1 (50%) 1(50%) persistent
hepatitis:
inactive
_
+
18 13 (72%) 36.4 k 10.2 57 f 61"
2 (67%) 39.7 + 18.8 27 2 7'
+
2 1 (5041) 39.0 L 5.0 55 2 9
19 (53%) 17 (48%)
CAH. chronic active hepatitis: CPH, chronic ” Mean + SD. ‘I p < 0.001.' p < 0.01.
3
HBeAg-negative
+ Male (%)
89, No.
initiation and persistence of HBV infection in the chimpanzee. Mason et al. (3) demonstrated appearance of supercoiled viral DNA before the synthesis of other replicative forms when DHBV infection was initiated in embryonic duck liver. Recently, we observed similar sequential appearances of replicative intermediates in l-day old ducklings (unpublished data). In the current study, two hybridization patterns of HBV DNA in the liver emerged; one with supercoiled viral DNA, indicating the presence of the template alone, and the other with replicative intermediates, indicating active replication of HBV DNA. Of the 38 HBeAg-seropositive patients, 36 showed the “active” pattern with high serum (95%) ALT levels. These results were concordant, because previous studies demonstrated that serum HBeAg was closely associated with high infectivity and active liver disease (15-20). Two patients with HBeAg in serum, but without detectable viral DNA, however, showed almost normal ALT values. Five patients who had active patterns, despite seronegativity for HBeAg, showed the highest mean ALT level (403 mu/ml). In contrast, 3 patients with the “inactive” form showed very low serum ALT levels (Table 3). The former had more advanced liver diseases, whereas all 3 patients of the latter group showed chronic persistent hepatitis histologically [Table 3). Thus, our results suggested that episomal viral DNA could be divided into two types, and that the active form was closely related to on-going viral replication and active liver disease, whereas the inactive form was related to minimal viral replication and quiescent liver disease. A long-term followup study is currently under way to see the relation of active viral replication and prognosis in such patients. Recently, Fowler et al. (14) studied replicative intermediates of viral DNA in a human HBV carrier, and failed to show the supercoiled form of HBV DNA. Our study, which included a digestion kinetic study of DNA using Eco RI and hot-phenol extrac-
tious cycle of DHBV DNA (1-3): (a] initiation of the cycle by transcription of pregenomic full-length plus-strand RNA from supercoiled or integrated viral DNA, (b) reverse transcription of minus-strand DNA from the RNA intermediate, (c) synthesis of plusstrand DNA with endogenous DNA polymerase reaction, using minus-strand DNA as a template, and (d) packaging and exportation of partially doublestranded DNA. Ruiz-Opazo et al. (10) suggested that the supercoiled form might play an important role in the Table
Vol.
9 (50%) 9 (50%,)
1 (20%) 4 (80%) HBeAg.
hepatitis
B e antigen:
SALT.
serum
alanine
3
3 [ 1OO%] 0 (0%) aminotransferase.
September 1985
ACTIVE
tion of liver DNA, supported the previous results in chimpanzees (10)and ducks (31, and clearly indicated the presence of the supercoiled form in humans as well. Gerlich and Robinson (21) described a protein covalently attached to the 5’ terminus of a complete HBV DNA strand that could be extracted into the organic solvent phase with phenol before, but not after, proteinase digestion. Thus, in selected samples we used an extraction procedure without prior proteinase treatment to remove DNA-protein complex, and to extract protein-free DNA. A hot phenol-SDS procedure without prior proteinase treatment gave a discrete band at 2.0 kb (Figure 4), whereas a procedure with pronase treatment gave a variety of viral forms that obscured the band at 2.0 kb (Figure 3A). Failure to reveal the supercoiled form was probably due to the presence of other radioactive signals that might submerge a discrete band, or mechanical nicking of the DNA or nuclease contamination, or both. Recently, we observed that the supercoiled form persisted in patients during treatment with large doses of recombinant a-interferon, whereas other replicative forms disappeared in posttreatment biopsy specimens [unpublished observation). It was later found that active virus replication recurred in these patients. Our data in humans strongly suggest that the supercoiled form plays a key role in initiation and maintenance of the carrier state as suggested in animals (3,10).
I .O% 3.2kb
m
Relaxed Circular
gel -
Linear
+Q
2.8kb
1
2.0kb
_
-
Partially Double Stranded
+OJ
m
Supercoiled
I .35kb .13-
-
Single Stranded
f
---J-J Figure
5. Schematic representation of bands and smears obtained by blot hybridization on 1.0% agarose gel and their corresponding replicative forms.
AND
INACTIVE
REPLICATION
OF HBV DNA
615
As with DHBV replication, episomal replicative forms could be resolved by Southern blot analysis. Summarizing our data and that of others (1,2,10), a schema given in Figure 5 may be proposed to correlate the radioactive bands and smears on 1.0% agarose gel electrophoresis with various viral DNA forms.
References 1. Summers J, Mason WS. Replication of the genome of a hepatitis B like virus by reverse transcription of an RNA intermediate. Cell 1982;29:403-15. 2. Mason WS, Aldrich C, Summers 1, et al. Asymmetrical replication of duck hepatitis B virus DNA in liver cells: free minus-strand DNA. Proc Nat1 Acad Sci IJSA 1982;79:39974001. 3. Mason WS, Halpern MS, England JM, et al. Experimental transmission of duck hepatitis B virus. Virology 1983:131: 375-84. 4. Chakraborty PR, Ruiz-Opazo N. Shouval D, et al. Identification of integrated hepatitis B virus DNA and expression of viral DNA in an HBsAg-producing human hepatocellular carcinoma cell line. Nature 1980;286:531-3. 5. Brechot C, Pourcel C, Louise A, et al. Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma. Nature 1980:286:533-5. 6. Edman JC, Gray P, Valenzuela P. et al. Integration of hepatitis B virus sequences and their expression in a human hepatoma cell. Nature 1980;286:535-8. 7. Brechot C. Hadchouel M, Scotto J. et al. Detection of hepatitis B virus DNA in liver and serum: a direct appraisal of the chronic carrier state. Lancet 1981;ii:765-8. 8. Shafritz D, Shouval D, Shermann HI, et al. Integration of hepatitis B virus DNA into the genome of the liver cells in chronic liver disease and hepatocellular carcinoma. N Engl J Med 1981;305:1067-73. 9. Kam W, Rall LB, Smuckler EA, et al. Hepatitis B viral DNA in liver and serum of asymptomatic carriers. Proc Nat1 Acad Sci USA 1982;79:7522-6. 10. Ruiz-Opazo N. Chakraborty PR, Shafritz DA. Evidence for supercoiled hepatitis B virus DNA in chimpanzee liver and serum Dane particles: possible implications in persistent HBV infection. Cell 1982;29:129-38, 11. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975: 98:503-17. 12. Cummings IW, Browne JK, Salser WA, et al. Isolation, characterization and comparison of recombinant DNAs derived from genomes of human hepatitis B virus and woodchuck hepatitis virus. Proc Nat1 Acad Sci USA 1980;77:1842-6. 13. Rigby PWJ, Dieckmann M, Rhodes C. et al. Labeling of deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase 1. J Mol Biol 1977: 113:237-51. 14. Fowler MJF, Monjardino J. Tsiquaye KN. et al. The mechanism of replication of hepatitis B virus, evidence of asymmetric replication of the two DNA strands. 1 Med Virol 1984; 13:83-91. 15. Seeff LB, Wright EC, Zimmerman HJ, et al. Type B hepatitis after needle-stick exposure: prevention with hepatitis B immune globulin. Final report of the Veterans Administration cooperative study. Ann Intern Med 1978:88:288-93. 16. Grady GF. Lee VA, Prince AM, et al. Hepatitis B immune
616
YOKOSUKA ET AL.
globulin for accidental exposures among medical personnel: final report of a multicentric controlled trial J Infect Dis 1978;138:625-38. 17. Okada K, Kamiyama I, Inomata M, et al. e Antigen and anti-e in the serum of asymptomatic carrier mothers as indicators of positive and negative transmission of hepatitis B virus to their infants. N Engl J Med 1976;294:746-9. 18. Shikata T, Karasawa T, Abe K, et al. Hepatitis B e antigen and infectivity of hepatitis B virus. J Infect Dis 1977;136:571-6.
GASTROENTEROLOGY Vol. 89, No. 3
19. Nielsen JO, Dietrichkson 0, Juh E. Incidence and meaning of the “e” determinant among hepatitis B antigen positive patients with acute and chronic liver disease. Lancet 1974; ii;913-5. 20. Hoofnagle JH. Type B hepatitis: virology, serology, and clinical course. Semin Liver Dis 1981;1:7-14. 21. Gerlich WH, Robinson WS. Hepatitis B virus contains protein attached to the 5’ terminus of its complete DNA strand. Cell 1980;21:801-9.
1985;89:610-6
Active and Inactive Replication of Hepatitis B Virus Deoxyribonucleic Acid in Chronic Liver Disease OSAMU YOKOSUKA, KUNIO OKUDA First Department
MASAO
of Internal Medicine,
OMATA,
Chiba University
Little is known about the replicative forms of hepatitis B virus (HBV) DNA in the liver in chronic liver disease. We therefore analyzed HBV DNA and the changes in DNA signals after endonuclease digestion in liver tissues taken from 64 patients with hepatitis B surface antigen-positive chronic liver disease. The “active” replication pattern, which included various replicative intermediates, was seen in 36 of 38 (95%) hepatitis B e antigen-seropositive patients. This pattern was also found in 5 of 26 (19%) hepatitis B e antigen-seronegative patients who showed the highest mean serum alanine aminotransferase level (403 ? 184 mU/ml]. Most of them had advanced liver disease. Episomal viral DNA of an “inactive” type having only the supercoiled form was found in 3 patients; they showed the lowest mean serum alanine aminotransferase level (27 t 7 mU/ml] and only mild liver disease. As with duck HBV infection, episomal replicative forms of human HBV could be resolved by Southern blot analysis and seem to have clinical implications in human HBV infection.
FUMIO IMAZEKI, School of Medicine,
and
Chiba, Japan
the minus strand of DNA, and synthesis of the plus strand by DNA polymerase reaction. In an experimental transmission study, Mason et al. (3) showed formation of supercoiled viral DNA that preceded the reverse transcription phase of viral DNA, suggesting the importance of this particular species in the initiation of infection. Studies of human liver tissue by Southern blot hybridization have demonstrated integration of HBV DNA into genomic DNA in neoplastic (4-6) and nonneoplastic liver disease (7-9). Information on episomal replicative forms in human liver disease, however, is still limited. We therefore analyzed episomal HBV DNA by the method of Southern blot hybridization and studied changes after endonuclease digestion in liver tissues taken from 64 patients with hepatitis B surface antigen-positive chronic liver disease, and correlated the patterns with clinical features. Materials and Methods Patients
Recently, Summers and Mason proposed a replicative pathway of duck hepatitis B virus (DHBV) DNA, a virus that shares many properties with human HBV (1,2). They demonstrated the presence of a pregenomic RNA intermediate, reverse transcription of
Received November Address
requests
13, 1984. Accepted March 4, 1985. for reprints to: Masao Omata, M.D., First
Department of Medicine, Chiba, Japan 280.
Chiba University
School of Medicine,
This research was supported in part by Japanese Ministry of Education grant (B) 58480215, a grant from the Japan Medical Research Foundation, and a grant from the Japanese Ministry of Health and Welfare. The authors thank Dr. Jesse Summers (Fox Chase, Pennsylvania] for his generous gift of cloned HBV DNA and helpful discussion. 0 1985 by the American Gastroenterological Association
0016-5085/85/$3.30
Percutaneous liver biopsy specimens were taken from 64 patients with chronic liver disease who were seen at the First Department of Medicine, Chiba University. They had been seropositive for hepatitis B surface antigen for at least 6 mo before liver biopsy. None of them had received antiviral treatments or immunosuppressive agents. The ages of patients ranged from 19 to 70 yr (mean 34 yr). Of the 64 patients, 38 were seropositive for hepatitis B e antigen (HBeAg), 7 were negative for both HBeAg and hepatitis B e antibody (anti-HBe), and 19 were positive for anti-HBe. The demographic data on these patients are shown in Table 1. Two needle cores were taken from all patients with a Tru-Cut biopsy needle. One core was processed for routine histologic examination, and the other was immediately frozen and stored at -80°C.
Abbreviation
used in this paper:
DHBV, duck hepatitis
B virus.
September
Table
1985
ACTIVE AND INACTIVE
1. Demographic
Data on 64 Patients
Sex (male/female) Age (mean + SD, yr) Liver histology Chronic persistent hepatitis Chronic active hepatitis Cirrhosis HBeAg status HBeAg (+)/anti-HBe (-) HBeAg (-)/anti-HBe (-) HBeAg (-)/anti-HBe (+) Serum alanine aminotransferase mU/ml: normal 5 40)
Studied 51113 34.2 5 11.1
33 20 11 38 7 19 146 + 204
(mean + SD,
Anti-HBe, hepatitis B e antibody: HBeAg, hepatitis B e antigen: +. positive: -, negative.
Isolation Liver
of Deoxyribonucleic
Acid
From
the
REPLICATION
OF HBV DNA
applied as a sensitivity as well as a size marker. Electrophoresed A-phage DNA digested with Hind III and endlabeled with 32P-dCTP using T,-polymerase was used as another size marker. Autoradiograms were analyzed by a Sakura PDS 15 densitometer (Sakura Co., Tokyo, Japan).
Restriction Endonuclease Deoxyribonucleic Acid
Analysis
of Liver
Ten micrograms of nucleic acid extracted from liver was digested with 10 U of Hind III or Eco RI for 2 h at 37°C with a mixture of 0.02 M Tris-HCl (pH 7.5), 0.05 M NaCl, 0.01% triton, and 1 mM dithiothreitol. To study the kinetics of Eco RI digestion, 20 Fg of liver DNA extracted with a hot phenol-SDS method without prior pronase treatment was digested with 20 U of Eco RI for 15, 30, 45, 60, 90, 120, and 180 min.
A biopsy core (10-25 mg) was homogenized in a mixture of 0.5 ml of 10 mM Tris-HCl (pH 7.4) and 10 mM
ethylenediaminetetraacetic acid (EDTA) by a loose-fitting Dounce homogenizer. To this was added 0.5 ml of 0.2 M NaCl, 0.02 M Tris-HCl (pH 7.4), 2 mM EDTA, 1 mg/ml pronase, and 1% sodium dodecyl sulfate (SDS) and the mixture was incubated for 30 min at 37°C. Extraction of DNA was done twice with 1 ml phenol/chloroform (l:l), and the aqueous phase was then precipitated and washed three times with absolute ethanol chilled to -20°C. The nucleic acid pellets were resuspended in 100 ~1 of a mixture of 5 mM Tris-HCl (pH 7.4) and 1 mM EDTA. The average yield of total nucleic acids was 7.2 t 3.4 pg/mg
Serum Hepatitis B Surface Hepatitis B e Antigen
Antigen
kb
E
uH
’*
-
9.4
-
6.7
-
4.4
4
3.2
-
2.3
-
2.0
??
Analysis of Hepatitis B Virus Deoxyribonucleic Acid Ten micrograms of nucleic acids from liver were electrophoresed on 1.0% or 1.5% agarose gel (Seakam Co., Rockland, Me.) and transferred to nitrocellulose filter (Schleicher & Schuell, Dassel, Federal Republic of Germany) by the Southern blot technique (11). The DNA immobi-
lized on nitrocellulose filter was hybridized with ‘*Plabeled cloned HBV DNA inserted into pBH-20 plasmid (12). The HBV DNA insert was repurified, and the excised HBV DNA segment was used for labeling. The labeling of DNA with “‘P-deoxycytidine triphosphate (dCTP) was done by the nick translation method (13). The specific activity of the HBV DNA probe was 3-9 X lOa cpm/pg DNA. After hybridization, the filter paper was washed twice with 2 x SSC (0.15 M NaCl, 0.015 M sodium citrate)/ 0.1% SDS at room temperature, and washed again with 0.1 X SSCiO.l% SDS at 56°C for 30 min each. After washing, was dried and autoradiographed with (Eastman Kodak Co., Rochester, N.Y.) Du Pont lightning (DuPont Co., Wilscreen at -70°C for l-3 days. Three DNA in 2 pg of calf thymus DNA was
and
Hepatitis B surface antigen was assayed by reversed passive hemagglutination, and HBeAg by radioimmunoassay (Abbott Laboratories, North Chicago, Ill.).
tissue (mean ? SD). In selected samples, a hot phenol-SDS extraction procedure before pronase treatment was used to isolate proteinfree DNA as described by Ruiz-Opazo et al. (10).
nitrocellulose filter Kodak X-Omat film in the presence of mington, Del.) plus picograms of HBV
611
.
Figure
*
1. “Inactive” free hepatitis B virus DNA on 1% agarose gel. Supercoiled (SC) form at 2.0 kb in lane LJ (uncut DNA] shifted to a band at 3.2 kb after Eco RI digestion (Jane E), but remained tion (Jane H).
unchanged
after Hind III diges-
612
Figure
YOKOSUKA
ET AL.
2. Digestion kinetic study of the 2.0-kb form (lane 1) with Eco RI. Liver DNA was extracted with the hot phenol-SDS procedure, and electrophoresed on 1.5% agarose gel. Liver DNA (20 pg) was digested with 20 U of Eco RI for 15 min (lane 2), 30 min (lane 31, 45 min (lane 4). 60 min (lane 51, 90 min (lane 6), 120 min (lane 7), and 180 min (lane 8). After 15 min of digestion, two bands (4.0 kb and 3.2 kb) started to appear. After 180 min, the 2.0-kb form was complete-
GASTROENTEROLOGY
1’2
3
4
56
Vol. 89. No. 3
7
?
RC L SC
ly converted to a 3.2-kb form. AC, relaxed circular; L, linear duplex; SC, supercoiled.
smears from 2.8 kb to 2.0 kb, and below 1.35 kb, and a band at 2.0 kb on 1.0% agarose gel
Results
included
Detection of Episomal Hepatitis B Virus Deoxyribonucleic Acid in Liver Tissue
(Figure 3A). A densitogram of the autoradiogram is shown in Figure 3B. Because the 2.0-kb form was not distinct in the active form (probably due to smears from above), hot phenol-SDS extraction was performed to isolate protein-free DNA. This revealed a distinct band at 2.0 kb with uncut liver DNA, and a 3.2-kb form after Eco RI digestion (Figure 4). With phenol-chloroform extraction, a band appeared at 3.2 kb after Eco RI digestion (Figure 3A). These results indicated that the supercoiled form was present in the active form, but was simply obscured by other replicative intermediate signals. This situation was somewhat similar to that reported with serum (10). As all the radioactive signals were converted after heat treatment to a smear below 1.35 kb, which was resistant to Eco RI (Figure 3A) but was sensitive to Sl nuclease, the smear below 1.35 kb appeared to represent single-stranded DNA. The smear from 2.8 to 2.0 kb was compatible with partially double-stranded DNA (1,2,14). Thus, the active form included various replicative intermediates, and indicated active on-going viral replication.
Two Southern blot hybridization patterns of free viral DNA were recognized on 1.0% agarose gel. One pattern showed a single band at 2.0 kb with uncut liver DNA (Figure 1). After digestion with Hind III, which has no cutting site within HBV DNA, the band remained at 2.0 kb, whereas it moved to 3.2 kb after digestion with Eco RI, which recognizes one site within HBV DNA (Figure 1). A digestion kinetic study of the 2.0-kb form using 20 U of Eco RI and 1.5% agarose gel revealed faint bands at 4.0 kb and 3.2 kb after 15 min of digestion. One band at 4.0 kb became weaker, and one at 3.2 kb became more intense after longer periods of digestion. After digestion for 3 h, only one band was recognized at 3.2 kb (Figure 2). Digestion kinetics of DNA as studied with Sl nuclease showed a similar conversion of the 2.0kb form to a 3.2-kb form. These findings suggested that the 2.0-kb form was supercoiled HBV DNA and the 4.0-kb form was relaxed circular fully doublestranded DNA. A band at 3.2 kb corresponded to a cloned, fully double-stranded linear HBV DNA. These findings in human liver were compatible with previous reports in ducks (2,3) and in chimpanzees (10). Summers and Mason postulated that supercoiled DNA would act as a template for viral replication when infection was initiated in the duck liver (1,3). Because the sole presence of the supercoiled form seems to indicate that no active transcription of viral DNA is occurring, despite the presence of the template, we tentatively designated this Southern blot pattern as “inactive.” In contrast to inactive replication, various radioactive signals were observed in an “active” form that
Patterns of Viral Replication Features
and Clinical
Of the 38 HBeAg-seropositive patients, 36 showed the active pattern with various replicative forms, and none showed the inactive pattern (Table 2). Of the 7 patients negative for HBeAg and anti-HBe, 1 patient showed the active and 1 patient showed the inactive pattern. Of the 19 patients positive for anti-HBe, 4 (21%) and 2 (11%) showed the active and the inactive pattern, respectively (Table 2). (95%)
September
1985
ACTIVE
AND
INACTIVE
A
REPLICATION
OF HBV DNA
613
r-4 (1)
w
(2)
4.4 Figure
3.2
2.32.0
1.35
kb
3. A. “Active” form of hepatitis B virus DNA on 1.0% agarose gel electrophoresis. Various replicative forms including singlestranded (SS), partially double-stranded (PDS), and supercoiled (SC) DNA are present. After Eco RI digestion (lane E). a band at 3.2 kb (relaxed circular or linear duplex, or both) emerged, and the radioactive signal at 2.0 kb became less than that in lane Lr (uncut DNA). The change may be better appreciated on densitograms [Figure 3B). Heating converted all double-stranded DNAs to single-stranded DNAs (lane H). B. Densitograms of uncut (lane U), Eco RI-digested (lane E), and heated (lane H) autoradiograms in A corroborated well the changes observed visually. They showed conversion of a peak at 2.0 kb [w, top (l)], which corresponded to the supercoiled form, to a peak at 3.2 kb [o, middle (2)], which corresponded to relaxed circular or linear duplex. or both, after Eco RI digestion. Heating converted all double-stranded DNAs to single-stranded DNAs [bottom
(311
Of the 38 HBeAg-positive patients, 36 with the active pattern had high serum alanine aminotransferase levels (ALT) (mean 183 mu/ml), whereas 2 patients without viral DNA in liver tissue showed near normal ALT values (mean 55 mu/ml) (Table 3). Of the 26 patients who were either negative for Table
2.
Relationship Between Serum Hepatitis B e Antigen/Hepatitis B e Antibody and Molecular State of Hepatitis B Virus Deoxyribonucleic Acid in the Liver No.
HBeAg (-t )/anti-HBe HBeAg (-)/anti-HBe HBeAg (-)/anti-HBe
(-1 (-) (+)
Total Anti-HBe, hepatitis B e antibody; positive: -, negative.
Active
Inactive
38 7 19
36 1 4
0 1 2
64
41
3
HBeAg,
hepatitis
B e antigen:
HBeAg or positive for anti-HBe, 5 patients with active replication showed the highest mean ALT values (403 mu/ml) and advanced liver disease (chronic active hepatitis or cirrhosis, or both, in 4 patients), whereas 3 patients with the inactive pattern showed normal ALT values (mean 27 mu/ml) and mild liver disease (chronic persistent hepatitis in 3 patients) (Table 3). Eighteen patients without demonstrable viral DNA in the liver showed near normal ALT values (mean 57 mu/ml).
Discussion
f,
Previous Southern blot hybridization analyses in human liver disease put much emphasis on integration of viral DNA into genomic DNA, but little on replicative intermediates of episomal viral DNA. Summers and Mason proposed the following infec-
614
YOKOSUKA
Figure
ET AL.
GASTROENTEROLOGY
4. Southern blot analysis of liver DNA extracted by the hot phenol-SDS procedure without prior pronase treatment revealed a distinct band at 2.0 kb. In this patient, the “active” form with various replicative forms was shown by the ordinary phenol-chloroform extraction method with prior pronase treatment. A band at 2.0 kb (supercoiled) (Jane 1, uncut] moved to 3.2 kb after Eco RI digestion (lane 2).
3.
Demography
and Their
of Patients
Laboratory
With an Active Data
and Inactive
Hepatitis
B Virus Deoxyribonucleic
HBeAg-positive Active
Number Age W SALT (mu/ml) Histology CPH CAH/cirrhosis
36 30 (83%) 31.9 + 10.6" 183 2 236
Acid Replication
Active
_
5 5 (lOO"~,) 38.0 + 9.4 403 2 184"'
1 (50%) 1(50%) persistent
hepatitis:
inactive
_
+
18 13 (72%) 36.4 k 10.2 57 f 61"
2 (67%) 39.7 + 18.8 27 2 7'
+
2 1 (5041) 39.0 L 5.0 55 2 9
19 (53%) 17 (48%)
CAH. chronic active hepatitis: CPH, chronic ” Mean + SD. ‘I p < 0.001.' p < 0.01.
3
HBeAg-negative
+ Male (%)
89, No.
initiation and persistence of HBV infection in the chimpanzee. Mason et al. (3) demonstrated appearance of supercoiled viral DNA before the synthesis of other replicative forms when DHBV infection was initiated in embryonic duck liver. Recently, we observed similar sequential appearances of replicative intermediates in l-day old ducklings (unpublished data). In the current study, two hybridization patterns of HBV DNA in the liver emerged; one with supercoiled viral DNA, indicating the presence of the template alone, and the other with replicative intermediates, indicating active replication of HBV DNA. Of the 38 HBeAg-seropositive patients, 36 showed the “active” pattern with high serum (95%) ALT levels. These results were concordant, because previous studies demonstrated that serum HBeAg was closely associated with high infectivity and active liver disease (15-20). Two patients with HBeAg in serum, but without detectable viral DNA, however, showed almost normal ALT values. Five patients who had active patterns, despite seronegativity for HBeAg, showed the highest mean ALT level (403 mu/ml). In contrast, 3 patients with the “inactive” form showed very low serum ALT levels (Table 3). The former had more advanced liver diseases, whereas all 3 patients of the latter group showed chronic persistent hepatitis histologically [Table 3). Thus, our results suggested that episomal viral DNA could be divided into two types, and that the active form was closely related to on-going viral replication and active liver disease, whereas the inactive form was related to minimal viral replication and quiescent liver disease. A long-term followup study is currently under way to see the relation of active viral replication and prognosis in such patients. Recently, Fowler et al. (14) studied replicative intermediates of viral DNA in a human HBV carrier, and failed to show the supercoiled form of HBV DNA. Our study, which included a digestion kinetic study of DNA using Eco RI and hot-phenol extrac-
tious cycle of DHBV DNA (1-3): (a] initiation of the cycle by transcription of pregenomic full-length plus-strand RNA from supercoiled or integrated viral DNA, (b) reverse transcription of minus-strand DNA from the RNA intermediate, (c) synthesis of plusstrand DNA with endogenous DNA polymerase reaction, using minus-strand DNA as a template, and (d) packaging and exportation of partially doublestranded DNA. Ruiz-Opazo et al. (10) suggested that the supercoiled form might play an important role in the Table
Vol.
9 (50%) 9 (50%,)
1 (20%) 4 (80%) HBeAg.
hepatitis
B e antigen:
SALT.
serum
alanine
3
3 [ 1OO%] 0 (0%) aminotransferase.
September 1985
ACTIVE
tion of liver DNA, supported the previous results in chimpanzees (10)and ducks (31, and clearly indicated the presence of the supercoiled form in humans as well. Gerlich and Robinson (21) described a protein covalently attached to the 5’ terminus of a complete HBV DNA strand that could be extracted into the organic solvent phase with phenol before, but not after, proteinase digestion. Thus, in selected samples we used an extraction procedure without prior proteinase treatment to remove DNA-protein complex, and to extract protein-free DNA. A hot phenol-SDS procedure without prior proteinase treatment gave a discrete band at 2.0 kb (Figure 4), whereas a procedure with pronase treatment gave a variety of viral forms that obscured the band at 2.0 kb (Figure 3A). Failure to reveal the supercoiled form was probably due to the presence of other radioactive signals that might submerge a discrete band, or mechanical nicking of the DNA or nuclease contamination, or both. Recently, we observed that the supercoiled form persisted in patients during treatment with large doses of recombinant a-interferon, whereas other replicative forms disappeared in posttreatment biopsy specimens [unpublished observation). It was later found that active virus replication recurred in these patients. Our data in humans strongly suggest that the supercoiled form plays a key role in initiation and maintenance of the carrier state as suggested in animals (3,10).
I .O% 3.2kb
m
Relaxed Circular
gel -
Linear
+Q
2.8kb
1
2.0kb
_
-
Partially Double Stranded
+OJ
m
Supercoiled
I .35kb .13-
-
Single Stranded
f
---J-J Figure
5. Schematic representation of bands and smears obtained by blot hybridization on 1.0% agarose gel and their corresponding replicative forms.
AND
INACTIVE
REPLICATION
OF HBV DNA
615
As with DHBV replication, episomal replicative forms could be resolved by Southern blot analysis. Summarizing our data and that of others (1,2,10), a schema given in Figure 5 may be proposed to correlate the radioactive bands and smears on 1.0% agarose gel electrophoresis with various viral DNA forms.
References 1. Summers J, Mason WS. Replication of the genome of a hepatitis B like virus by reverse transcription of an RNA intermediate. Cell 1982;29:403-15. 2. Mason WS, Aldrich C, Summers 1, et al. Asymmetrical replication of duck hepatitis B virus DNA in liver cells: free minus-strand DNA. Proc Nat1 Acad Sci IJSA 1982;79:39974001. 3. Mason WS, Halpern MS, England JM, et al. Experimental transmission of duck hepatitis B virus. Virology 1983:131: 375-84. 4. Chakraborty PR, Ruiz-Opazo N. Shouval D, et al. Identification of integrated hepatitis B virus DNA and expression of viral DNA in an HBsAg-producing human hepatocellular carcinoma cell line. Nature 1980;286:531-3. 5. Brechot C, Pourcel C, Louise A, et al. Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma. Nature 1980:286:533-5. 6. Edman JC, Gray P, Valenzuela P. et al. Integration of hepatitis B virus sequences and their expression in a human hepatoma cell. Nature 1980;286:535-8. 7. Brechot C. Hadchouel M, Scotto J. et al. Detection of hepatitis B virus DNA in liver and serum: a direct appraisal of the chronic carrier state. Lancet 1981;ii:765-8. 8. Shafritz D, Shouval D, Shermann HI, et al. Integration of hepatitis B virus DNA into the genome of the liver cells in chronic liver disease and hepatocellular carcinoma. N Engl J Med 1981;305:1067-73. 9. Kam W, Rall LB, Smuckler EA, et al. Hepatitis B viral DNA in liver and serum of asymptomatic carriers. Proc Nat1 Acad Sci USA 1982;79:7522-6. 10. Ruiz-Opazo N. Chakraborty PR, Shafritz DA. Evidence for supercoiled hepatitis B virus DNA in chimpanzee liver and serum Dane particles: possible implications in persistent HBV infection. Cell 1982;29:129-38, 11. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975: 98:503-17. 12. Cummings IW, Browne JK, Salser WA, et al. Isolation, characterization and comparison of recombinant DNAs derived from genomes of human hepatitis B virus and woodchuck hepatitis virus. Proc Nat1 Acad Sci USA 1980;77:1842-6. 13. Rigby PWJ, Dieckmann M, Rhodes C. et al. Labeling of deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase 1. J Mol Biol 1977: 113:237-51. 14. Fowler MJF, Monjardino J. Tsiquaye KN. et al. The mechanism of replication of hepatitis B virus, evidence of asymmetric replication of the two DNA strands. 1 Med Virol 1984; 13:83-91. 15. Seeff LB, Wright EC, Zimmerman HJ, et al. Type B hepatitis after needle-stick exposure: prevention with hepatitis B immune globulin. Final report of the Veterans Administration cooperative study. Ann Intern Med 1978:88:288-93. 16. Grady GF. Lee VA, Prince AM, et al. Hepatitis B immune
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YOKOSUKA ET AL.
globulin for accidental exposures among medical personnel: final report of a multicentric controlled trial J Infect Dis 1978;138:625-38. 17. Okada K, Kamiyama I, Inomata M, et al. e Antigen and anti-e in the serum of asymptomatic carrier mothers as indicators of positive and negative transmission of hepatitis B virus to their infants. N Engl J Med 1976;294:746-9. 18. Shikata T, Karasawa T, Abe K, et al. Hepatitis B e antigen and infectivity of hepatitis B virus. J Infect Dis 1977;136:571-6.
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19. Nielsen JO, Dietrichkson 0, Juh E. Incidence and meaning of the “e” determinant among hepatitis B antigen positive patients with acute and chronic liver disease. Lancet 1974; ii;913-5. 20. Hoofnagle JH. Type B hepatitis: virology, serology, and clinical course. Semin Liver Dis 1981;1:7-14. 21. Gerlich WH, Robinson WS. Hepatitis B virus contains protein attached to the 5’ terminus of its complete DNA strand. Cell 1980;21:801-9.