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Detection of hepatitis B virus X gene protein and antibody in type B chronic liver disease
GASTROENTEROLOGY
1989:97:990-6
Detection of Hepatitis B Virus X Gene Protein and Antibody in Type B Chronic Liver Disease KAZUHIRO KATAYAMA, NOR10 HAYASHI, YUTAKA SASAKI, AKINORI KASAHARA, KEIJI UEDA, HIDEYUKI FUSAMOTO, NOBUHIRO SATO, OSAMU CHISAKA, KENICHI MATSUBARA, and TAKENOBU KAMADA First Department of Medicine, Osaka University Medical School, Osaka: and Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan
The genome of the hepatitis B virus contains a sequence (X gene) whose role is unclear. The almost complete region of the hepatitis B virus X gene was expressed in Escherichia coli, with the resulting protein being -17 kilodaltons in molecular weight. Sera from 139 subjects were analyzed by Western blot analysis. Of the hepatitis B surface antigenpositive patients, anti-X was not found in 4 patients with acute hepatitis and in 12 healthy carriers, but was present in 41% (21/51)of the patients with chronic hepatitis, 63% (15/24) of those with liver cirrhosis, and 46% (12/26) of those with hepatocellular carcinoma. The expression of the X product in the liver tissues (43 hepatitis B surface antigenpositive patients) was investigated using an indirect immunohistochemical method. The X protein was observed in 64% (21/33) of the patients with chronic hepatitis and 50% (~/IO) of those with liver cirrhosis, and was found when the serum was negative for anti-X. Hepatitis B core antigen was frequently expressed together with the X protein in the liver. The conclusions reached were that the frequency of anti-X increases with the length of chronic hepatitis B virus infection, that anti-X may suppress the expression of the X protein in the liver, and that the X protein may be related to hepatitis B virus replication.
found a 28-kilodalton protein in HBV-infected livers that was recognized by antiserum prepared against a synthetic peptide from the X open reading frame, and have also detected the antibody against the synthetic peptide and the recombinant protein including the X open reading frame in sera of HBVinfected individuals (2,3). Authentic X polypeptides have also been found in infected liver (4). Although these studies demonstrate the coding potential of the X open reading frame and its association with HBV infection, the expressions of the X protein and anti-X in type B viral infection have not been fully elucidated. The putative viral polypeptide can be produced in Escherichia coli with expression vectors to aid attempts to define the proteins synthesized during viral infection. Chisaka et al. (5) reported the construction of a plasmid encoding a fusion protein consisting of E. coli plasmid pOCTZ-5 and HBV X gene, designated the trpE-X protein. In the present study, we investigated the expression of the X protein in type B liver diseases using this trpE-X protein and detected anti-X antibody in sera and the Xrelated protein in liver tissue. Materials
and
Methods
Subjects
H
epatitis B virus (HBV) is a causative agent for type B viral hepatitis, which has been linked to hepatocellular carcinoma in humans (1).Molecular cloning and nucleotide sequencing have indicated that the genome of HBV contains at least four open reading frames: S, C, P, and X. The roles of the S, C, and P regions have been reported, but the function of the X region is still unclear. Previous studies have
This study examined 156 were hepatitis B surface antigen hepatitis 4, asymptomatic carrier liver cirrhosis 24, hepatocellular
subjects, 129 of whom (HBsAg)-positive (acute 12, chronic hepatitis 63, carcinoma 26), with the
0 1989 by the American Gastroenterological 0016-5065/89/$3.50
Association
October 1989
Table
HBV X PROTEIN AND ANTI-X IN TYPE B HEPATITIS
1. Profile of 129 Patients
With Hepatitis
w n
B Surface
Antigen
(1857) Total serum bilirubin (mgldlj
Serum albumin (g/d0
Age (yrj
ALT (U/L)
26 * 9
537 t 897
3.5 + 3.1
4.3 ? 0.3
25 2 6
18 + 5
0.6 2 0.3
4.3 2 0.2
215 2 316
1.0 2 0.8
4.0 2 0.5
56 2 8
76 5 58
1.7 t 1.2
3.4 2 0.8
51 2 10
64 t 38
4.1 + 8.2
3.4 + 0.5
Disease Acute hepatitis (n = 4) Healthy carrier (n = 12) Chronic hepatitis (n = 63) Liver cirrhosis (n = 24) Hepatocellular carcinoma (n = 26)
991
Figure
39 ? 11
ALT, alanine aminotransferase. SD.
Values are expressed
as mean 2
serologic profiles shown in Table 1. Fourteen subjects were positive for antibody to hepatitis B surface antigen and had chronic liver diseases (chronic hepatitis 4, liver cirrhosis 3, primary biliary cirrhosis 2, hepatocellular 1). Thirteen subjects were negcarcinoma 4, cholangioma ative for HBV markers (normal 3, lupoid hepatitis 1, alcoholic liver injury 1, drug-induced hepatitis 1,chronic hepatitis 5, hepatocellular carcinoma 1, cholangioma 1). Sera from 117 HBsAg-positive and 22 HBsAg-negative subjects were tested for anti-X by Western blot analysis. Liver tissue samples of 43 HBsAg-positive subjects (chronic inactive hepatitis 14, chronic active hepatitis 17, liver cirrhosis 10, chronic active hepatitis with hepatocellular carcinoma 2) and 8 HBsAg-negative subjects (chronic hepatitis 5, liver cirrhosis 2, hepatocellular carcinoma 1) were examined to search for the expression of X protein using an indirect immunohistochemical method. Informed written consent was obtained from all patients examined for X protein in the liver.
Hepatitis
B Viral
Markers
1 Structure of pOCTX. A 584 base pairs fragment (from Barn HI to Bgl II] was excised from pBRadr4 and inserted after the seventh codon of pOCT2-5. This fragment included 145 amino acids [from the 10th to the 154th) of the X gene. The nucleotide number marked along the HBV genome starts from the Xho I site. Ptrp, tryptophan operon promoter; TrpE, TrpE gene.
tryptophan operon, and pBRHBadr4, which carries the entire HBV genome, and constructed a recombinant plasmid pOCTX, which contained the Barn HI-Bgl II fragment (1273-1857, 584-bp) of HBV genome (Figure 1). The plasmid was propagated and transferred to the E. coli strain (KY1603), and produced a fusion protein consisting of N-terminal 7 amino acids of the trpE gene product and C-terminal 145 amino acids of the HBV X gene product. This protein, designated the trpE-X protein, was purified as described previously (5). From 20 g of induced KY1603 wet cells, the trpE-X protein was purified by ion-exchange chromatography. Rabbit polyclonal antisera against the purified trpE-X protein were prepared by the usual method using Freund’s complete adjuvant (5). The purified trpE-X protein was subjected to 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis according to Laemmli (7). The gel was stained with Coomassie brilliant blue. This protein had a molecular weight of -17 kilodaltons, which the HBV X gene could encode (Figure 2), and
in Serum
Hepatitis B surface antigen, antibody to hepatitis B surface antigen, hepatitis B e antigen (HBeAg), antibody to hepatitis B e antigen (anti-HBe), and antibody to hepatitis B core antigen were determined using commercial radioimmunoassays (Abbott Laboratories, North Chicago, Ill.). Serum HBV deoxyribonucleic acid (DNA) was tested by a spot hybridization technique using a commercial kit (Dainabot, Tokyo, Japan). Hepatitis B virus DNA polymerase activity was measured according to Kaplan et al. (6).
Expression
of Hepatitis
B Virus
X Protein
A recombinant plasmid pOCTX was constructed as described previously (5) to express almost the complete region of the HBV X open reading frame. We used the plasmid pOCT2-5, which carries a promoter of E. coli
Figure 2. Identification of HBV X protein. Purified X protein was subjected to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Size markers are indicated in kilodaltons.
992
GASTROENTEROLOGY Vol. 97. No. 4
KATAYAMA ET AL.
anti-X positive human sera were preincubated for 2 h at 37°C with the trpE-X protein, and then analyzed.
indirect
Figure 3. Detection of antibodies against the HBV X protein using Western blot analysis. Lane 1, HBV marker-negative normal subject; lanes z and 3, chronic hepatitis; lanes 4 and 5, liver cirrhosis: lanes 6 and 7, hepatocellular carcinoma; lanes 8 and 9, same patient with chronic hepatitis (anti-X positive); lane 9, the sample had been reacted with the serum preincubated with trpE-X.
reacted peptides
strongly with corresponding
the antisera against synthesized to some parts of the HBV X protein
(5).
Western Blot Analysis To identify the anti-X in the serum, the trpE-X protein was subjected to 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and electrophoretitally transferred to nitrocellulose sheets (50 V, 3 h) according to Towbin et al. (8). The strips were made to react with 1:50 dilution of patient sera (37”C, 2 h), incubated with biotinylated goat antiserum to human immunoglobulin, and then incubated with avidin-biotinylated peroxidase complex (Vectastain ABC kit; Vector Laboratories, Inc., Burlingame, Calif.). The strips were finally developed with 4-chloro-l-naphtol. For 2 cases of acute hepatitis, serial sera were examined. To determine binding specificity, the
Table
2. Occurrence
of Antibody
to Hepatitis
B Virus X Antigen
lmmunohistochemical
Method
Liver tissue samples from liver biopsies or operations were fixed with Zamboni’s fixative for -30 min at 4°C (9). Frozen sections were cut on a cryostat to a thickness of 10 pm and stored at -20°C until use. The sections were incubated with 1:500 dilution of rabbit antiserum against the trpE-X protein (4”C, 48 h). Next, in the indirect immunofluorescence method, the sections were incubated with fluorescein isothiocyanate-conjugated goat antiserum against rabbit immunoglobulin G and observed under fluorescent photomicroscopy at 520 nm with excitation at 495 nm (10). When using peroxidaselabeled antibody, after incubation of the sections with anti-trpE-X, they were incubated with biotinylated goat antiserum against rabbit immunoglobulin G, then with avidin-biotinylated peroxidase complex (Vectastain ABC kit). The sections subjected to testing with the Vectastain ABC kit were developed with diaminobenzidine tetrahydrochloride. To determine the specificity of binding, rabbit antiserum (anti-trpE-X) was preincubated with the trpE-X protein for 2 h at 37°C. To examine for hepatitis B core antigen (HBcAg) in the liver, a liver sample was incubated with mouse monoclonal antibody to hepatitis B core antigen (Institute of Immunology, Tokyo, Japan) at 1:20 dilution for 48 h at 4”C, then allowed to react with peroxidase-labeled antimouse immunoglobulin G (Vectastain ABC kit) as described above. The expression of the X protein and HBcAg was graded as follows: (2+), positive hepatocytes accounted for >lO%; (+), staining was
Statistical The statistical uated
using
Fisher’s
Analysis significance exact test.
in Sera From Patients
of the results
With Hepatitis
was eval-
B Virus Infection
HBeAg/anti-HBe Diagnosis HBsAg-positive Acute hepatitis Healthy carrier Chronic hepatitis Liver cirrhosis Hepatocellular carcinoma HBsAg-negative Chronic liver diseaseb HBV markers negative”
+/o/4 o/7 lo/32 (31%) 7110 (70%) 3/7 (43%]
-/-
l/l (100%) l/2 (50%)
-/+ O/2" 015 11/19 (58%) 7/13 (54%) 8117 (47%)
Total
O/6 (0%) o/12 (0%) 21/51 (41%) 15124 (63%) 12/26 (46%) 3/14 (21%) O/8 (0%)
anti-HBe, antibody to hepatitis B e antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen: HBV, hepatitis B virus. n Of 4 patients with acute hepatitis, the serial sera of 2 patients were studied. b These patients with chronic liver disease had developed antibody to hepatitis B surface antigen. ’ These patients did not have hepatitis B surface antigen, antibody to hepatitis B surface antigen, or antibody to hepatitis B core antigen.
October 1989
HBV X PROTEIN AND ANTI-X IN TYPE B HEPATITIS
993
-e 4. Detection of the HBV X protein in liver tissue using an indirect immunohistochemical method. A. Positive staining appeared in the cytoplasm of the liver with chronic hepatitis (x400). B. This section was allowed to react with rabbit amtiserum (anti-trpE-X) preincubated with the trpE-X protein (x400). Positive staining was completely absorbed. Bars = 30 /Jtm.
Results Figure 3 shows representative Western blot analysis results for anti-trpE-X (anti-X) in the sera. Lanes 2, 4, and 6 show cases positive for anti-X, in which the antibody reacted with the 17-kilodalton trpE-X protein. Lanes 1,3,5,and 7 show the negative cases. Lanes 8 and 9 show the strips that had been allowed to react with the serum of 1 patient having anti-X, with the strip in lane 9 having been reacted with the serum preincubated with the trpE-X protein When 117 HBsAg-positive patients were checked for the presence of anti-X (Table 2), it was not found in patients with acute hepatitis and
healthy carriers, but was identified in 21 (41%) of the 51 patients with chronic hepatitis, 15 (63%) of the 24 with liver cirrhosis, and 12 (46%) of the 26 with hepatocellular carcinoma. In chronic hepatitis, antiX was found in 10 (31%) of the 32 HBeAg-positive patients and more often (58%) in patients positive for anti-HBe. The frequency of anti-X was lower in earlier phases of HBV infection, such as in patients with acute hepatitis, healthy carriers, and HBeAgpositive patients with chronic hepatitis than in later phases. Thus, the frequency of anti-X seems to increase with the length of HBV infection, although no significant correlation existed between the fre-
994
GASTROENTEROLOGY Vol. 97. No. 4
KATAYAMA ET AL.
Table 3. Expression of the X Protein in Livers From Patients With Hepatitis B Virus Infection HBeAg/anti-HBe Diagnosis HBsAg-positive Chronic hepatitis Liver cirrhosis Hepatocellular carcinoma HBsAg-negative Chronic hepatitisb Liver cirrhosis” Hepatocellular carcinoma”
-it
t/-
17l23
(74%)
o/4 (0%) o/2 (0%)
4110 (40%) 5/6 (83%)
Total
21133 (64%) 5/10 (50%) o/2 (0%)”
o/5 (0%)
l/2 (50%) O/l (0%)
anti-HBe, antibody to hepatitis B e antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen. ’ The X protein was detected in the noncancerous region (chronic hepatitis) of these 2 patients. b These patients did not have hepatitis B surface antigen, antibody to hepatitis B surface antigen, or antibody to hepatitis B core antigen. ’ These patients had developed antibody to hepatitis B surface antigen.
quency of anti-X and the HBeAg/anti-HBe system. Anti-X was found in 21% of the antibody to hepatitis B surface antigen-positive patients with chronic liver disease, but was not found in subjects who had no HBV markers. The X-positive structures stained by the antiserum against the trpE-X protein were identified in the cytoplasm of hepatocytes (Figure 4A). The X-positive liver sample, which had reacted with the anti-X incubated with the trpE-X protein, showed no staining (Figure 4B); this indicates the specificity for the binding. Clustering positive cells were seen more frequently around the portal area than the central vein area. The X protein was identified in 21 (64%) of 33 patients with chronic hepatitis, which was slightly more than for liver cirrhosis (50%). In chronic hepatitis, the X protein was found more often in HBeAg-positive cases (74%) than in antiHBe-positive cases (40%) (Table 3). In 2 patients with hepatocellular carcinoma, the X protein was found in the noncancerous region (Figure 5B), but not in the cancerous region (Figure 5A). Table 4 shows the appearance of the X protein in the liver and anti-X in the serum in each patient and also the relationship between X/anti-X and the other HBV markers. The anti-X in the serum was negative except in 1 case (No. 19) where the X protein in the liver was positive. The X protein in the liver was not expressed when the anti-X in the serum was positive, except in 1 case (No. 25). Thus, the X protein was found when the serum was negative for anti-X. There did not appear to be a direct relationship between X/anti-X and other HBV markers (Table 4). Thirty-two HBsAg-positive patients (26 with
chronic hepatitis and 6 with liver cirrhosis) were examined for X protein and HBcAg in the liver (Table 5). With chronic hepatitis, the positive rate of HBcAg in the liver with X protein (14/16,87.5%) was significantly higher than that without X protein (l/10, 10%) (p < 0.01). With liver cirrhosis, HBcAg was detected in 3 (75%) of the 4 cases with the X protein, and in 1 (50%) of the 2 cases without the X protein. Furthermore, in the liver with the X protein, HBcAg was found in the 2 cases of chronic hepatitis and in 3 (75%) of 4 cases with liver cirrhosis, in spite of the serum being negative for HBeAg. In some cases, consecutive sections were stained for the X protein and HBcAg. Hepatitis B core antigen-positive cells were mainly found among the X-positive hepatocytes, which frequently showed a wider distribution (Figure 6). Table 4. Expression of Hepatitis B Virus X Protein, Anti-X, and Hepatitis B Virus Markers Patient 1 2 3 4 5
6 7 8
9 10 11
12 13 14
15 16 17 18
19 20 21 22 23 24
25 26 27 28
29 30 31
HBeAgl anti-HBe
DNA-P (cpm)
ilil-
4637
2+/2t/-
t/k
856
2t/-
ti-
420
2t/-
t/-
147
2+/-
t/-
Histology
X/Anti-X
CIH CIH CAH CAH CAH CIH CAH CAH CIH CIH CAH CIH CIH CAH + HCC” LC LC LC LC LC
2t/-
674
2t/-
t/-
2t/-
tt-
2-k/-
tt-
2+1-
-/t
2+/-
t/-
+I-
-/t
24
+I-
t/-
416
2t/-
320 9 9
-/t
3
-Ii
2+/-
-/t
10
2+1-
-/t
71
2+1-
-Ii -Ii
8 8
CIH LC LC LC
-l-
-/t
41
-l-
-II-
0
-l-
+/-
12
-l-
-/t
0
CAH CIH CIH CAH CIH CAH LC LC
-Ii
+I-
-F/t
-1-I
495 4
-Ii
-Ii
0
-I+
+I-
169
-Ii
tt-
-Ii -it -Ii
titi&I-
t t + t t -
400
2t/-
2ilf
HBVDNA
t _ t t + _ _ t + -
16 4173
102 10
anti-HBe, antibody to hepatitis B e antigen: CAH, chrcnic active hepatitis; CIH, chronic inactive hepatitis: DNA-P, deoxyribonucleic acid polymerase; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LC, liver cirrhosis. a X protein was expressed in hepatocytes of noncancerous region. 2+, positive hepatocytes accounted for >lO%; t, staining was
October 1989
HBV X PROTEIN AND ANTI-X IN TYPE B HEPATITIS
995
Figure 5. Expression of the X protein. The X protein was not expressed in the region of hepatocellular carcinoma (A, x400). but was in the noncancerous region with chronic hepatitis adjacent to the hepatocellular carcinoma (B, ~400). Bars = 30 pm.
Discussion Conservation of the X gene among mammalian hepadnaviruses suggests that it serves a vital function. There have been some reports about the expression of the X gene, but its role has not yet been determined clearly. The most interesting finding in the present study is that the X protein was found in the liver when the serum was negative for anti-X. We also found that anti-X did not appear in the early stage of HBV infection, such as in patients with acute hepatitis and HBeAg-positive asymptomatic carriers. The frequency of anti-X being found in HBeAgpositive patients with chronic hepatitis was less than in the later stage of HBV carriers, such as in
cases of anti-HBe-positive chronic hepatitis or liver cirrhosis. These findings suggested that the frequency of anti-X in serum increased with the length of HBV infection, and the X protein was expressed in the early stage of HBV infection. Indeed, in chronic hepatitis, the frequency of X protein decreased as HBeAg was converted to anti-HBe, but this trend was not seen in liver cirrhosis. As we were only able to examine a limited number of patients with acute hepatitis, healthy carriers, and patients with liver cirrhosis, more patients need to be studied before firm conclusions can be drawn. The presence of anti-X (2,3,11-13)and of the X protein (4) in HBV infection has been noted, but
996
GASTROENTEROLOGY Vol. 97, No. 4
KATAYAMA ET AL.
Figul -e 6. Detection of the X protein [A, x400) and HBcAg (B, x400) in consecutive than that of HBcAg. Bars = 30 pm..
there has been no mention of any relationship between them. Pfaff et al. (11)reported finding anti-X in patients who had been exposed for an extended period to HBV gene products, but they found no X-related proteins in the liver using immunoprecipitation. Meyers et al. (12)also detected the anti-X antibody in 9 of 26 asymptomatic HBV carriers, and indicated that there was no direct relationship between the anti-X and other HBV markers. Pfaff et al. and Meyers et al. used almost the complete region of the HBV X gene (Barn HI-Bgl II) (11,12), which was the part we used, and our results for anti-X agreed with theirs. Moriarty et al. (2) found anti-X antibody predominantly in the sera from patients with hepa-
sections. The distribution
of the X protein was W,ider
tocellular carcinoma when they used synthesized peptides corresponding to certain parts of the HBV X gene. Elfassi et al. (3)also detected anti-X in 3 of 11 patients with chronic active hepatitis using the fusion protein corresponding to 133 amino acids of HBV X gene. These results seem to differ from ours, partly because of the difference of antigenicity between the X protein that we used and previously used peptides (2) or protein (3). In our study, the X protein corresponded to almost the complete region of the HBV X gene, whereas Moriarty et al. used synthesized peptide and Elfassi et al. used fusion protein that did not contain the C terminus. Our work also showed that the X protein was not ex-
October
1989
HBV X PROTEIN AND ANTI-X IN TYPE B HEPATITIS
Table 5. Relationship Between the X Protein and Hepatitis B Core Antigen
Diagnosis Chronic hepatitis
Positive rate of HBcAg
HBcAg
X (liver)
HBeAg (serum)
2+
+
-
Total
+
+
9
3
2
14'
-
0
20
2.
-
+
0 1
04 05
46 I
+
_ +
03
01 00
4 0
+
0
11
-
000
2 0I
14/16 (87.5)"
cirrhosis Liver
(%I
_
1
l/l0 (lO.O)b
314 (75.0)
l/2 (50.0)
HBcAg, hepatitis B core antigen; HBeAg, hepatitis B e antigen. 2 +, positive hepatocytes accounted for >lO%; +, staining was
pressed in the cancerous region, but was in the noncancerous region around it in 2 cases of hepatocellular carcinoma. This implies that the protein is not required for the maintenance of this neoplastic state, but may be required for carcinogenesis. Siddiqui et al. (14) prepared the plasmid vector that contained the HBV X gene, and COS cells were used to express it. They then indicated that the X protein was expressed in the cytoplasm of the COS cell, and suggested that the X protein might be associated with cytoskeletal components. These results are similar to our findings that the X protein was expressed in the cytoplasm of the hepatocyte. Thus, in the early stage of HBV infection, the X protein seems to be expressed in the cytoplasm of hepatocytes, and anti-X seems to occur during HBV infection, accompanied by the disappearance of the X protein from the liver. The expression of the X protein in the early stage of infection might indicate some relationship of it to HBV replication, which is active in the early stage. Spandau and Lee (15) suggested that the HBV X protein was the transactivating factor of the HBV enhancer in vitro. Furthermore, we found the expression of HBcAg, which is a reliable marker of HBV DNA replication (16), where the X protein was expressed. We also noted that HBcAg could be frequently found in patients with the X protein, even with negative serum HBeAg. Thus, our results indicated some relationship of the X protein to HBV replication in vivo. One possibility is that the presence of the X protein in the liver might promote the expression of HBcAg there. Findings reported by Spandau and Lee could support this possibility (15). Frequently, there was a wider distribution of X
997
protein-positive hepatocytes than HBcAg-positive ones, possibly because HBcAg-positive cells might be lysed by cytotoxic T cells but not the X-positive cells. The presence of anti-X seemed to inhibit the expression of the X protein in the liver, but did not directly affect the serum HBV DNA polymerase activity. Further study is needed on the relationship between X and HBV replication. The X protein was sometimes found in liver not containing HBcAg, and also in that containing serum anti-HBe. As the absence of HBcAg in the liver usually indicates the absence of free HBV DNA (17), the X protein might be translated from HBV DNA integrated into the host genome, or the amount of free HBV DNA might have been too small to detect. If the former is the case, the X protein does not necessarily have the same function as authentic X. Generally, the frequency of X protein decreased and that of anti-X increased as HBeAg was converted to anti-HBe. However, in liver cirrhosis, the opposite trend appeared. One possible reason is the small number of patients with cirrhosis that we examined. Twenty-four patients with liver cirrhosis were tested for anti-X, but only the liver tissue from 10 patients could be used. Another strong possibility is that the X protein in anti-HBe-positive liver cirrhosis might be translated from the integrated HBV DNA into the host genome as described above. Indeed, HBV DNA is integrated into the host chromosome in type B chronic liver disease (17,18). In summary, we detected expression of the X protein in the liver and anti-X in the sera in type B liver disease, and suggested that the X gene might have some relationship with HBV replication. References 1. Tiollais P, Pourcel C, Dejean A. The hepatitis B virus. Nature 1985;317:489-95. 2. Moriarty AM, Alexander H, Lerner RA. Antibodies to peptides detect new hepatitis B antigen: serological correlation with hepatocellular carcinoma. Science 1985;227:429-33. 3. Elfassi E, Haseltine WA, Dienstag L. Detection of hepatitis B virus X product using an open reading frame Escherichia coli expression vector. Proc Nat1 Acad Sci USA 1986;83:2219-22. 4. Feitelson MA. Products of the “X” gene in hepatitis B and related viruses. Hepatology 1986;6:191-8. 5. Chisaka 0, Araki K, Ochiya T, et al. Purification of hepatitis B virus X gene product synthesized in Escherichio coli and its detection in a human hepatoblastoma cell line producing hepatitis B virus. Gene 1987;60:183-9. 6. Kaplan PM, Greenman RL, Gerin JL, Purcell RH, Robinson WS. DNA polymerase associated with human hepatitis B antigen. J Virol 1973;12:995-1005. 7. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-5. 8. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of
998
9.
10.
11.
12.
13.
14.
GASTROENTEROLOGY Vol. 97. No. 4
KATAYAMA ET AL.
proteins from polyacrylamide gels to nitrocellulose sheets. Proc Nat1 Acad Sci USA 1979;76:4350-4. Zamboni L, Martin0 CD. Buffered picric acid-formaldehyde: a new, rapid fixative for electron microscopy. J Cell Biol 1967; 35:148A. Coons A. Fluorescent antibody methods. In: Danielli J, eds. General cytochemical method. New York: Academic, 1958: 399-422. Pfaff E, Salfeld J, Gmelin K, Schaller H, Theilmann L. Synthesis of the X protein of hepatitis B virus in vitro and detection of anti-X antibodies in human sera. Virology 1987; 158:456-60. Meyers ML, Trepo LV, Nath N, Sninsky JJ. Hepatitis B virus polypeptide X: expression in Escherichia coli and identification of specific antibodies in sera from hepatitis B virus infected humans. J Virol 1986;57:101-9. Feitelson MA, Detolla LJ, Zhou X. A chronic carrierlike state is established in nude mice injected with cloned hepatitis B virus DNA. J Virol 1988;62:1408-15. Siddiqui A, Jameel S, Mapoles J. Expression of hepatitis B virus X gene in mammalian cells. Proc Nat1 Acad Sci USA 1987;84:2513-7.
15. Spandau
DF, Lee CH. Trans-activation of viral enhancers by the hepatitis B virus X protein. J Virol 1988;62:427-34. 16. Gowans EJ, Burrell CJ, Jilbert AR, Marmion BP. Cytoplasmic (but not nuclear] hepatitis B core antigen reflects HBV DNA synthesis at the level of the infected hepatocyte. Intervirology 1985;24:220-5. 17. Omata M, Yokosuka 0, Imazeki F, et al. Correlation of hepatitis B virus DNA and antigens in the liver. A study in chronic liver disease. Gastroenterology 1987;92:192-6. 18. Shafritz D, Shouval D, Shermann HI, Hadziyannis SJ, Kew MC. 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;79:7522-6.
Received February 15, 1988. Accepted April 14, 1989. Address requests for reprints to: Norio Hayashi, M.D., First Department of Medicine, Osaka University Medical School, Fukushima l-l-50, Fukushima-ku, Osaka 553, Japan. This work was supported by a Grant-in-Aid from the Ministry of Education, Science, and Culture of Japan.
1989:97:990-6
Detection of Hepatitis B Virus X Gene Protein and Antibody in Type B Chronic Liver Disease KAZUHIRO KATAYAMA, NOR10 HAYASHI, YUTAKA SASAKI, AKINORI KASAHARA, KEIJI UEDA, HIDEYUKI FUSAMOTO, NOBUHIRO SATO, OSAMU CHISAKA, KENICHI MATSUBARA, and TAKENOBU KAMADA First Department of Medicine, Osaka University Medical School, Osaka: and Institute for Molecular and Cellular Biology, Osaka University, Suita, Japan
The genome of the hepatitis B virus contains a sequence (X gene) whose role is unclear. The almost complete region of the hepatitis B virus X gene was expressed in Escherichia coli, with the resulting protein being -17 kilodaltons in molecular weight. Sera from 139 subjects were analyzed by Western blot analysis. Of the hepatitis B surface antigenpositive patients, anti-X was not found in 4 patients with acute hepatitis and in 12 healthy carriers, but was present in 41% (21/51)of the patients with chronic hepatitis, 63% (15/24) of those with liver cirrhosis, and 46% (12/26) of those with hepatocellular carcinoma. The expression of the X product in the liver tissues (43 hepatitis B surface antigenpositive patients) was investigated using an indirect immunohistochemical method. The X protein was observed in 64% (21/33) of the patients with chronic hepatitis and 50% (~/IO) of those with liver cirrhosis, and was found when the serum was negative for anti-X. Hepatitis B core antigen was frequently expressed together with the X protein in the liver. The conclusions reached were that the frequency of anti-X increases with the length of chronic hepatitis B virus infection, that anti-X may suppress the expression of the X protein in the liver, and that the X protein may be related to hepatitis B virus replication.
found a 28-kilodalton protein in HBV-infected livers that was recognized by antiserum prepared against a synthetic peptide from the X open reading frame, and have also detected the antibody against the synthetic peptide and the recombinant protein including the X open reading frame in sera of HBVinfected individuals (2,3). Authentic X polypeptides have also been found in infected liver (4). Although these studies demonstrate the coding potential of the X open reading frame and its association with HBV infection, the expressions of the X protein and anti-X in type B viral infection have not been fully elucidated. The putative viral polypeptide can be produced in Escherichia coli with expression vectors to aid attempts to define the proteins synthesized during viral infection. Chisaka et al. (5) reported the construction of a plasmid encoding a fusion protein consisting of E. coli plasmid pOCTZ-5 and HBV X gene, designated the trpE-X protein. In the present study, we investigated the expression of the X protein in type B liver diseases using this trpE-X protein and detected anti-X antibody in sera and the Xrelated protein in liver tissue. Materials
and
Methods
Subjects
H
epatitis B virus (HBV) is a causative agent for type B viral hepatitis, which has been linked to hepatocellular carcinoma in humans (1).Molecular cloning and nucleotide sequencing have indicated that the genome of HBV contains at least four open reading frames: S, C, P, and X. The roles of the S, C, and P regions have been reported, but the function of the X region is still unclear. Previous studies have
This study examined 156 were hepatitis B surface antigen hepatitis 4, asymptomatic carrier liver cirrhosis 24, hepatocellular
subjects, 129 of whom (HBsAg)-positive (acute 12, chronic hepatitis 63, carcinoma 26), with the
0 1989 by the American Gastroenterological 0016-5065/89/$3.50
Association
October 1989
Table
HBV X PROTEIN AND ANTI-X IN TYPE B HEPATITIS
1. Profile of 129 Patients
With Hepatitis
w n
B Surface
Antigen
(1857) Total serum bilirubin (mgldlj
Serum albumin (g/d0
Age (yrj
ALT (U/L)
26 * 9
537 t 897
3.5 + 3.1
4.3 ? 0.3
25 2 6
18 + 5
0.6 2 0.3
4.3 2 0.2
215 2 316
1.0 2 0.8
4.0 2 0.5
56 2 8
76 5 58
1.7 t 1.2
3.4 2 0.8
51 2 10
64 t 38
4.1 + 8.2
3.4 + 0.5
Disease Acute hepatitis (n = 4) Healthy carrier (n = 12) Chronic hepatitis (n = 63) Liver cirrhosis (n = 24) Hepatocellular carcinoma (n = 26)
991
Figure
39 ? 11
ALT, alanine aminotransferase. SD.
Values are expressed
as mean 2
serologic profiles shown in Table 1. Fourteen subjects were positive for antibody to hepatitis B surface antigen and had chronic liver diseases (chronic hepatitis 4, liver cirrhosis 3, primary biliary cirrhosis 2, hepatocellular 1). Thirteen subjects were negcarcinoma 4, cholangioma ative for HBV markers (normal 3, lupoid hepatitis 1, alcoholic liver injury 1, drug-induced hepatitis 1,chronic hepatitis 5, hepatocellular carcinoma 1, cholangioma 1). Sera from 117 HBsAg-positive and 22 HBsAg-negative subjects were tested for anti-X by Western blot analysis. Liver tissue samples of 43 HBsAg-positive subjects (chronic inactive hepatitis 14, chronic active hepatitis 17, liver cirrhosis 10, chronic active hepatitis with hepatocellular carcinoma 2) and 8 HBsAg-negative subjects (chronic hepatitis 5, liver cirrhosis 2, hepatocellular carcinoma 1) were examined to search for the expression of X protein using an indirect immunohistochemical method. Informed written consent was obtained from all patients examined for X protein in the liver.
Hepatitis
B Viral
Markers
1 Structure of pOCTX. A 584 base pairs fragment (from Barn HI to Bgl II] was excised from pBRadr4 and inserted after the seventh codon of pOCT2-5. This fragment included 145 amino acids [from the 10th to the 154th) of the X gene. The nucleotide number marked along the HBV genome starts from the Xho I site. Ptrp, tryptophan operon promoter; TrpE, TrpE gene.
tryptophan operon, and pBRHBadr4, which carries the entire HBV genome, and constructed a recombinant plasmid pOCTX, which contained the Barn HI-Bgl II fragment (1273-1857, 584-bp) of HBV genome (Figure 1). The plasmid was propagated and transferred to the E. coli strain (KY1603), and produced a fusion protein consisting of N-terminal 7 amino acids of the trpE gene product and C-terminal 145 amino acids of the HBV X gene product. This protein, designated the trpE-X protein, was purified as described previously (5). From 20 g of induced KY1603 wet cells, the trpE-X protein was purified by ion-exchange chromatography. Rabbit polyclonal antisera against the purified trpE-X protein were prepared by the usual method using Freund’s complete adjuvant (5). The purified trpE-X protein was subjected to 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis according to Laemmli (7). The gel was stained with Coomassie brilliant blue. This protein had a molecular weight of -17 kilodaltons, which the HBV X gene could encode (Figure 2), and
in Serum
Hepatitis B surface antigen, antibody to hepatitis B surface antigen, hepatitis B e antigen (HBeAg), antibody to hepatitis B e antigen (anti-HBe), and antibody to hepatitis B core antigen were determined using commercial radioimmunoassays (Abbott Laboratories, North Chicago, Ill.). Serum HBV deoxyribonucleic acid (DNA) was tested by a spot hybridization technique using a commercial kit (Dainabot, Tokyo, Japan). Hepatitis B virus DNA polymerase activity was measured according to Kaplan et al. (6).
Expression
of Hepatitis
B Virus
X Protein
A recombinant plasmid pOCTX was constructed as described previously (5) to express almost the complete region of the HBV X open reading frame. We used the plasmid pOCT2-5, which carries a promoter of E. coli
Figure 2. Identification of HBV X protein. Purified X protein was subjected to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Size markers are indicated in kilodaltons.
992
GASTROENTEROLOGY Vol. 97. No. 4
KATAYAMA ET AL.
anti-X positive human sera were preincubated for 2 h at 37°C with the trpE-X protein, and then analyzed.
indirect
Figure 3. Detection of antibodies against the HBV X protein using Western blot analysis. Lane 1, HBV marker-negative normal subject; lanes z and 3, chronic hepatitis; lanes 4 and 5, liver cirrhosis: lanes 6 and 7, hepatocellular carcinoma; lanes 8 and 9, same patient with chronic hepatitis (anti-X positive); lane 9, the sample had been reacted with the serum preincubated with trpE-X.
reacted peptides
strongly with corresponding
the antisera against synthesized to some parts of the HBV X protein
(5).
Western Blot Analysis To identify the anti-X in the serum, the trpE-X protein was subjected to 15% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and electrophoretitally transferred to nitrocellulose sheets (50 V, 3 h) according to Towbin et al. (8). The strips were made to react with 1:50 dilution of patient sera (37”C, 2 h), incubated with biotinylated goat antiserum to human immunoglobulin, and then incubated with avidin-biotinylated peroxidase complex (Vectastain ABC kit; Vector Laboratories, Inc., Burlingame, Calif.). The strips were finally developed with 4-chloro-l-naphtol. For 2 cases of acute hepatitis, serial sera were examined. To determine binding specificity, the
Table
2. Occurrence
of Antibody
to Hepatitis
B Virus X Antigen
lmmunohistochemical
Method
Liver tissue samples from liver biopsies or operations were fixed with Zamboni’s fixative for -30 min at 4°C (9). Frozen sections were cut on a cryostat to a thickness of 10 pm and stored at -20°C until use. The sections were incubated with 1:500 dilution of rabbit antiserum against the trpE-X protein (4”C, 48 h). Next, in the indirect immunofluorescence method, the sections were incubated with fluorescein isothiocyanate-conjugated goat antiserum against rabbit immunoglobulin G and observed under fluorescent photomicroscopy at 520 nm with excitation at 495 nm (10). When using peroxidaselabeled antibody, after incubation of the sections with anti-trpE-X, they were incubated with biotinylated goat antiserum against rabbit immunoglobulin G, then with avidin-biotinylated peroxidase complex (Vectastain ABC kit). The sections subjected to testing with the Vectastain ABC kit were developed with diaminobenzidine tetrahydrochloride. To determine the specificity of binding, rabbit antiserum (anti-trpE-X) was preincubated with the trpE-X protein for 2 h at 37°C. To examine for hepatitis B core antigen (HBcAg) in the liver, a liver sample was incubated with mouse monoclonal antibody to hepatitis B core antigen (Institute of Immunology, Tokyo, Japan) at 1:20 dilution for 48 h at 4”C, then allowed to react with peroxidase-labeled antimouse immunoglobulin G (Vectastain ABC kit) as described above. The expression of the X protein and HBcAg was graded as follows: (2+), positive hepatocytes accounted for >lO%; (+), staining was
Statistical The statistical uated
using
Fisher’s
Analysis significance exact test.
in Sera From Patients
of the results
With Hepatitis
was eval-
B Virus Infection
HBeAg/anti-HBe Diagnosis HBsAg-positive Acute hepatitis Healthy carrier Chronic hepatitis Liver cirrhosis Hepatocellular carcinoma HBsAg-negative Chronic liver diseaseb HBV markers negative”
+/o/4 o/7 lo/32 (31%) 7110 (70%) 3/7 (43%]
-/-
l/l (100%) l/2 (50%)
-/+ O/2" 015 11/19 (58%) 7/13 (54%) 8117 (47%)
Total
O/6 (0%) o/12 (0%) 21/51 (41%) 15124 (63%) 12/26 (46%) 3/14 (21%) O/8 (0%)
anti-HBe, antibody to hepatitis B e antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen: HBV, hepatitis B virus. n Of 4 patients with acute hepatitis, the serial sera of 2 patients were studied. b These patients with chronic liver disease had developed antibody to hepatitis B surface antigen. ’ These patients did not have hepatitis B surface antigen, antibody to hepatitis B surface antigen, or antibody to hepatitis B core antigen.
October 1989
HBV X PROTEIN AND ANTI-X IN TYPE B HEPATITIS
993
-e 4. Detection of the HBV X protein in liver tissue using an indirect immunohistochemical method. A. Positive staining appeared in the cytoplasm of the liver with chronic hepatitis (x400). B. This section was allowed to react with rabbit amtiserum (anti-trpE-X) preincubated with the trpE-X protein (x400). Positive staining was completely absorbed. Bars = 30 /Jtm.
Results Figure 3 shows representative Western blot analysis results for anti-trpE-X (anti-X) in the sera. Lanes 2, 4, and 6 show cases positive for anti-X, in which the antibody reacted with the 17-kilodalton trpE-X protein. Lanes 1,3,5,and 7 show the negative cases. Lanes 8 and 9 show the strips that had been allowed to react with the serum of 1 patient having anti-X, with the strip in lane 9 having been reacted with the serum preincubated with the trpE-X protein When 117 HBsAg-positive patients were checked for the presence of anti-X (Table 2), it was not found in patients with acute hepatitis and
healthy carriers, but was identified in 21 (41%) of the 51 patients with chronic hepatitis, 15 (63%) of the 24 with liver cirrhosis, and 12 (46%) of the 26 with hepatocellular carcinoma. In chronic hepatitis, antiX was found in 10 (31%) of the 32 HBeAg-positive patients and more often (58%) in patients positive for anti-HBe. The frequency of anti-X was lower in earlier phases of HBV infection, such as in patients with acute hepatitis, healthy carriers, and HBeAgpositive patients with chronic hepatitis than in later phases. Thus, the frequency of anti-X seems to increase with the length of HBV infection, although no significant correlation existed between the fre-
994
GASTROENTEROLOGY Vol. 97. No. 4
KATAYAMA ET AL.
Table 3. Expression of the X Protein in Livers From Patients With Hepatitis B Virus Infection HBeAg/anti-HBe Diagnosis HBsAg-positive Chronic hepatitis Liver cirrhosis Hepatocellular carcinoma HBsAg-negative Chronic hepatitisb Liver cirrhosis” Hepatocellular carcinoma”
-it
t/-
17l23
(74%)
o/4 (0%) o/2 (0%)
4110 (40%) 5/6 (83%)
Total
21133 (64%) 5/10 (50%) o/2 (0%)”
o/5 (0%)
l/2 (50%) O/l (0%)
anti-HBe, antibody to hepatitis B e antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen. ’ The X protein was detected in the noncancerous region (chronic hepatitis) of these 2 patients. b These patients did not have hepatitis B surface antigen, antibody to hepatitis B surface antigen, or antibody to hepatitis B core antigen. ’ These patients had developed antibody to hepatitis B surface antigen.
quency of anti-X and the HBeAg/anti-HBe system. Anti-X was found in 21% of the antibody to hepatitis B surface antigen-positive patients with chronic liver disease, but was not found in subjects who had no HBV markers. The X-positive structures stained by the antiserum against the trpE-X protein were identified in the cytoplasm of hepatocytes (Figure 4A). The X-positive liver sample, which had reacted with the anti-X incubated with the trpE-X protein, showed no staining (Figure 4B); this indicates the specificity for the binding. Clustering positive cells were seen more frequently around the portal area than the central vein area. The X protein was identified in 21 (64%) of 33 patients with chronic hepatitis, which was slightly more than for liver cirrhosis (50%). In chronic hepatitis, the X protein was found more often in HBeAg-positive cases (74%) than in antiHBe-positive cases (40%) (Table 3). In 2 patients with hepatocellular carcinoma, the X protein was found in the noncancerous region (Figure 5B), but not in the cancerous region (Figure 5A). Table 4 shows the appearance of the X protein in the liver and anti-X in the serum in each patient and also the relationship between X/anti-X and the other HBV markers. The anti-X in the serum was negative except in 1 case (No. 19) where the X protein in the liver was positive. The X protein in the liver was not expressed when the anti-X in the serum was positive, except in 1 case (No. 25). Thus, the X protein was found when the serum was negative for anti-X. There did not appear to be a direct relationship between X/anti-X and other HBV markers (Table 4). Thirty-two HBsAg-positive patients (26 with
chronic hepatitis and 6 with liver cirrhosis) were examined for X protein and HBcAg in the liver (Table 5). With chronic hepatitis, the positive rate of HBcAg in the liver with X protein (14/16,87.5%) was significantly higher than that without X protein (l/10, 10%) (p < 0.01). With liver cirrhosis, HBcAg was detected in 3 (75%) of the 4 cases with the X protein, and in 1 (50%) of the 2 cases without the X protein. Furthermore, in the liver with the X protein, HBcAg was found in the 2 cases of chronic hepatitis and in 3 (75%) of 4 cases with liver cirrhosis, in spite of the serum being negative for HBeAg. In some cases, consecutive sections were stained for the X protein and HBcAg. Hepatitis B core antigen-positive cells were mainly found among the X-positive hepatocytes, which frequently showed a wider distribution (Figure 6). Table 4. Expression of Hepatitis B Virus X Protein, Anti-X, and Hepatitis B Virus Markers Patient 1 2 3 4 5
6 7 8
9 10 11
12 13 14
15 16 17 18
19 20 21 22 23 24
25 26 27 28
29 30 31
HBeAgl anti-HBe
DNA-P (cpm)
ilil-
4637
2+/2t/-
t/k
856
2t/-
ti-
420
2t/-
t/-
147
2+/-
t/-
Histology
X/Anti-X
CIH CIH CAH CAH CAH CIH CAH CAH CIH CIH CAH CIH CIH CAH + HCC” LC LC LC LC LC
2t/-
674
2t/-
t/-
2t/-
tt-
2-k/-
tt-
2+1-
-/t
2+/-
t/-
+I-
-/t
24
+I-
t/-
416
2t/-
320 9 9
-/t
3
-Ii
2+/-
-/t
10
2+1-
-/t
71
2+1-
-Ii -Ii
8 8
CIH LC LC LC
-l-
-/t
41
-l-
-II-
0
-l-
+/-
12
-l-
-/t
0
CAH CIH CIH CAH CIH CAH LC LC
-Ii
+I-
-F/t
-1-I
495 4
-Ii
-Ii
0
-I+
+I-
169
-Ii
tt-
-Ii -it -Ii
titi&I-
t t + t t -
400
2t/-
2ilf
HBVDNA
t _ t t + _ _ t + -
16 4173
102 10
anti-HBe, antibody to hepatitis B e antigen: CAH, chrcnic active hepatitis; CIH, chronic inactive hepatitis: DNA-P, deoxyribonucleic acid polymerase; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; LC, liver cirrhosis. a X protein was expressed in hepatocytes of noncancerous region. 2+, positive hepatocytes accounted for >lO%; t, staining was
October 1989
HBV X PROTEIN AND ANTI-X IN TYPE B HEPATITIS
995
Figure 5. Expression of the X protein. The X protein was not expressed in the region of hepatocellular carcinoma (A, x400). but was in the noncancerous region with chronic hepatitis adjacent to the hepatocellular carcinoma (B, ~400). Bars = 30 pm.
Discussion Conservation of the X gene among mammalian hepadnaviruses suggests that it serves a vital function. There have been some reports about the expression of the X gene, but its role has not yet been determined clearly. The most interesting finding in the present study is that the X protein was found in the liver when the serum was negative for anti-X. We also found that anti-X did not appear in the early stage of HBV infection, such as in patients with acute hepatitis and HBeAg-positive asymptomatic carriers. The frequency of anti-X being found in HBeAgpositive patients with chronic hepatitis was less than in the later stage of HBV carriers, such as in
cases of anti-HBe-positive chronic hepatitis or liver cirrhosis. These findings suggested that the frequency of anti-X in serum increased with the length of HBV infection, and the X protein was expressed in the early stage of HBV infection. Indeed, in chronic hepatitis, the frequency of X protein decreased as HBeAg was converted to anti-HBe, but this trend was not seen in liver cirrhosis. As we were only able to examine a limited number of patients with acute hepatitis, healthy carriers, and patients with liver cirrhosis, more patients need to be studied before firm conclusions can be drawn. The presence of anti-X (2,3,11-13)and of the X protein (4) in HBV infection has been noted, but
996
GASTROENTEROLOGY Vol. 97, No. 4
KATAYAMA ET AL.
Figul -e 6. Detection of the X protein [A, x400) and HBcAg (B, x400) in consecutive than that of HBcAg. Bars = 30 pm..
there has been no mention of any relationship between them. Pfaff et al. (11)reported finding anti-X in patients who had been exposed for an extended period to HBV gene products, but they found no X-related proteins in the liver using immunoprecipitation. Meyers et al. (12)also detected the anti-X antibody in 9 of 26 asymptomatic HBV carriers, and indicated that there was no direct relationship between the anti-X and other HBV markers. Pfaff et al. and Meyers et al. used almost the complete region of the HBV X gene (Barn HI-Bgl II) (11,12), which was the part we used, and our results for anti-X agreed with theirs. Moriarty et al. (2) found anti-X antibody predominantly in the sera from patients with hepa-
sections. The distribution
of the X protein was W,ider
tocellular carcinoma when they used synthesized peptides corresponding to certain parts of the HBV X gene. Elfassi et al. (3)also detected anti-X in 3 of 11 patients with chronic active hepatitis using the fusion protein corresponding to 133 amino acids of HBV X gene. These results seem to differ from ours, partly because of the difference of antigenicity between the X protein that we used and previously used peptides (2) or protein (3). In our study, the X protein corresponded to almost the complete region of the HBV X gene, whereas Moriarty et al. used synthesized peptide and Elfassi et al. used fusion protein that did not contain the C terminus. Our work also showed that the X protein was not ex-
October
1989
HBV X PROTEIN AND ANTI-X IN TYPE B HEPATITIS
Table 5. Relationship Between the X Protein and Hepatitis B Core Antigen
Diagnosis Chronic hepatitis
Positive rate of HBcAg
HBcAg
X (liver)
HBeAg (serum)
2+
+
-
Total
+
+
9
3
2
14'
-
0
20
2.
-
+
0 1
04 05
46 I
+
_ +
03
01 00
4 0
+
0
11
-
000
2 0I
14/16 (87.5)"
cirrhosis Liver
(%I
_
1
l/l0 (lO.O)b
314 (75.0)
l/2 (50.0)
HBcAg, hepatitis B core antigen; HBeAg, hepatitis B e antigen. 2 +, positive hepatocytes accounted for >lO%; +, staining was
pressed in the cancerous region, but was in the noncancerous region around it in 2 cases of hepatocellular carcinoma. This implies that the protein is not required for the maintenance of this neoplastic state, but may be required for carcinogenesis. Siddiqui et al. (14) prepared the plasmid vector that contained the HBV X gene, and COS cells were used to express it. They then indicated that the X protein was expressed in the cytoplasm of the COS cell, and suggested that the X protein might be associated with cytoskeletal components. These results are similar to our findings that the X protein was expressed in the cytoplasm of the hepatocyte. Thus, in the early stage of HBV infection, the X protein seems to be expressed in the cytoplasm of hepatocytes, and anti-X seems to occur during HBV infection, accompanied by the disappearance of the X protein from the liver. The expression of the X protein in the early stage of infection might indicate some relationship of it to HBV replication, which is active in the early stage. Spandau and Lee (15) suggested that the HBV X protein was the transactivating factor of the HBV enhancer in vitro. Furthermore, we found the expression of HBcAg, which is a reliable marker of HBV DNA replication (16), where the X protein was expressed. We also noted that HBcAg could be frequently found in patients with the X protein, even with negative serum HBeAg. Thus, our results indicated some relationship of the X protein to HBV replication in vivo. One possibility is that the presence of the X protein in the liver might promote the expression of HBcAg there. Findings reported by Spandau and Lee could support this possibility (15). Frequently, there was a wider distribution of X
997
protein-positive hepatocytes than HBcAg-positive ones, possibly because HBcAg-positive cells might be lysed by cytotoxic T cells but not the X-positive cells. The presence of anti-X seemed to inhibit the expression of the X protein in the liver, but did not directly affect the serum HBV DNA polymerase activity. Further study is needed on the relationship between X and HBV replication. The X protein was sometimes found in liver not containing HBcAg, and also in that containing serum anti-HBe. As the absence of HBcAg in the liver usually indicates the absence of free HBV DNA (17), the X protein might be translated from HBV DNA integrated into the host genome, or the amount of free HBV DNA might have been too small to detect. If the former is the case, the X protein does not necessarily have the same function as authentic X. Generally, the frequency of X protein decreased and that of anti-X increased as HBeAg was converted to anti-HBe. However, in liver cirrhosis, the opposite trend appeared. One possible reason is the small number of patients with cirrhosis that we examined. Twenty-four patients with liver cirrhosis were tested for anti-X, but only the liver tissue from 10 patients could be used. Another strong possibility is that the X protein in anti-HBe-positive liver cirrhosis might be translated from the integrated HBV DNA into the host genome as described above. Indeed, HBV DNA is integrated into the host chromosome in type B chronic liver disease (17,18). In summary, we detected expression of the X protein in the liver and anti-X in the sera in type B liver disease, and suggested that the X gene might have some relationship with HBV replication. References 1. Tiollais P, Pourcel C, Dejean A. The hepatitis B virus. Nature 1985;317:489-95. 2. Moriarty AM, Alexander H, Lerner RA. Antibodies to peptides detect new hepatitis B antigen: serological correlation with hepatocellular carcinoma. Science 1985;227:429-33. 3. Elfassi E, Haseltine WA, Dienstag L. Detection of hepatitis B virus X product using an open reading frame Escherichia coli expression vector. Proc Nat1 Acad Sci USA 1986;83:2219-22. 4. Feitelson MA. Products of the “X” gene in hepatitis B and related viruses. Hepatology 1986;6:191-8. 5. Chisaka 0, Araki K, Ochiya T, et al. Purification of hepatitis B virus X gene product synthesized in Escherichio coli and its detection in a human hepatoblastoma cell line producing hepatitis B virus. Gene 1987;60:183-9. 6. Kaplan PM, Greenman RL, Gerin JL, Purcell RH, Robinson WS. DNA polymerase associated with human hepatitis B antigen. J Virol 1973;12:995-1005. 7. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-5. 8. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of
998
9.
10.
11.
12.
13.
14.
GASTROENTEROLOGY Vol. 97. No. 4
KATAYAMA ET AL.
proteins from polyacrylamide gels to nitrocellulose sheets. Proc Nat1 Acad Sci USA 1979;76:4350-4. Zamboni L, Martin0 CD. Buffered picric acid-formaldehyde: a new, rapid fixative for electron microscopy. J Cell Biol 1967; 35:148A. Coons A. Fluorescent antibody methods. In: Danielli J, eds. General cytochemical method. New York: Academic, 1958: 399-422. Pfaff E, Salfeld J, Gmelin K, Schaller H, Theilmann L. Synthesis of the X protein of hepatitis B virus in vitro and detection of anti-X antibodies in human sera. Virology 1987; 158:456-60. Meyers ML, Trepo LV, Nath N, Sninsky JJ. Hepatitis B virus polypeptide X: expression in Escherichia coli and identification of specific antibodies in sera from hepatitis B virus infected humans. J Virol 1986;57:101-9. Feitelson MA, Detolla LJ, Zhou X. A chronic carrierlike state is established in nude mice injected with cloned hepatitis B virus DNA. J Virol 1988;62:1408-15. Siddiqui A, Jameel S, Mapoles J. Expression of hepatitis B virus X gene in mammalian cells. Proc Nat1 Acad Sci USA 1987;84:2513-7.
15. Spandau
DF, Lee CH. Trans-activation of viral enhancers by the hepatitis B virus X protein. J Virol 1988;62:427-34. 16. Gowans EJ, Burrell CJ, Jilbert AR, Marmion BP. Cytoplasmic (but not nuclear] hepatitis B core antigen reflects HBV DNA synthesis at the level of the infected hepatocyte. Intervirology 1985;24:220-5. 17. Omata M, Yokosuka 0, Imazeki F, et al. Correlation of hepatitis B virus DNA and antigens in the liver. A study in chronic liver disease. Gastroenterology 1987;92:192-6. 18. Shafritz D, Shouval D, Shermann HI, Hadziyannis SJ, Kew MC. 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;79:7522-6.
Received February 15, 1988. Accepted April 14, 1989. Address requests for reprints to: Norio Hayashi, M.D., First Department of Medicine, Osaka University Medical School, Fukushima l-l-50, Fukushima-ku, Osaka 553, Japan. This work was supported by a Grant-in-Aid from the Ministry of Education, Science, and Culture of Japan.