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Expression of pre-S1, pre-S2, and C proteins in duck hepatitis B virus infection
VIROLOGY
167,82-86
(1988)
Expression
of Pre-Sl,
Pre-S2, and C Proteins
OSAMU First Department
YOKOSUKA, of Medicine, Received
in Duck Hepatitis
MASAO OMATA,’ Chiba
September
University
School
18, 1987; accepted
AND
YOSHIMI
of Medicine, April
Chiba,
B Virus Infection IT0
280, Japan
27, 1988
We have examined the expression of duck hepatitis B virus (DHBV)-associated proteins in experimentally infected ducks by an immunoblot (Western) method. The DHBV-related core protein, C protein, was identified at the position of 35,000 Da (P35). Pre-S proteins were recognized as two major bands (P37 and P28), the former representing pre-Sl and the latter pre-SP protein. Expression of the proteins was examined in the early phase of infection in ducklings sequentially sacrificed from 6 hr postinoculation to 10 days. C protein (P35) was detected as early as 24 hr postinoculation. This timing coincided with the exponential increase of RNA transcripts and double-stranded viral DNA. Pre-Sl/ S2 proteins were detected at 3 days postinoculation. The early appearance of C protein suggested that the proteins were utilized for nucleic acid packaging. On the other hand, the late appearance of pre-Sl/S2 proteins suggested that they were utilized in the production of virions near the end of the replication cycle. o 1999Academic press. inc.
that contained 5 X 10’ genome eq/ml, estimated by spot hybridization using cloned DHBV DNA as standard (Tagawa et al., 1986). Serum and liver tissue were obtained at 6, 12, 24, and 36 hr and 3 and 6 days postinoculation by sacrificing three ducklings each time, and two ducklings at 10 days. The serum and liver tissue were immediately frozen and stored at -20 and -8O”C, respectively, until use.
In 1982, Summers and Mason described the presence of a unique replication cycle of duck hepatitis B virus (DHBV) (Summers and Mason, 1982). They extracted nucleocapsid cores from persistently infected liver and showed that viral minus-strand DNA utilized an RNA template (reverse transcription), and plusstrand synthesis occurred on the minus-strand DNA. They proposed a pathway for replication of the genome of hepatitis B-like viruses (Summers and Mason, 1982). In a kinetic study of experimental transmission of duck embryos, Mason et a/. (1983) observed the appearance of supercoiled viral DNA preceding the reverse transcription phase of viral DNA synthesis. We conducted similar studies using 1-day-old ducklings, and studied the kinetics of viral DNA and RNA transcript synthesis (Tagawa eta/., 1985, 1986). We found an exponential increase of 3.5-kb RNA transcripts (“pre-genome” RNA) preceding the appearance of singlestranded DNA. However, there has been little information regarding viral protein synthesis. In this study, we examined the sequential appearance of viral proteins by using an immunoblotting method in the early phase of DHBV infection, and correlated the results with the synthesis of viral DNA and messenger RNA (mRNA). MATERIALS Ducklings
AND
Ducks
We also obtained serum and liver tissue from 19 ducks who were similarly infected and kept for 2 to 135 weeks in the laboratory. Analysis of C, pre-Sl, or Western blotting)
METHODS
Twenty 1-day-old ducklings were obtained from a virus-free flock (Omata et a/., 1984). They were intravenously inoculated with 500 ~1 of DHBV-positive serum requests
0042-6822188
$3.00
for reprints
Copyright Q 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
should
and pre-S2 proteins
(immuno
We homogenized 50 mg of liver tissue in 1% Nonidet-P40, 0.5% deoxycholic acid, and 20 mMTris-HCI (pH 7.5), and kept the homogenate on ice for 10 min. The homogenate was spun at 10,000 rpm for 10 min, and the supernatant was saved for protein quantitation by Lowry’s method (Lowry et al., 195 1). The protein in the homogenate supernatant was denatured in 1%I sodium dodecyl sulfate (SDS) and 2.5% mercaptoethanol for 5 min at 1 OO”C, and 50 pg of denatured protein was applied to a SDS-PAGE (polyacrylamide gel electrophoresis) gel, and electrophoresed for 2 hr at 10 mA. Proteins were electrotransferred to a nitrocellulose membrane (Schleicher & Schuell, BA 85). To demonstrate viral proteins on the nitrocellulose membrane, we used three different antibodies. For nucleocapsid (C protein) staining, we used polyclonal antibody against the core particle derived from liver tissue
(Anas domesticus)
’ To whom
(Anas domesticus)
be addressed. 82
PRE-S/C
PROTEIN
FIG. 1. C protein in the liver homogenate, immunoblotted with antiserum against core particles derived from the liver. A distinct band was observed at the position of 35,000 Da (P35) (lane 1, arrow). No such band was observed in noninfected liver (lane 2).
(a gift of W. S. Mason, Fox Chase Cancer Institute, Philadelphia) (Halpern et a/., 1984). To demonstrate pre-S and S proteins, we used two different antisera: anti-virion antibody and anti-denatured major surface protein antibody. The former antibody was raised in guinea pigs by inoculating purified DHBV particles from serum by rate zonal centrifugation in sucrose and by isopycnic banding in CsCI. The latter antibody was a generous gift from J. Pugh (Pugh et al., 1987). We incubated nitrocellulose papers with each antiserum for 60 min. To prevent nonspecific binding of serum to nitrocellulose papers, they were immersed in 1% gelatin, 20 mM Tris-HCI, 500 mM NaCI, 0.059/o Tween 20 (pH 7.5)for 2 hr before the primary antiserum was applied. After incubation with appropriate secondary antiserum conjugated with horseradish peroxidase, diaminobenzidine was used as substrate. Brownish bands were observed on nitrocellulose paper. To establish the specificity of the staining, thefollowing were confirmed in the preliminary experiments: absence of specific bands with preimmune serum, absence of specific bands in the liver extracts from noninfected animals, and absence of specific bands with antisera previously absorbed with immunogens. RESULTS Nucleocapsid
protein (C protein)
With antiserum against immature core, immunoblotting revealed a sharp band at the position of 35,000 Da (P35) with protein extracts from the infected liver (Fig. 1). Such a band was not observed in the blots with preimmune serum or with protein extracts from noninfected liver (data not shown). Surface
proteins
(S, pre-Sl,
pre-S2)
With antiserum against DHBV-DNA-rich particles derived from serum, immunoblotting revealed two major
IN DHBV
83
INFECTION
bands, P37 and P28; these bands appeared only with protein extracts from the infected liver (Fig. 2a). To clarify that the anti-virion antibody does not cross-react with anti-core protein, a parallel immunoblot was performed on a DHBV-positive liver specimen with anticore antibody and with anti-virion antibody (Fig. 2b). The two bands of P37 and P28, demonstrated by antivirion antibody, were not abolished by prior incubation of anti-core antibody serum (Fig. 2b, lanes 1 and 2). The P35 band which was demonstrated by anti-core antibody was not blocked by prior incubation of antivirion antibody (Fig. 2b, lanes 3 and 4). These results indicated that our anti-virion antibody did not contain anti-core antibody activity. The predicted molecular weight of pre-Sl and pre-S2 from the encoding sequence of DHBV DNA indicated that the P37 protein seems to represent pre-Sl and P28 protein pre-S2 protein (Mandardt er al., 1984). With anti-serum against denatured major surface protein, three bands, P37, P28, and P17, were recognized (Fig. 2~). These data indicated that P37 and P28, demonstrated by anti-virion antibody, have the major surface protein component (Fig. 2~). No such bands were observed in the immunoblot with preimmune serum or in the homogenate from noninfected liver. The intensity of P37 was generally weaker than that of P28. SDS-PAGE analysis of DHBV particles derived from serum revealed a major protein band at the position of 17,000 Da (P17) (Fig. 2d, lane 1). This was consistent with the size of major surface protein (167 amino acids, no potential glycosylation site predicted from nucleotide sequence) (Mandardt et al., 1984). However, immunoblotting with anti-virion antibody did not reveal a specific band at the position of 17,000 Da (Figs. 2a, b, c). The immunoreactive P17 band was recognized only by anti-denatured major surface antibody (Fig. 2c, lane 2). These data indicated that the anti-surface (major protein) antibody is a “conformational” antibody as reported for hepatitis B virus (Mishiro et a/., 1980) and could not detect the major surface protein once reduced and denatured into component proteins. Viral proteins
in early phase of infection
Appearance of Cprotein (P35). The appearance of C protein was serially studied in the liver from 6 hr to 10 days postinoculation. C protein was detected as early as 24 hr postinoculation (Fig. 3). The P35 band was markedly intensified at Day 3. Multiple smaller bands (P30, P28, P27, P19, P16) were detected after 3 days postinoculation (Fig. 3). Appearance of pre-S 1 and pre-S2 protein (P37 and P28). The appearance of pre-Sl and pre-S2 protein was serially studied in the liver from 6 hr to 10 days postinoculation. P37 and P28 were first detected at 3
84
YOKOSUKA,
OMATA,
AND
ITO
P37 -
P37--,
P28 -
P28 -
-
P37
~27 P22
P28 Pl7
-PI7
FIG. 2. (a) Pre-Sl/S2 proteins in the liver homogenate, immunoblotted with antiserum against DHBV-DNA-rich particles in the serum, Two bands, P37 and P28. were observed in the infected liver (lane l), but no specific band was observed in noninfected liver (lane 2). (b) Immunoblotting of infected liver tissue. P37 and P28 bands were observed by anti-virion antibody (lane 1). These bands were not abolished by prior incubation of nitrocellulose paper with anti-core serum (lane 2). A strong band of P35 was observed by anti-core antibody (lane 3). This band was not abolished by prior incubation of nitrocellulose paper with anti-virion antibody (lane 4). These results indicated that there was no cross-reactivity between anti-core and anti-virion antisera. (c) Pre-Sl , pre-S2, and S proteins in the infected liver homogenate, immunoblotted with anti-virion antibody (lane 1) and with anti-denatured major surface antibody (a gift of 1. Pugh) (lane 2). Anti-denatured major surface antibody could recognize bands at the positions of 37,000, 28,000, and 17,000 Da (lane 2) whereas anti-virion antibody immunoblotted bands at the positions of 37,000 and 28,000 Da (lane 1). (d) SDS-PAGE protein profile of major S proteins of duck hepatitis B virus (lane 1) and hepatitis B virus (lane 2). DHBV and HBV were purified from serum (10 ml) by ultracentrifugation with a sucrose cushion and CsCl density gradient. A single band (P17, arrow) was observed in purified DHBV preparation by Coomassie blue staining (lane 1). However this P17 band was not reactive with anti-virionantibody by immunoblotting. In lane 2, two major S protein bands of HBV (P22 and P27, arrowheads) were shown by Coomassie blue staining.
days postinoculation (Fig. 4). The intensity of P28 increased at 6 days. However, the intensity of P37 was unchanged. After 6 days postinoculation, multiple smaller bands (P26, P23, P21, P19) were identified.
S2 protein (P28) was found in 16 of 19 (84%). The preSl band was generally weaker than that of pre-S2 in its intensity. DISCUSSION
Viral proteins
in chronic
C, pre-Sl, and liver of 19 ducks weeks. C protein protein (P37) was
infection
pre-S2 proteins were studied in the with chronic infection of 2 to 135 was detected in all (Table 1). Pre-Sl detected in 12 of 19 (63%) and pre-
123458789
FIG. 3. lmmunoblotting of DHBVcore antigen protein in livertissue, serially obtained from 6 hr postinoculation to 10 days. Lane 1, noninfected duck (negative control); lane 2, 6 hr; lane 3, 12 hr; lane 4. 24 hr; lane 5, 36 hr; lane 6, 3 days; lane 7, 6 days; lane 8, 10 days postinoculation. Lane 9, persistently infected 1 B-week-old duck liver (positive control). Thick arrow indicates a 35,000-Da core protein (P35). Thin arrows indicate 30.000, 28,000, 27,000, 19,000 and 16,000 Da.
Although knowledge about DHBV DNA and RNA is increasing, information regarding viral proteins of DHBV is still limited. Marion et a/. (1983) reported that SDS-PAGE revealed the major protein of DHBV virus particles derived from serum to be a single 17,500-Da
1
2
3
458789
FIG. 4. lmmunoblotting of DHBV pre-S proteins in liver tissue, serially obtained from 6 hr postinoculation to 10 days. Lane 1, noninfected duck liver (negative control); lane 2, 6 hr; lane 3, 12 hr; lane 4, 24 hr; lane 5. 36 hr; lane 6,3 days; lane 7.6 days; lane 8, 10 days; lane 9, infected 16-week-old duck liver (positive control). Two thick arrows indicate 37,000 and 28,000 Da, respectively. Thin arrows indicate 26,000, 23,000,21,000 and 19,000 Da.
PRE-S/C TABLE C, PRE-Sl,
No. positive/No.
PROTEIN
1
AND PRE-S2 PROTEINS DETECTED BY IMMUNOBLOTTING THE LIVER OF DUCKS WITH CHRONIC INFECTION
examined
IN
C protein (P35)
Pre-S 1 protein
Pre-S2 protein
19/19 (100%)
12119 (63%)
(84%)
16/19
nonglycosylated polypeptide. Halpern et a/. (1984) using immunoblotting estimated that the molecular size of the DHBV core protein derived from liver tissue was 35,000 Da (P35). Regarding pre-S protein, Pugh et al. (1987) by Western blotting found one band at the position of 36,000 Da. In this study, we extracted protein from liver tissue and denatured and electroblotted it to a nitrocellulose membrane. For immunoblotting we used three antisera: one against surface component of the virus pat-ticle of serum origin (anti-virion antibody), one against the denatured major surface protein (Pugh eta/., 1987), and another against the immature core derived from liver tissue (Halpern et a/., 1984). With regard to the DHBV core protein, we detected a single band at the position of 35,000 Da (P35) in the liver of infected ducks. This result was consistent with the core protein molecular weight value estimated from the encoding region of DHBV DNA for core antigen (338 amino acids) (Mandardt et al., 1984) and with the results obtained by Halpern et al. (1984). With regard to the DHBV major surface protein, we were able to obtain a band at the position of 17,000 Da (P17) by SDS-PAGE analysis and with antiserum against the denatured major surface protein by immunoblotting. However, we were unable to obtain a band at the position of 17,000 Da with anti-virion antibody by immunoblotting. This suggests that the antigenicity of the major surface protein (P17) is conformation dependent, and our antibody raised against virions of serum origin was a conformational antibody and could not recognize the denatured surface protein by immunoblotting (Mishiro et a/., 1980). In contrast to the major surface protein, we observed two specific bands (P37 and P28) in infected liver by immunoblotting with anti-virion antiserum. The study using antibody against the denatured major surface protein revealed that these two bands have the major surface protein as their component (Fig. 2~). As predicted from the viral DNA sequence, pre-Sl and preS2 proteins could be composed of 364 and 276 amino acids, respectively (Mandardt et a/., 1984). Thus, P37 and P28 may represent pre-Sl and pre-S2, respec-
IN DHBV
INFECTION
85
tively. In a previous study, Pugh eta/. reported the presence of a 36,000-Da polypeptide by immunoblotting using antibody raised against the fusion protein produced by recombinant DNA containing the pre-S coding sequence (Pugh et al., 1987). However, there was no mention about a smaller protein, corresponding to our P28, in their article (Pugh et a/., 1987). In human and woodchuck hepatitis B-like viruses, two classes of proteins, pre-Sl and pre-S2, are well documented (Heerman et al., 1984; Schaeffer et al., 1986). Thus, the evidence suggests that P28 is pre-S2 protein of the DHBV surface protein. Since we were able to demonstrate P28 band in our infected liver by anti-denatured major surface protein antibody, kindly supplied by Dr. Pugh, one possible explanation for discrepancy about pre-S2 protein is the difference in virus strains used. We used inoculum of Chinese origin. A critical test of these possibilities will require sequence analysis of individual proteins or immunoblotting with antiserum against individual segments of viral proteins. Our previous kinetic study of viral DNA and RNA transcripts in the liver of one-day-old ducklings inoculated with 500 ~1 of 5 x 10’ genome eq/ml revealed the following: (1) generation of supercoiled DNA at 6 hr postinoculation; (2) transcription of “pre-genome” mRNA at 12 hr; (3) increase of pregenome and synthesis of double-stranded DNA at 24 hr; (4) secretion of virion at 3 days (Tagawa et al., 1986). In this experiment, we utilized frozen liver tissue obtained at the same time as the previous work (Tagawa et a/., 1986) and studied the kinetics of viral protein synthesis. The synthesis of C protein was detected in the relatively early phase of infection (24 hr postinoculation). Pre-genome mRNA is exponentially increased and single- and doublestranded DNA begins to be synthesized at 24 hr postinoculation. Thus, it is highly suggestive that C protein (P35) was utilized for nucleic acid packaging. In contrast to the production of C protein, pre-Sl and pre-S2 proteins were produced relatively late in the infection (3 days postinoculation). Recent data suggested that pre-Sl and pre-S2 proteins were important components of the virion (Dane particle) and pre-Sl and pre-S2 proteins were more concentrated in the virion than in the small spherical particles (Heerman et al., 1984). Thus, it is tempting to speculate that the production of pre-Sl and pre-S2 proteins in hepatocytes coincided with the production of virions. In fact, preSl and pre-S2 were detected at 3 days postinoculation when viral DNA (virion) was first secreted in the serum (Tagawa et al., 1986). Thus, it appears that viral protein synthesis in the early phase of DHBV infection starts with core protein production and the maturation of the virion is complete when the pre-Sl and S2 are synthesized.
86
YOKOSUKA,
In chronic infection, C protein was detected in all 19 ducks by immunoblotting. By conventional immunohistological methods with horseradish peroxidase, we were able to demonstrate positive staining in only 77% of these cases (unpublished data), confirming the immunoblot method is fairly sensitive in detecting a small amount of protein in the tissue. With this method, preSl protein was usually found less frequently and less intensely than pre-S2 protein in liver tissue. The significance of the difference is yet to be determined. REFERENCES HALPERN, M. S., ENGLAND, J. M., FLORES, L., EGAN, J., NEWBOLD, J., and MASON, W. S. (1984). Individual cells in tissues of DHBV-infected ducks express antigens crossreactive with those on virus surface antigen particles and immature viral cores. Virology 137, 408413, HEERMAN, K. H., GOLDMANN, U., SCHWARTZ, W., SEYFFARTH, T.. BAUMGARTEN, H., and GERLICH, W. H. (1984). Large surface proteins of hepatitis B virus containing the pre-S sequence. 1. Viral. 52,396-402. LOWRY, 0. H., ROSENBROUGH, N. J., FORR, A. L., and RANDALL, R. J. (1951). Protein measurement with the phenol reagent. J. Biol. Chem. 193,265-273. MANDARDT, E., KAY, A., and GALIBERT, F. (1984). Nucleotide sequence of a cloned duck hepatitis B virus genome: Comparison with woodchuck and human hepatitis B virus sequence. 1. Viral. 49,782-792.
OMATA,
AND
IT0
MARION, P. L., KNIGHT, S. S., FEITELSON, M. A., OSHIRO, L. S., and ROBINSON, W. S. (1983). Major polypeptide of duck hepatitis B surface antigen particles. J. Viral. 48, 534-541. MASON, W. S., HALPERN, M. S., ENGLAND, 1. M.. SEAL, G., EGAN, J., COATES, L., ALDRICH, C., and SUMMERS, J. (1983). Experimental transmission of duck hepatitis B virus. Virology 131, 375-384. MISHIRO, S., IMAI, M., TAKAHASHI, K., MACHIDA, A., GOTANDA, T., MIYAKAWA, T., and MAYUMI, M. (1980). A 49,000-dalton polypeptide bearing all antigenic determinants and full immunogenicity of 22nm hepatitis B surface antigen particles. J. Immunol. 124, 15891593. OMATA, M., YOKOSUKA, O., IMAZEKI, F., MATSUYAMA, Y., UCHIUMI, K., ITO, Y., MORI, J., and OKUDA, K. (1984). Transmission of duck hepatitis B virus from Chinese carrier ducks to Japanese ducklings: A study of viral DNA in serum and tissue. Hepafology4, 603-607. PUGH, J. C., SNINSKY, J. J., SUMMERS, J., and SCHAEFFER, E. (1987). Characterization of a pre-S polypeptide on the surface of infectious avian hepadnavirus particles. J. Viral. 81, 1384-l 390. SCHAEFFER, E., SNYDER, R. L., and SNINSKY, J. J. (1986). Identification and localization of pre-S-encoded polypeptides from woodchuck and ground squirrel hepatitis viruses. J. Viral. 57, 173-l 82. SUMMERS, J., and MASON, W. S. (1982). Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 29,403-415. TAGAWA, M., OMATA, M., and OKUDA, K. (1986). Appearance of viral RNA transcripts in the early stage of duck hepatitis B virus infection. Virology 152,477-482. TAGAWA, M., OMATA, M., YOKOSUKA. O., UCHIUMI, K., IMAZEKI, F., and OKUDA, K. (1985). Early events in duck hepatitis B virus infection: Sequential appearance of viral deoxyribonucleic acid in the liver, pancreas, kidney and spleen. Gasrroenferology89, 1224-l 229.
167,82-86
(1988)
Expression
of Pre-Sl,
Pre-S2, and C Proteins
OSAMU First Department
YOKOSUKA, of Medicine, Received
in Duck Hepatitis
MASAO OMATA,’ Chiba
September
University
School
18, 1987; accepted
AND
YOSHIMI
of Medicine, April
Chiba,
B Virus Infection IT0
280, Japan
27, 1988
We have examined the expression of duck hepatitis B virus (DHBV)-associated proteins in experimentally infected ducks by an immunoblot (Western) method. The DHBV-related core protein, C protein, was identified at the position of 35,000 Da (P35). Pre-S proteins were recognized as two major bands (P37 and P28), the former representing pre-Sl and the latter pre-SP protein. Expression of the proteins was examined in the early phase of infection in ducklings sequentially sacrificed from 6 hr postinoculation to 10 days. C protein (P35) was detected as early as 24 hr postinoculation. This timing coincided with the exponential increase of RNA transcripts and double-stranded viral DNA. Pre-Sl/ S2 proteins were detected at 3 days postinoculation. The early appearance of C protein suggested that the proteins were utilized for nucleic acid packaging. On the other hand, the late appearance of pre-Sl/S2 proteins suggested that they were utilized in the production of virions near the end of the replication cycle. o 1999Academic press. inc.
that contained 5 X 10’ genome eq/ml, estimated by spot hybridization using cloned DHBV DNA as standard (Tagawa et al., 1986). Serum and liver tissue were obtained at 6, 12, 24, and 36 hr and 3 and 6 days postinoculation by sacrificing three ducklings each time, and two ducklings at 10 days. The serum and liver tissue were immediately frozen and stored at -20 and -8O”C, respectively, until use.
In 1982, Summers and Mason described the presence of a unique replication cycle of duck hepatitis B virus (DHBV) (Summers and Mason, 1982). They extracted nucleocapsid cores from persistently infected liver and showed that viral minus-strand DNA utilized an RNA template (reverse transcription), and plusstrand synthesis occurred on the minus-strand DNA. They proposed a pathway for replication of the genome of hepatitis B-like viruses (Summers and Mason, 1982). In a kinetic study of experimental transmission of duck embryos, Mason et a/. (1983) observed the appearance of supercoiled viral DNA preceding the reverse transcription phase of viral DNA synthesis. We conducted similar studies using 1-day-old ducklings, and studied the kinetics of viral DNA and RNA transcript synthesis (Tagawa eta/., 1985, 1986). We found an exponential increase of 3.5-kb RNA transcripts (“pre-genome” RNA) preceding the appearance of singlestranded DNA. However, there has been little information regarding viral protein synthesis. In this study, we examined the sequential appearance of viral proteins by using an immunoblotting method in the early phase of DHBV infection, and correlated the results with the synthesis of viral DNA and messenger RNA (mRNA). MATERIALS Ducklings
AND
Ducks
We also obtained serum and liver tissue from 19 ducks who were similarly infected and kept for 2 to 135 weeks in the laboratory. Analysis of C, pre-Sl, or Western blotting)
METHODS
Twenty 1-day-old ducklings were obtained from a virus-free flock (Omata et a/., 1984). They were intravenously inoculated with 500 ~1 of DHBV-positive serum requests
0042-6822188
$3.00
for reprints
Copyright Q 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
should
and pre-S2 proteins
(immuno
We homogenized 50 mg of liver tissue in 1% Nonidet-P40, 0.5% deoxycholic acid, and 20 mMTris-HCI (pH 7.5), and kept the homogenate on ice for 10 min. The homogenate was spun at 10,000 rpm for 10 min, and the supernatant was saved for protein quantitation by Lowry’s method (Lowry et al., 195 1). The protein in the homogenate supernatant was denatured in 1%I sodium dodecyl sulfate (SDS) and 2.5% mercaptoethanol for 5 min at 1 OO”C, and 50 pg of denatured protein was applied to a SDS-PAGE (polyacrylamide gel electrophoresis) gel, and electrophoresed for 2 hr at 10 mA. Proteins were electrotransferred to a nitrocellulose membrane (Schleicher & Schuell, BA 85). To demonstrate viral proteins on the nitrocellulose membrane, we used three different antibodies. For nucleocapsid (C protein) staining, we used polyclonal antibody against the core particle derived from liver tissue
(Anas domesticus)
’ To whom
(Anas domesticus)
be addressed. 82
PRE-S/C
PROTEIN
FIG. 1. C protein in the liver homogenate, immunoblotted with antiserum against core particles derived from the liver. A distinct band was observed at the position of 35,000 Da (P35) (lane 1, arrow). No such band was observed in noninfected liver (lane 2).
(a gift of W. S. Mason, Fox Chase Cancer Institute, Philadelphia) (Halpern et a/., 1984). To demonstrate pre-S and S proteins, we used two different antisera: anti-virion antibody and anti-denatured major surface protein antibody. The former antibody was raised in guinea pigs by inoculating purified DHBV particles from serum by rate zonal centrifugation in sucrose and by isopycnic banding in CsCI. The latter antibody was a generous gift from J. Pugh (Pugh et al., 1987). We incubated nitrocellulose papers with each antiserum for 60 min. To prevent nonspecific binding of serum to nitrocellulose papers, they were immersed in 1% gelatin, 20 mM Tris-HCI, 500 mM NaCI, 0.059/o Tween 20 (pH 7.5)for 2 hr before the primary antiserum was applied. After incubation with appropriate secondary antiserum conjugated with horseradish peroxidase, diaminobenzidine was used as substrate. Brownish bands were observed on nitrocellulose paper. To establish the specificity of the staining, thefollowing were confirmed in the preliminary experiments: absence of specific bands with preimmune serum, absence of specific bands in the liver extracts from noninfected animals, and absence of specific bands with antisera previously absorbed with immunogens. RESULTS Nucleocapsid
protein (C protein)
With antiserum against immature core, immunoblotting revealed a sharp band at the position of 35,000 Da (P35) with protein extracts from the infected liver (Fig. 1). Such a band was not observed in the blots with preimmune serum or with protein extracts from noninfected liver (data not shown). Surface
proteins
(S, pre-Sl,
pre-S2)
With antiserum against DHBV-DNA-rich particles derived from serum, immunoblotting revealed two major
IN DHBV
83
INFECTION
bands, P37 and P28; these bands appeared only with protein extracts from the infected liver (Fig. 2a). To clarify that the anti-virion antibody does not cross-react with anti-core protein, a parallel immunoblot was performed on a DHBV-positive liver specimen with anticore antibody and with anti-virion antibody (Fig. 2b). The two bands of P37 and P28, demonstrated by antivirion antibody, were not abolished by prior incubation of anti-core antibody serum (Fig. 2b, lanes 1 and 2). The P35 band which was demonstrated by anti-core antibody was not blocked by prior incubation of antivirion antibody (Fig. 2b, lanes 3 and 4). These results indicated that our anti-virion antibody did not contain anti-core antibody activity. The predicted molecular weight of pre-Sl and pre-S2 from the encoding sequence of DHBV DNA indicated that the P37 protein seems to represent pre-Sl and P28 protein pre-S2 protein (Mandardt er al., 1984). With anti-serum against denatured major surface protein, three bands, P37, P28, and P17, were recognized (Fig. 2~). These data indicated that P37 and P28, demonstrated by anti-virion antibody, have the major surface protein component (Fig. 2~). No such bands were observed in the immunoblot with preimmune serum or in the homogenate from noninfected liver. The intensity of P37 was generally weaker than that of P28. SDS-PAGE analysis of DHBV particles derived from serum revealed a major protein band at the position of 17,000 Da (P17) (Fig. 2d, lane 1). This was consistent with the size of major surface protein (167 amino acids, no potential glycosylation site predicted from nucleotide sequence) (Mandardt et al., 1984). However, immunoblotting with anti-virion antibody did not reveal a specific band at the position of 17,000 Da (Figs. 2a, b, c). The immunoreactive P17 band was recognized only by anti-denatured major surface antibody (Fig. 2c, lane 2). These data indicated that the anti-surface (major protein) antibody is a “conformational” antibody as reported for hepatitis B virus (Mishiro et a/., 1980) and could not detect the major surface protein once reduced and denatured into component proteins. Viral proteins
in early phase of infection
Appearance of Cprotein (P35). The appearance of C protein was serially studied in the liver from 6 hr to 10 days postinoculation. C protein was detected as early as 24 hr postinoculation (Fig. 3). The P35 band was markedly intensified at Day 3. Multiple smaller bands (P30, P28, P27, P19, P16) were detected after 3 days postinoculation (Fig. 3). Appearance of pre-S 1 and pre-S2 protein (P37 and P28). The appearance of pre-Sl and pre-S2 protein was serially studied in the liver from 6 hr to 10 days postinoculation. P37 and P28 were first detected at 3
84
YOKOSUKA,
OMATA,
AND
ITO
P37 -
P37--,
P28 -
P28 -
-
P37
~27 P22
P28 Pl7
-PI7
FIG. 2. (a) Pre-Sl/S2 proteins in the liver homogenate, immunoblotted with antiserum against DHBV-DNA-rich particles in the serum, Two bands, P37 and P28. were observed in the infected liver (lane l), but no specific band was observed in noninfected liver (lane 2). (b) Immunoblotting of infected liver tissue. P37 and P28 bands were observed by anti-virion antibody (lane 1). These bands were not abolished by prior incubation of nitrocellulose paper with anti-core serum (lane 2). A strong band of P35 was observed by anti-core antibody (lane 3). This band was not abolished by prior incubation of nitrocellulose paper with anti-virion antibody (lane 4). These results indicated that there was no cross-reactivity between anti-core and anti-virion antisera. (c) Pre-Sl , pre-S2, and S proteins in the infected liver homogenate, immunoblotted with anti-virion antibody (lane 1) and with anti-denatured major surface antibody (a gift of 1. Pugh) (lane 2). Anti-denatured major surface antibody could recognize bands at the positions of 37,000, 28,000, and 17,000 Da (lane 2) whereas anti-virion antibody immunoblotted bands at the positions of 37,000 and 28,000 Da (lane 1). (d) SDS-PAGE protein profile of major S proteins of duck hepatitis B virus (lane 1) and hepatitis B virus (lane 2). DHBV and HBV were purified from serum (10 ml) by ultracentrifugation with a sucrose cushion and CsCl density gradient. A single band (P17, arrow) was observed in purified DHBV preparation by Coomassie blue staining (lane 1). However this P17 band was not reactive with anti-virionantibody by immunoblotting. In lane 2, two major S protein bands of HBV (P22 and P27, arrowheads) were shown by Coomassie blue staining.
days postinoculation (Fig. 4). The intensity of P28 increased at 6 days. However, the intensity of P37 was unchanged. After 6 days postinoculation, multiple smaller bands (P26, P23, P21, P19) were identified.
S2 protein (P28) was found in 16 of 19 (84%). The preSl band was generally weaker than that of pre-S2 in its intensity. DISCUSSION
Viral proteins
in chronic
C, pre-Sl, and liver of 19 ducks weeks. C protein protein (P37) was
infection
pre-S2 proteins were studied in the with chronic infection of 2 to 135 was detected in all (Table 1). Pre-Sl detected in 12 of 19 (63%) and pre-
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FIG. 3. lmmunoblotting of DHBVcore antigen protein in livertissue, serially obtained from 6 hr postinoculation to 10 days. Lane 1, noninfected duck (negative control); lane 2, 6 hr; lane 3, 12 hr; lane 4. 24 hr; lane 5, 36 hr; lane 6, 3 days; lane 7, 6 days; lane 8, 10 days postinoculation. Lane 9, persistently infected 1 B-week-old duck liver (positive control). Thick arrow indicates a 35,000-Da core protein (P35). Thin arrows indicate 30.000, 28,000, 27,000, 19,000 and 16,000 Da.
Although knowledge about DHBV DNA and RNA is increasing, information regarding viral proteins of DHBV is still limited. Marion et a/. (1983) reported that SDS-PAGE revealed the major protein of DHBV virus particles derived from serum to be a single 17,500-Da
1
2
3
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FIG. 4. lmmunoblotting of DHBV pre-S proteins in liver tissue, serially obtained from 6 hr postinoculation to 10 days. Lane 1, noninfected duck liver (negative control); lane 2, 6 hr; lane 3, 12 hr; lane 4, 24 hr; lane 5. 36 hr; lane 6,3 days; lane 7.6 days; lane 8, 10 days; lane 9, infected 16-week-old duck liver (positive control). Two thick arrows indicate 37,000 and 28,000 Da, respectively. Thin arrows indicate 26,000, 23,000,21,000 and 19,000 Da.
PRE-S/C TABLE C, PRE-Sl,
No. positive/No.
PROTEIN
1
AND PRE-S2 PROTEINS DETECTED BY IMMUNOBLOTTING THE LIVER OF DUCKS WITH CHRONIC INFECTION
examined
IN
C protein (P35)
Pre-S 1 protein
Pre-S2 protein
19/19 (100%)
12119 (63%)
(84%)
16/19
nonglycosylated polypeptide. Halpern et a/. (1984) using immunoblotting estimated that the molecular size of the DHBV core protein derived from liver tissue was 35,000 Da (P35). Regarding pre-S protein, Pugh et al. (1987) by Western blotting found one band at the position of 36,000 Da. In this study, we extracted protein from liver tissue and denatured and electroblotted it to a nitrocellulose membrane. For immunoblotting we used three antisera: one against surface component of the virus pat-ticle of serum origin (anti-virion antibody), one against the denatured major surface protein (Pugh eta/., 1987), and another against the immature core derived from liver tissue (Halpern et a/., 1984). With regard to the DHBV core protein, we detected a single band at the position of 35,000 Da (P35) in the liver of infected ducks. This result was consistent with the core protein molecular weight value estimated from the encoding region of DHBV DNA for core antigen (338 amino acids) (Mandardt et al., 1984) and with the results obtained by Halpern et al. (1984). With regard to the DHBV major surface protein, we were able to obtain a band at the position of 17,000 Da (P17) by SDS-PAGE analysis and with antiserum against the denatured major surface protein by immunoblotting. However, we were unable to obtain a band at the position of 17,000 Da with anti-virion antibody by immunoblotting. This suggests that the antigenicity of the major surface protein (P17) is conformation dependent, and our antibody raised against virions of serum origin was a conformational antibody and could not recognize the denatured surface protein by immunoblotting (Mishiro et a/., 1980). In contrast to the major surface protein, we observed two specific bands (P37 and P28) in infected liver by immunoblotting with anti-virion antiserum. The study using antibody against the denatured major surface protein revealed that these two bands have the major surface protein as their component (Fig. 2~). As predicted from the viral DNA sequence, pre-Sl and preS2 proteins could be composed of 364 and 276 amino acids, respectively (Mandardt et a/., 1984). Thus, P37 and P28 may represent pre-Sl and pre-S2, respec-
IN DHBV
INFECTION
85
tively. In a previous study, Pugh eta/. reported the presence of a 36,000-Da polypeptide by immunoblotting using antibody raised against the fusion protein produced by recombinant DNA containing the pre-S coding sequence (Pugh et al., 1987). However, there was no mention about a smaller protein, corresponding to our P28, in their article (Pugh et a/., 1987). In human and woodchuck hepatitis B-like viruses, two classes of proteins, pre-Sl and pre-S2, are well documented (Heerman et al., 1984; Schaeffer et al., 1986). Thus, the evidence suggests that P28 is pre-S2 protein of the DHBV surface protein. Since we were able to demonstrate P28 band in our infected liver by anti-denatured major surface protein antibody, kindly supplied by Dr. Pugh, one possible explanation for discrepancy about pre-S2 protein is the difference in virus strains used. We used inoculum of Chinese origin. A critical test of these possibilities will require sequence analysis of individual proteins or immunoblotting with antiserum against individual segments of viral proteins. Our previous kinetic study of viral DNA and RNA transcripts in the liver of one-day-old ducklings inoculated with 500 ~1 of 5 x 10’ genome eq/ml revealed the following: (1) generation of supercoiled DNA at 6 hr postinoculation; (2) transcription of “pre-genome” mRNA at 12 hr; (3) increase of pregenome and synthesis of double-stranded DNA at 24 hr; (4) secretion of virion at 3 days (Tagawa et al., 1986). In this experiment, we utilized frozen liver tissue obtained at the same time as the previous work (Tagawa et a/., 1986) and studied the kinetics of viral protein synthesis. The synthesis of C protein was detected in the relatively early phase of infection (24 hr postinoculation). Pre-genome mRNA is exponentially increased and single- and doublestranded DNA begins to be synthesized at 24 hr postinoculation. Thus, it is highly suggestive that C protein (P35) was utilized for nucleic acid packaging. In contrast to the production of C protein, pre-Sl and pre-S2 proteins were produced relatively late in the infection (3 days postinoculation). Recent data suggested that pre-Sl and pre-S2 proteins were important components of the virion (Dane particle) and pre-Sl and pre-S2 proteins were more concentrated in the virion than in the small spherical particles (Heerman et al., 1984). Thus, it is tempting to speculate that the production of pre-Sl and pre-S2 proteins in hepatocytes coincided with the production of virions. In fact, preSl and pre-S2 were detected at 3 days postinoculation when viral DNA (virion) was first secreted in the serum (Tagawa et al., 1986). Thus, it appears that viral protein synthesis in the early phase of DHBV infection starts with core protein production and the maturation of the virion is complete when the pre-Sl and S2 are synthesized.
86
YOKOSUKA,
In chronic infection, C protein was detected in all 19 ducks by immunoblotting. By conventional immunohistological methods with horseradish peroxidase, we were able to demonstrate positive staining in only 77% of these cases (unpublished data), confirming the immunoblot method is fairly sensitive in detecting a small amount of protein in the tissue. With this method, preSl protein was usually found less frequently and less intensely than pre-S2 protein in liver tissue. The significance of the difference is yet to be determined. REFERENCES HALPERN, M. S., ENGLAND, J. M., FLORES, L., EGAN, J., NEWBOLD, J., and MASON, W. S. (1984). Individual cells in tissues of DHBV-infected ducks express antigens crossreactive with those on virus surface antigen particles and immature viral cores. Virology 137, 408413, HEERMAN, K. H., GOLDMANN, U., SCHWARTZ, W., SEYFFARTH, T.. BAUMGARTEN, H., and GERLICH, W. H. (1984). Large surface proteins of hepatitis B virus containing the pre-S sequence. 1. Viral. 52,396-402. LOWRY, 0. H., ROSENBROUGH, N. J., FORR, A. L., and RANDALL, R. J. (1951). Protein measurement with the phenol reagent. J. Biol. Chem. 193,265-273. MANDARDT, E., KAY, A., and GALIBERT, F. (1984). Nucleotide sequence of a cloned duck hepatitis B virus genome: Comparison with woodchuck and human hepatitis B virus sequence. 1. Viral. 49,782-792.
OMATA,
AND
IT0
MARION, P. L., KNIGHT, S. S., FEITELSON, M. A., OSHIRO, L. S., and ROBINSON, W. S. (1983). Major polypeptide of duck hepatitis B surface antigen particles. J. Viral. 48, 534-541. MASON, W. S., HALPERN, M. S., ENGLAND, 1. M.. SEAL, G., EGAN, J., COATES, L., ALDRICH, C., and SUMMERS, J. (1983). Experimental transmission of duck hepatitis B virus. Virology 131, 375-384. MISHIRO, S., IMAI, M., TAKAHASHI, K., MACHIDA, A., GOTANDA, T., MIYAKAWA, T., and MAYUMI, M. (1980). A 49,000-dalton polypeptide bearing all antigenic determinants and full immunogenicity of 22nm hepatitis B surface antigen particles. J. Immunol. 124, 15891593. OMATA, M., YOKOSUKA, O., IMAZEKI, F., MATSUYAMA, Y., UCHIUMI, K., ITO, Y., MORI, J., and OKUDA, K. (1984). Transmission of duck hepatitis B virus from Chinese carrier ducks to Japanese ducklings: A study of viral DNA in serum and tissue. Hepafology4, 603-607. PUGH, J. C., SNINSKY, J. J., SUMMERS, J., and SCHAEFFER, E. (1987). Characterization of a pre-S polypeptide on the surface of infectious avian hepadnavirus particles. J. Viral. 81, 1384-l 390. SCHAEFFER, E., SNYDER, R. L., and SNINSKY, J. J. (1986). Identification and localization of pre-S-encoded polypeptides from woodchuck and ground squirrel hepatitis viruses. J. Viral. 57, 173-l 82. SUMMERS, J., and MASON, W. S. (1982). Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 29,403-415. TAGAWA, M., OMATA, M., and OKUDA, K. (1986). Appearance of viral RNA transcripts in the early stage of duck hepatitis B virus infection. Virology 152,477-482. TAGAWA, M., OMATA, M., YOKOSUKA. O., UCHIUMI, K., IMAZEKI, F., and OKUDA, K. (1985). Early events in duck hepatitis B virus infection: Sequential appearance of viral deoxyribonucleic acid in the liver, pancreas, kidney and spleen. Gasrroenferology89, 1224-l 229.