- Email: [email protected]
Expression of hepatitis B virus core and precore antigens in insect cells and characterization of a core-associated kinase activity
VIROLOGY
176,222-233
(1990)
Expression
of Hepatitis 6 Virus Core and Precore Antigens in Insect Cells and Characterization of a Core-Associated Kinase Activity’ ROBERT E. LANFORD’ AND LENA NOTVALL
Department
of Virology and Immunology,
Southwest
Foundation
Received November
for Biomedical Research, P.O. Box 28 147, San Antonio,
Texas 78284
15, 1989; accepted January f9, 1990
The hepatitis B virus core open reading frame with and without the precore domain was expressed in insect cells using a baculovirus expression system. Precore antigen was not properly processed in insect cells and was present in highly insoluble cytoplasmic aggregates. Core antigen without the precore domain formed core particles with a diameter of 28 nm that were secreted into the medium. Both core and precore antigens were phosphorylated in insect cells. The immune response in mice to both antigens yielded antibodies with a high degree of preferential reactivity for the homologous immunizing polypeptide. A kinase activity that phosphorylated core antigen was associated with highly purified core particles. The kinase activity resembled that previously demonstrated for core particles purified from the cytoplasm of infected hepatocytes and detergent-treated Dane particles. Partial resistance of the phosphate-label to phosphatase treatment suggested that some of the phosphorylated sites are in the interior of the particle. The presence of kinase activity in recombinant core particles demonstrated that this activity is not derived from another hepatitis B virus-encoded polvpeptide. and the lack of a kinase consensus sequence in the core open reading frame suggests _. 0 1990Academic PBS. IIIC that the kinase is of cellular origin.
INTRODUCTION
1987; Moroy et a/., 1985; Will et al., 1987). Initiation of translation at the internal AUG yields the 22,000 molecular weight core protein (~22) which assembles to form the nucleocapsid; whereas, use of the upstream AUG results in the synthesis of the 25,000 molecular weight precore protein (~25) (Weimer eta/., 1987; Standring eZ al., 1988; Jean-Jean et al., 1989; Ou et al., 1986; Roossinck et a/., 1986; Chang et al., 1987; Schlicht et al., 1987; McLachlan et al., 1987; Bruss and Gerlich, 1988). The highly hydrophobic amino-terminal domain of precore directs the protein to the endoplasmic reticulum. Proteolytic cleavages remove the signal sequence and the carboxyl-terminal domain, and a polypeptide, the HBeAg, with a molecularweight of approximately 15,000 is secreted. A function for this polypeptide has not been determined, and in duck hepatitis B virus this protein is not essential for replication (Chang et al., 1987; Schlicht et al., 1987). The immunology of the two polypeptides of the core lineage are quite distinct as well. The nucleocapsid is a very powerful immunogen that is capable of functioning as a T cell dependent and independent antigen (Milich and McLachlan, 1986). The use of monoclonal antibodies has defined a single immunodominant epitope on the core particle, while two distinct epitopes have been detected on HBeAg and denatured core particles (Ferns and Tedder, 1984; lmai er al., 1982; Waters et al., 1986; Salfeld eta/., 1989). The sequence specificity of the dominant T cell epitope varies with the H-2 haplotype when different strains of mice are immunized
The hepatitis B virion (HBV) possesses an outer lipoprotein coat containing the surface antigen (HBsAg) and an inner icosahedral capsid assembled from the core antigen (HBcAg). The core particle contains a partially double-stranded DNA genome with a protein covalently bound to the 5’terminus of the minus strand of DNA and a viral polymerase (for review see Ganem and Varmus, 1987; Tiollais et a/., 1985). In addition, core particles contain a kinase activity of undetermined origin that phosphorylates the core protein in an in vitro kinase assay (Albin and Robinson, 1980; Gerlich el a/., 1982; Feitelson e2 al,, 1982; Serrano and Hirschman, 1984; Petit and Pillot, 1985). The viral polymerase reverse transcribes a pregenomic RNA after encapsidation by core protein (Ganem and Varmus, 1987; Tiollais e2 al., 1985). The exact protein-protein and proteinnucleic acid interactions required for morphogenesis of the core particle remain to be elucidated. The 5’ terminus of the core gene contains two inframe AUG initiation codons such that two proteins with a common carboxy terminus and different amino termini are synthesized. Separate mRNAs with 5’ termini upstream of each AUG permit the use of both initiation condons (Buscher et a/., 1985; Enders et a/., ’ A preliminary report on the isolation of the recombinant baculoviruses used in this study has been published previously in a symposium proceedings (Lanford et a/., 1988). ‘To whom reprint requests should be addressed, 0042.6822/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproducton I” any form resewed.
222
223
HEW CORE AND PRECORE ANTIGENS
with synthetic peptides (Milich et al., 1987). The importance of the immunological response to the core polypeptides is emphasized by the finding that HBcAg represents an effective vaccine against viral infection (Murray et a/., 1987; Roos et al., 1989). The protection is presumably due to a cytotoxic T lymphocyte response to infected hepatocytes that bear an undetermined core determinant on the cell surface. Recombinant HBV core proteins have been expressed using a variety of cell systems including bacteria (Salfeld et a/., 1989; Stahl eta/., 1982; Lanford et a/., 1987), yeast (Miyanohara et al., 1986; Kniskern et al., 1986), Xenopus oocytes (Standring et a/., 1988), insect cells (Lanford et a/., 1988; Takehara era/., 1988), and mammalian cells (Weimer et al., 1987; Jean-Jean et al., 1989; Ou et a/., 1986; Roossinck et a/., 1986). In this investigation, the baculovirus Autographa californica was used to express the core and precore polypeptides. The baculovirus insect ceil expression system (Summers and Smith, 1987) provides the advantage of high levels of synthesis, coupled with post-translational modifications similar to that obtained in mammalian cells (Luckow and Summers, 1988). The core polypeptide was assembled into core particles which were secreted into the medium. Highly purified core particles exhibited a kinase activity that phosphorylated the core protein. The precore polypeptide was not properly processed by proteolytic: cleavage and was present in highly insoluble aggregates in the cytoplasm of insect cells. The immune response in mice to core and precore proteins was quantitatively and qualitatively different. MATERIALS
AND METHODS
Cells and viruses The Sf9 cell line (Summers and Smith, 1987) derived from the fall armyworm, Spodoptera frugiperda, was cultivated in TMN-FH medium (Hink, 1970) supplemented with 10% fetal bovine serum. The medium for suspension cultures was additionally supplemented with 0.1% Pluronic F-68 (BASF Corp., Parsippany, NJ). The methods for the growth, purification, and assay of AcNPV have been described (Volkman and Summers, 1975; Volkman et a/., 1976) and a detailed procedures manual for the use of AcNPV as an expression vector has been published (Summers and Smith, 1987). Construction
and isolation
of recombinant
viruses
The baculovirus transfer vector plasmid pAC373 was used in this investigation (Luckow and Summers, 1988). The plasmid consists of pUC8 with an insert of AcNPV DNA encompassing sequences both 3’ and 5’
of the polyhedrin gene. Two plasmids were constructed with HBV DNA sequences inserted into the transfer vector in such a manner as to encode the core polypeptide (p373C) and the precore polypeptide (p373PC). Recombinant, occlusion negative viruses were isolated following cotransfection of Sf9 cells with AcNPV DNA and the respective transfer vector using the published methodology (Summers and Smith, 1987). Details of the construction and isolation of these viruses have been published previously in symposium proceedings (Lanford et al., 1988). SDS-polyacrylamide gel electrophoresis and immunoblot analysis
(PAGE)
Samples for SDS-PAGE were disrupted in electrophoresis sample buffer containing 2% SDS and 2% 2mercaptoethanol and heated at 100” for 5 min. Proteins were separated by SDS-PAGE as previously described (Lanford and Butel, 1979), electrophoretically transferred (Towbin et al., 1979) to a GeneScreen Plus membrane (New England Nuclear, Boston, MA), and processed for the immunoblot procedure as previously described (Lanford et al., 1989a) using a rabbit antiserum prepared against liver-derived core particles (Lanford eta/., 1987) and ‘251-protein A (NEN). Analysis of phosphorylation Sf9 cells growing as adherent cultures (25 cm’ flask) were infected with the recombinant viruses 373C and 373PC. Cultures were labeled at 48 hr postinfection (p.i.) for 2 hr with 100 &i/ml carrier-free [32P]or-thophosphoric acid (NEN) or 50 &I/ml [35S]methionine (ICN, Irvine, CA, Trans 35 S-label; 1000 Ci/mmol). The cells were scraped from the flasks, pelleted, and disrupted with electrophoresis sample buffer. The total cell lysates were analyzed by SDS-PAGE, Coomassie blue staining, and autoradiography. Purification
of HBcAg core particles
Sf9 cells growing in 1 liter spinner cultures were harvested 5 days p.i. with 373C. The medium was clarified at 3000 g for 20 min and core particles were pelleted through a 30% sucrose cushion by ultracentrifugation for 4 hr at 26,000 rpm in an SW28 rotor (Beckman, Fullerton, CA). The pellet was resuspended by sonication and layered onto a CsCl gradient (density 1.20-l .40 gl cm3). Equilibrium sedimentation of HBcAg was performed by ultracentrifugation for 20 hr at 36,000 rpm in an SW41 rotor (Beckman). Fractions (1 ml) were diluted 1OO-fold and used to coat a microtiter plate (Corning, Corning, NY) and positive fractions were screened by ELISA using rabbit anti-core anti-serum and peroxidase-conjugated goat anti-rabbit IgG (Fisher, Pitts-
224
LANFORD
burgh, PA). Positive fractions were dialyzed against phosphate-buffered saline (PBS) and layered onto a sucrose gradient (15-60%). Equilibrium sedimentation was performed by ultracentrifugation for 14 hr at 30,000 rpm in an SW41 rotor (Beckman). HBcAg positive fractions were detected by ELISA as described above and were dialyzed against PBS. Electron microscopy Purified core particles were examined by electron microscopy using a modification of the pseudoreplication technique (Portnoy et a/., 1977). Briefly, 10 ~1of purified core particles was pipetted onto an agar disc (2% in 0.15 M NaCI, 0.01% Merthiolate), and the agar disc was coated with a film of Parlodion, 0.75% in amyl acetate (Mallinckrodt, Paris, KY). The Parlodion film was floated onto the surface of 1% phosphotungstic acid, pH 7.0, and the film was transferred to a 3-mm copper grid and dried before observation by electron microscopy. Solubilization
of precore polypeptide
(~25)
Sf9 cells growing as adherent cultures (25-cm* flasks) were infected with the 373PC recombinant virus and were harvested at 48 hr p.i. by scraping into PBS. The cell suspension was sonicated, divided into multiple aliquots, and separated into soluble and particulate fractions by centrifugation at 15,000 g for 2 min. The insoluble fraction of each aliquot was resuspended in 100 ~1 of one of the solubilizing agents and incubated for 20 min at 23”. The samples were divided into soluble and particulate fractions as above and the fractions were analyzed for the presence of p25 by the immunoblot procedure described above. Kinase assays Assays for the HBcAg-associated kinase activity were performed by a modification of a previously described procedure (Albin and Robinson, 1980; Gerlich et al., 1982) using core particles purified as described above. Briefly, purified core particles (136 ng) were diluted in 100 ~1of TNM buffer (50 mMTris-HCI, pH 7.5, 0.5% NP40, and 10 mM MgC12) and were incubated for 1 hr at 37” with 0.5 &i [Y-~~P]ATP (NEN; 3000 Ci/ mmol). Samples were disrupted with electrophoresis sample buffer and analyzed by SDS-PAGE and autoradiography. Sensitivity of the products of the kinase reaction to alkali and acid was examined by adjusting the kinase reaction to contain 0.1 A# NaOH or 0.1 n/l HCI, respectively, and incubation for 3 hr at 60” (Gerlich et a/., 1982). The samples were neutralized, disrupted in electrophoresis sample buffer, and analyzed by SDSPAGE and autoradiography. Alkaline phosphatase
AND NOTVALL
treatment of the products of the kinase reaction was performed by incubating the samples with 6 units of calf intestinal alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN) in 50 mM Tris-HCI, pH 7.5, 10 mM MgCI,, and 5 mM dithiothreitol for 18 hr at 30”. In some assays, HBcAg was isolated from culture media and cell lysates by immunoprecipitation. Sf9 cells growing as adherent cultures (75-cm’ flasks) were infected with 373C and harvested at 48-72 hr p.i. The culture medium was clarified at 1000 g for 15 min and adjusted to contain 0.5% NP40. The cell monolayer was washed three times with PBS and extracted with 1 ml of TNM. HBcAg was immunoprecipitated from the cell extracts and culture medium using 20 lg of rabbit anti-core IgG bound to protein A-agarose (BRL, Gaithersburg, MD). The immunoprecipitates were washed three times with TNM and resuspended in 100 yl of TNM containing 50 &i of [y-32P]ATP, and used directly in a kinase assay as described above. Antigenicity and immunogenicity precore antigens
of core and
Gradient-purified core particles and precore antigen partially purified by NaOH solubilization of the insoluble fraction of p25 were assayed for HBcAg/HBeAg reactivity using the HBe EIA kit (Abbott, North Chicago, IL) as described by the manufacturer. Protein concentrations were determined prior to assay using the Bio-Rad (Richmond, CA) protein assay. Denatured core and precore were obtained by treatment of the respective antigens with 2% SDS and 2% 2-mercaptoethanol at 100” for 2 min and removal of the excess detergent and reducing agent by gel filtration on Sephadex G-25 (Pharmacia, Piscataway, NJ). The immunogenicity of core and precore antigens was determined by immunization of groups of five Balb/c mice with a single dose of 5 pg of the respective antigens in complete Freund’s adjuvant administered intraperitoneally. Mice were bled from the tail vein at 28 days postinoculation and the sera from each group were pooled. The reactivity of the antisera was tested by ELISA. Briefly, microtiter plates were coated overnight at 4” with 200 ng of core or precore antigens, unoccupied protein binding sites were blocked with 10% normal goat serum (NGS) in PBS containing 0.3% Tween-20, and the microtiter wells were incubated with serial dilutions of the mouse antiserum in 10% NGS for 2 hr at 37”. Bound antibody was detected using peroxidase-conjugated goat antimouse IgG diluted in 10% NGS (1 hr at 37”) and the peroxidase substrate, 2,2’-azino-bis(3-ethylbenzthiazoline) sulfonic acid (30 min at 23”). Between each step the wells were washed three times with PBS-Tween. Optical density was measured at 4 10 nm.
225
HBV CORE AND PRECORE ANTIGENS
MEDIA
Days 11
2 3
CELLS
4
51 1 I 2 3
4
5)
-p22
FIG. 1. Time course of accumulatton of intracellular and extracellular p22 and ~25 polypepttdes. Sf9 cells infected with 373C and 373PC were harvested every day for 5 days p.i. The medium was clarified to remove cellular debris, and a total cellular extract was prepared by disruption of the cell pellet with electrophorests sample buffer. An equal fraction of the cell lysates and media were analyzed by SDSPAGE and immunoblot. The expression of p22 in 373C-infected cultures was first detected in the cells and the medium on Days 2 and 3, respectively. The p25 polypeptrde was first detected on Day 2 in the cells and was not detected In the medium.
RESULTS Expression
of core and precore polypeptides
HBV DNA fragments encoding the open reading frames for the core polypeptide and the core polypeptide including the precore domain were cloned into the baculovirus transfer vector pAC373 and the recombinant viruses 373C and 373PC were isolated by established procedures (Summers and Smith, 1987). Sf9 cells growing in 25.cm2 flasks were infected with the two recombinant viruses and one culture was harvested each day for 5 days and examined for HBcAgrelated polypeptides. An equivalent amount of the medium and a total cell lysate were analyzed by SDSPAGE and immunoblot using a rabbit antisera prepared against liver-derived HBV core particles. An immunoreactive polypeptide with an apparent molecular weight of 22,000 (~22) was detected in the cell lysate of 373Cinfected cells on Day 2 and the maximum accumulation occurred on Day 3 (Fig. 1). The core polypeptide was observed in the medium beginning on Day 3 and the level continued to increase through Day 5. The presence of p22 in the medium did not appear to be due to cell lysis, because greater than 95% of the cells excluded trypan blue on Days 3 and 4. A precore polypeptide with an apparent molecular weight of 25,000 (~25) was detected in the cell lysate of 373PC-infected cells on Day 2 and approximately equivalent amounts of p25 were detected through Day 5. The proteolytic
processing of the precore polypeptide to HBeAg-related polypeptides was not observed in insect cells. No immunoreactive material was detected in the medium by ELISA for HBeAg (data not shown), or by immunoblot with the exception of Day 4 when a very weak reaction was observed for p25 (Fig. 1). This low level of reactivity could be accounted for by the amount of cell death occurring by Day 4. The molecular weight of the p25 suggested that the precore polypeptide was not being targeted to the endoplasmic reticulum where cleavage of the signal sequence would occur. This was substantiated by the observation that p25 synthesized in Escherichia co/i (Lanford el al., 1987) comigrated by SDS-PAGE with p25 obtained from insect cells (data not shown). The solubility of p22 and p25 in the intracellular fraction was examined to better determine the fate of ~25. Sf9 cells infected with recombinant viruses 373C and 373PC were harvested at 48 hr p.i. and the cells were divided into soluble and particulate fractions by sonication in PBS and clarification at 15,000 g for 2 min. Equivalent samples of each fraction were analyzed by SDS-PAGE and immunoblot. The core polypeptide was detected almost exclusively in the soluble fraction, whereas the vast majority of p25 was in the particulate fraction (Fig. 2). Two immunoreactive polypeptides of lower molecular weight were detected in the particulate fraction of 373PC-infected cells. These polypeptides were not routinely observed in other fractionation experiments and probably represent proteolytic degradation rather than post-translational processing. The
373 PC
- p25
* p22
FIG. 2. Solubility of intracellular core and precore antigens. Sf9 cells infected with 373C and 373PC were harvested at 48 hr p.i. and were divided into cellular soluble (S) and particulate (P) fractions by sonication in PBS and centnfugation. Equivalent samples of each fraction were analyzed by SDS-PAGE and immunoblot. Core antigen (~22) was detected almost exclusively in the soluble fraction, while the precore antigen (~25) was predominantly rn the particulate fraction.
226
LANFORD TABLE 1 SOLUBILIZATION
OF PRECORE
POLYPEPTIDE
Precore antrgen actrvrtyb Solubilizrng agent”
Pellet
Supernatant
PBS NP40 (1%) Urea (7 M) GuHCl(6 n/l)’ NP40 (1%) + DOC (1%) + SDS (0.1%) Sarc (0.1%) Sarc (0.5%) NaOH (0.01 N)
+++ ++ t + + + -
t ++ ++ ++ ++ +++ +++
a Samples of the particulate fraction from 373PC-infected Sf9 cells were resuspended in the various solubilizrng agents for 20 min at 23°C. The samples were divided into pellet and supernatant fluid by centrifugatron at 15,000 g for 2 min. ’ Fracttons were analyzed for the presence of p25 by SDS-PAGE and immunoblot. c GuHCI, guanidrne hydrochloride; DOC, sodium deoxycholate; Sarc, sodrum lauroylsarcosine.
particulate fraction of 373PCinfected cells was treated with a variety of solubilizing and denaturing agents in an attempt to determine the nature of the insoluble material and to obtain soluble p25 for other experiments. A sample of the particulate fraction was resuspended in each solubilizing agent for 20 min at 23” and the resulting soluble and particulate fractions were examined by SDS-PAGE and immunoblot. Nonionic detergents were minimally effective at solubilizing ~25, and incomplete solubilization was also observed following treatment with the chaotropic agents 7 M urea and 6 M guanidine hydrochloride (Table 1). Complete solubilization of p25 was effected by treatment with ionic detergents (SDS or sodium N-lauroylsarcosine) at concentrations of 0.5% and by 0.01 N sodium hydroxide. These results suggest that p25 is not targeted to the endoplasmic reticulum of insect cells and that the polypeptide forms highly insoluble aggregates in the cytoplasm. The ability of insect cells to modify p22 and p25 by phosphorylation was examined in 373C and 373PC infected Sf9 cells. Infected cells were labeled for 2 hr at 48 hr p.i. with carrier-free [32P]orthophosphoric acid or [35S]methionine, and total cellular extracts were examined by SDS-PAGE and autoradiography. The core polypeptide was weakly labeled with 32P in 373Cinfected cells, while p25 represented the major phosphoprotein in the 373PCinfected cells (Fig. 3). Examination of [35S]methionine-labeled cell extracts and Coomassie blue staining of the same gel indicated that the greater incorporation of 32P-label into p25 was in
AND NOTVALL
part due to the higher level of intracellular p25 in comparison to ~22. These results demonstrate that insect cells are capable of post-translational modification of both p22 and p25 by phosphorylation. Purification of core particles precore polypeptide
and soluble
The formation of intact core particles in 373C-infected cells was examined by ultracentrifugation. Sf9 cells infected with the recombinant virus 373C were harvested 5 days p.i. The medium and a soluble cell extract prepared by sonication in PBS were clarified by centrifugation at 3000 g for 20 min and subjected to sedimentation by ultracentrifugation through a 30% sucrose cushion. Essentially all of the immunoreactive p22 was recovered in the pellet fraction with little or no reactivity remaining in the supernatant. Core particles from the medium were further characterized and purified by equilibrium sedimentation first in a CsCl gradient (density 1.20-l .40 g/cm3) and then in a 15-600/o sucrose gradient. Each fraction was diluted lOO-fold and used to coat microtiterwells and the HBcAg immunoreactive material was detected by ELISA using rabbit anti-core antiserum. The density of the core particles was 1.30 g/cm3 in CsCl (Fig. 4A) and 1.19 g/cm3 in sucrose (Fig. 4B). Particles resembling the nucleocapsid of HBV with a mean diameter of 28 nm were observed
32P r&-El
35S L-J
CB L-4
FIG. 3. Phosphorylation of core and precore antigens. Sf9 cells were infected with 373C and 373PC, and cultures were labeled from 48 to 50 hr p.i. with 100 &i/ml [3’P]orthophosphate or 50 &i/ml [?S]methionine. Total cell lysates were prepared by disruption in electrophoresis sample buffer and were analyzed by SDS-PAGE, Coomassie blue staining, and autoradiography. Precore antigen (~25) was the most abundant cellular protein by Coomassie blue staining (CB) and was the major cellular phosphoprotein “P. Core antigen (~22) represented a minor cellular protein by Coomassie blue staining but was a prominent cellular phosphoprotein.
HBV CORE AND PRECORE ANTIGENS 1.5
2.0
m c z 5 n 2
1.4
1.5
i u .
1.0
u E 0
0.5
1.1
0.0 1
2
3
4
5
6
7
8
9
101112
2.0
c 3; ;5 n 2
1.25
m
1.20
'E u
1.5 !9 1.15 1.0 1.10
E g
94* 68’
g z 5 n :
0.5 1.05
g 0
433Ob - P25 20’
- p22
14 *
2 1 .oo
0.0 1
3
5
7
9
FRACTION
11
13
15
17
19
NUMBERS
FIG. 4. Purification of core particles and soluble precore polypeptide. (A) Medium from 373C-Infected Sf9 cells (5 days p.1.) was clarified at 3000 g for 20 min. and core partrcles were sedimented through 30% sucrose at 26,000 rpm for 4 hr in an SW28 rotor. Pelleted particles were layered on a CsCl gradrent (density 1.20-l .40 g/cm3) and sedimentation was performed at 36,000 rpm for 20 hr in an SW41 rotor. The densrty of each fraction was determined by refractive index and fractions were diluted 1 OO-fold prior to determination of HBcAg reactivrty by ELISA (Optical Densrty). Peak HBcAg reactivity was at a density of 1.30 g/cm3. (B) The peak fractrons from the CsCl gradient were pooled, dralyzed against PBS, and layered on a 15-60% sucrose gradient. Sedimentation was at 30,000 rpm for 14 hr In an SW41 rotor. Density and HBcAg reactivity were determined as described above. Peak HBcAg reactivity was at 1.19 g/cm3. (C)The peak fractions from the sucrose gradient were pooled, dialyzed against PBS, and examined by electron microscopy by the pseudoreplication technique. Typical core-like structures were observed with a mean diameter of 28 nm. Bar equals 100 nm. (D) Core particles (~22) purified by CsCl and sucrose densrty gradient centnfugation were analyzed by SDS-PAGE and Coomassie blue staining (lane 1). Precore antigens (~25) partrally purified by solubilization of the detergentinsoluble cellular fraction with 0.01 N NaOH (lane 2) or 0.5% N-lauroylsarcosine (lane 3) were analyzed by SDS-PAGE and Coomassie blue staining. The positions of molecularweight standards (Xl 000) are indicated at the left.
by electron microscopic analysis of the immunoreactive material recovered from the sucrose gradients (Fig. 4C). Examination of the purified particles by SDS-PAGE and Coomassie blue staining indicated that p22 had been purified to apparent homogeneity (Fig. 4D, lane 1). Several different purification schemes were examined for the partial purification of ~25. The best preparations were obtained by extracting detergent-soluble
material with 1olo NP40 and solubilizing the particulate fraction with either 0.01 N NaOH or 0.5% N-lauroylsarcosine. Examination of the solubilized fractions by SDS-PAGE and Coomassie blue staining revealed that the NaOH (Fig. 4D, lane 2) and N-lauroylsarcosine (Fig. 4D, lane 3)-solubilized materials were highly enriched for p25 but contained minor amounts of several other polypeptides.
228
LANFORD TABLE 2 ANTIGENICIT~OFCOREAND
Antigen”
Core
PRECORE POLYPEPTIDES
d Coreb
d Precore
OptIcal density
PLQ 1 0.1 0.01 0.001
Precore
>2 1.14 0.14
0.12
>2 >2 1.32
0.31
1.89
1.28
0.63
0.41
0.17 0.10
0.13 0.10
“Core particles purified on CsCl and sucrose density gradients and precore anttgen solubilized with NaOH were assayed at various protein concentrations in the Abbott HBe EIA. ’ Core particles and precore antigen were denatured (d) with SDS prior to assay.
Antigenicity and immunogenicity precore polypeptides
AND NOTVALL
from Dane particles possess a kinase activity that phosphorylates the core polypeptides (Albin and Robinson, 1980; Feitelson eta/., 1982; Gerlich eta/., 1982; Petit and Pillot, 1985). To determine whether a similar activity was present in core particles derived from insect cells, a series of kinase reactions were performed. The core particles employed in kinase reactions were purified to near homogeneity from the medium of 373Cinfected Sf9 cells by differential centrifugation through a 30% sucrose cushion, and equilibrium sedimentation in CsCl and sucrose gradients as described above (Fig. 4). Kinase reactions were pet-formed in 100 ~1 of TNM buffer containing 136 ng of core particles and 0.5 &i of [y-32P]ATP. Samples were incubated for 1 hr at 37”, disrupted in electrophoresis sample buffer, and analyzed by SDS-PAGE and autoradiography.
of core and
The core and precore polypeptides were evaluated for reactivity in a commercial ELISA for HBcAg/HBeAg reactivity (HBe EIA, Abbott) and for immunogenicity in mice. The gradient-purified core particles and the NaOH solubilized precore antigen were titrated for reactivity in the HBeAg assay before and after denaturation with SDS and 2-mercaptoethanol. Denaturation increased the reactivity of core particles by 1O-fold, whereas the reactivity of precore antigen was not affected by denaturation and was 1O-fold and 1OO-fold lower in reactivity than native and denatured core particles, respectively (Table 2). To test the immunogenicity of the two preparations, groups of five Balb/c mice were immunized with a single dose of 5 fig of the respective antigens and were bled 28 days p.i. The sera from each group were pooled and titered by ELISA using both p22 and p25 as the solid-phase antigens. Sera from mice immunized with core particles were reactive with p22 out to a dilution of 1,024,000, whereas sera from mice immunized with precore antigen were reactive only to a dilution of 1600 in the same assay (Fig. 5A). In contrast, sera from mice immunized with precore were reactive against p25 at a dilution of 256,000, while sera from mice immunized with core particles were reactive against p25 at a dilution of 1600 (Fig. 5B). These results demonstrate that the immune response to p22 containing core particles and solubilized p25-precore antigen is quantitatively and qualitatively different, with the antisera being most reactive to the homologous antigen. Kinase reactions HBV core particles derived from the cytoplasm of infected hepatocytes or by removal of the lipid envelope
2.0A
o.o\ 2
3 DILUTION
OF
-
Pre immune
w
Anti-Core
bd
Pm? ullm”ne
L
Anta-Precore
4 ANTISERA
5 (log)
6
FIG. 5. lmmunogenicity of core and precore antigens. Groups of five Balbk mice were immunized with a single dose of 5 pg of CsCl and sucrose density-purified core particles of NaOH solubilized precore antigen. The respective antigens were mixed with complete Freund’s adjuvant and administered intraperitoneally, and mice were bled 28 days post-inoculation. Sera from each group were pooled and titered by ELISA using microtiter plates coated with either core particles (A) or precore antigen (B). Values are expressed as optical density at 410 nm for the ELISA versus the log of the dilution of the antisera. In each case the sera possessed the greatest reactivity for the homologous immunizing antigen.
HBV CORE AND PRECORE ANTIGENS A.
123
4
p22-’
FIG. 6. Characterization of the products of krnase reactions with purified core partrcles. Kinase reactions were performed using gradient purified core particles in a volume of 100 ~1 containing 136 ng of core antigen and 0.5 &i of [y-32P]ATP in TNM buffer. Samples were incubated for 1 hr at 37” and either analyzed directly by SDS-PAGE and autoradrography (A, lane 1) or subjected to various treatments prior to electrophoresis. Kinase samples were incubated at 60°C for 3 hr in PBS (A, lane 2) 0.1 N HCI (A, lane 3) or 0.1 N NaOH (A, lane 4). Krnase samples were incubated at 30” for 18 hr in the absence of alkalrne phosphatase (B, lane l), or in the presence of 6 units of alkaline phosphatase (B, lane 2). Alkaline phosphatase treatment was also conducted on a kinase sample after drsruptron of the core partrcles with SDS and removal of the excess SDS (B. lane 3).
Two major phosphorylated products were detected with apparent molecular weights of 22,000 and 14,000 (Fig. 6A, lane 1). The lower molecularweight phosphorylated polypeptide probably represents a degradation product of p22 as has been suggested previously (Albin and Robinson, 1980) and as discussed below. Sedimentation of the core particles after the kinase reaction in a 15-600/o sucrose gradient revealed that both species (~22 and ~14) still migrated with intact core particles (data not shown). The specificity of the kinase activity for 373C-infected Sf9 cells was demonstrated by using media purified by the same procedure from Sf9 cells infected with the recombinant baculovirus 941T which express SV40 large T antigen (Lanford, 1988). The medium from 941T-infected cells did not possess a detectable kinase activity even when heatdenatured p22 was added to the kinase reaction as a potential phosphate acceptor (data not shown). A series of kinase reactions demonstrated that the kinase activity could utilize both Mg’+ and Mn2+ for the diva-
229
lent cation requirement, GTP could not substitute for ATP as the phosphate donor, and a variety of proteins including casein, bovine serum albumin, and immunoglobulin G were not suitable phosphate acceptors (data not shown). To determine which amino acids were phosphorylated by the kinase activity, kinase reactions were incubated at 60” for 3 hr in PBS (Fig. 6A, lane 2) 0.1 N HCI (Fig. 6A, lane 3) and 0.1 N NaOH (Fig. 6A, lane 4) before analysis by SDS-PAGE. The phosphate linkages were removed by treatment with alkali but not acid. The phosphate linkages to serine and threonine are acid labile and alkali sensitive, while the linkage to tyrosine is stable to both alkali and acid. These results are consistent with previous findings using liver-derived HBV core particles (Gerlich era/., 1982). The accessibility of the phosphate linkages was examined by treatment of the kinase reactions with alkaline phosphatase. Kinase samples incubated at 30” for 18 hr in the absence of alkaline phosphatase displayed the typical pattern of labeled products (Fig. 6B, lane 2), whereas the phosphate label associated with the lower molecularweight polypeptide was completely removed by treatment with alkaline phosphatase (Fig. 6B, lane 2). In addition, a portion of the p22-associated label was removed by the alkaline phosphatase treatment which resulted in an increased mobility of the polypeptide by SDS-PAGE (Fig. 66, lane 2). The resistance of a portion of the label to treatment with alkaline phosphatase suggested that the phosphorylated residues were on the interior of the core particle. Treatment of the core particles with SDS following the kinase reaction but prior to phosphatase treatment significantly reduced the amount of 32P-label remaining on ~22. A portion of the label was resistant to phosphatase perhaps because p22 is able to reassembled into core-like structures when the excess SDS is removed. These results suggest that the kinase activity at least in part phosphorylates residues on the interior of the core particle. The complete removal of phosphate label from the lower molecular weight polypeptide suggests that it is either present in disrupted cores that are permeable to phosphatase or is a portion of core exposed on the exterior of the particle. The relationship of the lower molecular weight product of the kinase reaction to a degradation product of p22 was further evaluated by performing kinase reactions with p22 immunoprecipitated from the medium and cell lysates of 373C-infected cells at 48 and 72 hr p.i. A definitive trend was observed in which the products of the kinase reactions from newly synthesized and rapidly purified core particles contained less of the lower molecular weight phosphorylated species. Gradient-purified core particles that were derived from the medium of 373C-infected cells 5 days p.i. and in which
230
LANFORD
1
23
4
p22 -
FIG. 7. Relatronship of low molecular werght products of the kinase reaction to ~22. Kinase reactions were performed with purified or rmmunoprecrpitated core particles still bound to the immunoglobulin and protein A agarose as described under Materrals and Methods. Kinase reactions with core particles purified from the medium of 373C-infected cells 5 days p.i. predominantly yielded lower molecular weight products (lane 1). In contrast, core particles rapidly immu nopreciprtated from the medium (lane 2) and cell lysate (lane 3) of 373~.infected cells at 3 days p.i. contained increasing amounts of phosphorylated ~22. A kinase reaction with core particles rmmunoprecipitated from a cell lysate 2 days p.i. contained almost exclusively phosphorylated p22 (lane 4).
the purification required 48 hr yielded kinase reaction products that were predominantly the lower molecular weight species (Fig. 7, lane 1). A small increase in the relative amount of phosphorylated p22 was observed with core particles rapidly immunoprecipitated from the medium 3 days p.i. (Fig. 7, lane 2) and the intensity of the phosphorylated p22 band was greater than the pl4 band when the particles were immunoprecipitated from a cell lysate of the same culture harvested at 3 days p.i. (Fig. 7, lane 3). The kinase reaction from core particles immunoprecipitated from a cell lysate prepared from 373Cinfected cells 2 days p.i. contained exclusively phosphorylated p22 (Fig. 7, lane 4). Insufficient core particles were present in the medium to examine the kinase reaction from core particles in the medium of cultures 2 days p.i. These results suggest that pl4 is derived from p22 as a result of core particles remaining in the medium at 37” for extended periods of time.
AND NOTVALL
DISCUSSION The baculovirus expression vector system has provided a useful tool for the analysis of post-translational processing and biochemical function for numerous mammalian proteins which are not normally present in sufficient quantity to facilitate such studies. In this investigation, two recombinant baculoviruses were constructed for the expression of the HBV core polypeptide with and without the precore domain. In insect cells, the precore domain was not capable of targeting the ~25 polypeptide to the ER membrane and p25 formed highly insoluble aggregates in the cytoplasm of infected cells. This is in contrast to reports using mammalian cells (Jean-Jean et a/., 1989; Ou et a/., 1986; Roossinck et al., 1986; McLachlan et al., 1987), Xenopus oocytes (Standring et a/., 1988) and in vitro translation systems (Weimer et a/., 1987; Bruss and Gerlich, 1988) for the expression of the precore polypeptide. In these systems, the precore polypeptide is targeted to the ER membrane, the amino-terminal signal sequence is cleaved, and at least a portion of the polypeptides is further processed by proteolytic cleavage and secreted as HBeAg. The precore polypeptide represents a unique instance in which a mammalian signal sequence is not recognized by insect cells. Our previous studies with the synthesis of HBsAg in insect cells demonstrated that, although the HBsAg signal sequence was recognized, HBsAg particles were not secreted into the medium (Lanford et a/., 1989b). Further investigations will be required to determine the defective step for translocation of p25 in insect cells. Modification of the precore signal sequence may circumvent the problems with p25 processing in insect cells. The recombinant baculovirus encoding the core domain induced the synthesis of large quantities of the p22 polypeptide. The core polypeptide was assembled into recognizable core structures. The density of the core particles in CsCl (1.30 g/cm3) and their appearance by electron microscopy suggested that most of the core particles do not contain nucleic acid. This is in contrast to core particles synthesized in bacteria which contain nucleic acid (Cohen and Richmond, 1982). A major portion of the core particles synthesized in insect cells were detected in the culture medium. Several lines of evidence suggest that the core particles were not released into the medium by cell lysis. Large amounts of core particles could be detected in the medium by 3 days p.i. at which time less than 1% of the cells were permeable to tt-ypan blue. By Day 4 p.i., greater than 50% of total p22 polypeptide was present in the medium yet greater than 95% of the cells still excluded trypan blue. Although the secretion of core particles was not initially anticipated, the same phe-
HEW CORE AND PRECORE ANTIGENS
nomenon has been observed in mammalian cells. Core reactivity banding in CsCl at the density of immature core particles was observed in HepG2 cells transfected with a complete HBV genome (Sureau eta/., 1986) and in HBV-infected primary chimpanzee hepatocyte cultures (Jacob et a/., 1989). Recently, a carefully controlled study indicated that the appearance of immature core particles in the culture medium of mammalian cells was not due to cell lysis. In cells expressing both core particles and /3-galactosidases only HBcAg reactivity was detected in the culture medium (Jean-Jean et a/., 1989). The secretion of a naked capsid structure is not unique to the HBV core particle. A recent electron microscopy investigation visualized the secretion of SV40 virions (a naked capsid) via large cytoplasmic vesicular structures (Clayson eta/., 1989). In addition, the use of an improved baculovirus transfer vector, pVL941 (Luckow and Summers, 1988; Lanford, 1988), which yielded increased core mRNA levels resulted in reduced accumulation of p22 in the medium due to early cell death. Although circumstantial in nature, we interpreted these findings to indicate that toxic levels of core particles were accumulating in the cells, because the rate of synthesis became greater than the rate of secretion. Again, this would imply that core is secreted from living cells, otherwise increased cell death would result in an increased level of core in the medium. The immunogenicity studies with precore and core indicated that the immune response to the two polypeptides was qualitatively different, with the reactivity of antiserum being greatest to the immunizing polypeptide. These findings are similar to those reported by others for the immunogenicity of core particles and HBeAg. Although the precore antigen was not properly processed in insect cells, the immunogenicity studies indicate that p25 may be suitable for studies comparing the immunogenicity of HBeAg and HBcAg. Core particles derived from infected hepatocytes and detergent-treated Dane particles possess an associated kinase activity that phosphorylates HBcAg (Albin and Robinson, 1980; Gerlich eT al., 1982; Feitelson et al., 1982; Petit and Pillot, 1985). In interest of determining the nature of this kinase activity and the usefulness of the baculovirus system for the analysis of this function, kinase assays were performed with highly purified core particles. Insect cell-derived core particles exhibited a kinase activity similar to the activity previously observed with core particles produced during active HBV replication. The phosphorylated products of the kinase reaction were polypeptides with molecular weights of 22,000 and 14,000. These same products were previously observed in core kinase reactions and the 14,000 molecularweight polypeptide was con-
231
sidered to be a degradation product of p22 (Albin and Robinson, 1980). Our results confirmed this relationship. Newly synthesized intracellular core antigen contained no pl4 following a kinase reaction, yet core particles accumulating in the medium have progressively increasing amounts of ~14. Following a 2-day purification scheme, pl4 was the predominant product of the kinase reaction. The events leading to the degradation of p22 are not currently apparent, but the possibility exists that the presence of a lipid envelope or encapsidation of the pregenome may stabilize the core particle. The degradation observed may resemble the process by which HBV DNA is released after penetration of the cell. Kinase reactions followed by alkaline phosphatase treatment suggested that particles possessing pl4 were permeable. All of the phosphate-label on pl4 and a portion of the p22 phosphate-label was removed by alkaline phosphatase. The accessible phosphates of p14 and p22 may be present on disrupted core particles which would be consistent with pl4 being a degradation product of ~22. An alternative hypothesis would envision a copurifying kinase that phosphory lates p14 and p22 on the exterior of the particle. The remainder of the p22 phosphate-label was completely resistant to phosphatase treatment which implies that some the phosphate moieties reside on the interior of the core particle (Gerlich et a/., 1982). An internal localization of the kinase is consistent with the observations that the activity cannot be separated from core particles after numerous purification steps and no similar activity can be purified from the medium of uninfected cells (data not shown). The presence of kinase activity in recombinant core particles demonstrates that in this system the activity is not derived from another HBVencoded protein. However, no direct evidence was obtained that would suggest that the activity or the phosphorylation sites present in Dane particles and insect cell-derived cores is the same. Comparison of the amino acid sequence of the core open reading frame with the consensus sequence for protein kinases (Hunter, 1987; Hanks et al., 1988) indicates that p22 does not possess a domain similar to known kinases. Thus, the kinase activity in insect cell-derived cores appears to be a cellular protein. Several mechanisms can be envisioned forthe encapsidation of a cellular kinase. The random entrapment of soluble cellular proteins at the site of particle morphogenesis is one such possibility. However, if a cellular kinase plays a role in HBV replication, a mechanism may have evolved for specific encapsidation. The ability to purify large quantities of core particles from insect cells should be useful in the characterization of the encapsidated kinase. In addition, the bacu-
232
LANFORD
lovirus system may be useful for the analysis of other functional characteristics potentially required of ~22 for virion morphogenesis, such as recognition of the pregenome and polymerase and association with HBsAg. Furthermore, large quantities of highly purified core particles will be useful in the preparation of a two component HBsAg-HBcAg vaccine that will provide both humoral and cellular immunity. ACKNOWLEDGMENTS We gratefully acknowledge Max D. Summers for the generous gift of the baculovirus expression system, Max D. Summers and Verne A. Luckow for helpful advtce on the use of the system, and Ana Elias for assistance In the preparation of this manuscript. This work was supported in part by the Texas Advanced Technology Program (grant 3220).
REFERENCES ALBIN, C., and ROBINSON,W. S. (1980). Protein kinase activity in hepa-
titis B virus. /. Viral. 34, 297-302. BRUSS, V., and GERLICH, W. H. (1988). Formation of transmembranous hepatitis B e-antigen by cotranslatlonal in vitro processing of the viral precore protein. \/iro/ogy 163, 268-275. BUSCHER, M., REISER, W., WILL, H., and SCHALLER, H. (1985). Transcripts and the putative RNA pregenome of duck hepatitis B virus: Implications for reverse transcription. Cell40, 717-724. CHANG, C., ENDERS,G.. SPRENGEL,R., PETERS,N., VARMUS, H. E., and GANEM, D. (1987). Expresslon of the precore region of an avian hepatitis B virus is not required for viral replication. /. viral. 61, 3322-3325. CLAYSON, E. T., BRANDO, L. V. J., and COMPANS, R. W. (1989). Release of simian virus 40 vlrlons from eplthelial cells is polarized and occurs without cell lysis. 1. Viral. 63, 2278-2288. COHEN, B. J., and RICHMOND,J. E. (1982). Electron microscopy of hepatitis B core antigen synthesized in f. co/i Nature (London) 296, 677-678. ENDERS, G. H., GANEM, D., and VARMUS, H. E. (1987). 5’.Terminal sequences Influence the segregation of ground squirrel hepatitis virus RNAs Into polynbosomes and viral core particles. /. L’irol. 61, 35-41. FEITELSON, M. A., MARION, P. L.. and ROBINSON,W. S. (1982). Core particles of hepatitis B virus and ground squirrel hepatitis virus. /. Viroi. 43, 741-748. FERNS,R. B., and TEDDER, R. S. (1984). Monoclonal antibodies to hepatitis Be antigen (HBeAg) derived from hepatitis B core antigen (HBcAg): Their use in characterization and detection of HBeAg. /. Gen. Wrol. 65, 899-908. GANEM, D., and VARMUS, H. E. (1987). The molecular biology of the hepatitis B viruses. Annu. Rev. Biochem. 56, 651-693. GERLICH, W. H., GOLDMANN, U., MULLER, R., STIBBE. W., and WOLFF, W. (1982). Specificity and localization of the hepatitis B virus-assoelated protein kinase. /. Viral. 42, 761-766. HANKS, S. K.. QUINN, A. M., and HUNTER,T. (1988). The protein kinase family: Conserved features and deduced phylogeny of the catalytic domains. Science 241,42-52. HINK, W. F. (1970). Established Insect cell line from the cabbage looper, Trxhoplusia ni. Nature (London) 226, 466-467. HUNTER, T. (1987). A thousand and one protein kinases. Cell 50, 823-829. IMAI, M., NOMURA, M., GOTANDA, T., SANO. T., TACHIBANA, K., MIYAMOTO, H., TAKAHASHI, K., TOYAMA, S.. MIYAKAWA. Y., and MAYUMI,
AND NOTVALL M. (1982). Demonstration of two distinct antlgenic determinants on hepatitis Be antigen by monoclonal antibodies. J. lmmunol. 128, 69-72. JACOB,J. R., EICHBERG.J. W., and LANFORD, R. E. (1989). In vitro repllcation and expressjon of hepatitis B virus from chronically infected primary chimpanzee hepatocytes. HepatoiogylO. 921-927. JEAN-JEAN,O., LEVRERO,M., WILL, H., PERRICAUDET,M., and ROSSIGNOL, J.-M. (1989). Expression mechanism of the hepatitis B virus (HBV) C gene and biosynthesis of HBe antigen. Virology 170, 99106. KNISKERN,P. J., HAGOPIAN,A., MONTGOMERY, D. L., BURKE,P., DUNN, N. R., HOFMANN, K. J., MILLER, W. J., and ELLIS, R. W. (1986). Unusually high-level expresslon of a foreign gene (hepatltls B virus core antigen) in Saccharomyces cerevisiae. Gene 46, 135-l 41. LANFORD, R. E. (1988). Expression of simian virus 40 T antigen In insect cells using a baculovirus expression vector. !I;ro/ogy 167, 72-81. LANFORD, R. E., and BUTEL, J. S. (1979). Antigenic relatlonshtp of SV40 early protetns to purified large T polypeptide. L/iro/ogy 97, 295-306. LANFORD, R. E., CAREY, K. D., ESTLACK, L. E.. SMITH, G. C., and HAY, R. V. (1989a). Analysts of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained In serum-free medium. ln Vitro Cell. Dev. Biol. 25, 174-l 82. LANFORD, R. E., KENNEDY, R. C., DREESMAN, G. R., EICHBERG,J. W., NOTVALL, L., LUCKOW,V. A., and SUMMERS, M. D. (1988). Expression of hepatitis B virus surface and core antigens using a baculovirus expression vector. ln “International Symposium on Viral Hepatitis and Liver Disease, London, 1987, Proceedings: Vtral Hepatltis and Liver Disease” (A. J. Zuckerman, Ed.), pp. 372-378. A. R. Liss, New York. LANFORD, R. E., LUCKOW,V., KENNEDY, R. C., DREESMAN, G. R., NOTE VALL, L., and SUMMERS, M. D. (198913). Expression and characterization of hepatitis B virus surface antigen polypeptldes In insect cells with a baculovlrus expresston system. /. Viral. 63, 15491557. LANFORD, R. E., NOT~ALL, L. M., DREESMAN, G. R., HARRISON,C. R., LOCKWOOD,D., and BURK, K. H. (1987). Expression and charactertzation of hepatitis B virus precore-core antigen in f. co/i Viral lmmunol. 1, 97-l 09. LUCKOW,V. A., and SUMMERS, M. D. (1988). Trends In the development of baculovirus expression vectors. Bioflechnology6,47P55. MCLACHLAN, A., MILICH, D. R., RANEY, A. K.. RIGGS, M. G., HUGHES, 1. L., SORGE, L., and CHISARI, F. V. (1987). Expression of hepatitis B virus surface and core antigens: Influences of pre-S and precore sequences. /. Viral. 61, 683-692. MILICH, D. R.. MCLACHLAN, A., MORIARPI, A., and THORNTON, G. B. (1987). Immune response to hepatitis b virus core antigen (HBcAg): Localization of T cell recognition sites within HBcAg/HBeAg J.lmmunol. 139, 1223-1231. MIYANOHARA,A., IMAMURA, T., ARAKI, M., SUGAWARA,K.. OHTOMO, N., and MATSUBARA,K. (1986). Expression of hepatitis B virus core antigen gene in Saccharomyces cerevisiae: Synthesis of two polypeptides translated from different initiation codons. /. Viral. 59, 176-180. MOROY, T., ETIEMBLE,J., TREPO, C., TIOLLAIS, P., and BUENDIA, M.-A. (1985). Transcription of woodchuck hepatitts virus In the chronically infected liver. fMBO/. 4, 1507-l 514. MURRAY, K., BRUCE, S. A., WINGFIELD, P., VAN EERD, P., DE REUS, A., and SCHELLEKENS,H. (1987). Protective lmmunisatlon against hepatitis B with an internal antigen of the virus. 1. Med. Viral. 23, 10 l107. Ou, J. H., LAuB, O., and RU~TER,W. J. (1986). Hepatitis B virus gene function: The precore region targets the core antigen to cellular
HBV CORE AND PRECORE ANTIGENS membranes and causes the secretion of the e antigen. Proc. Nat/. Acad. SC;. USA 83,1578-l 582. PETIT, M.-A., and PILLOT, 1. (1985). HBc and HBe antigenicity and DNA-binding activity of major core protein P22 in hepatitis B virus core particles isolated from the cytoplasm of human liver cells. J. Viral. 53, 543-551. PORTNOY, B. L., CONKLIN, R. H., MENN. M., OLARTE. J., and DUPONT, H. L. (1977). Reliable identification of reovirus-like agent in diarrhea1 stools. J. Lab. C/in. Med. 89, 560-563. Roos, S., FUCHS, K., and ROGGENDORF.M. (1989). Protection of woodchucks from InfectIon with woodchuck hepatitis virus by immunization with recombinant core protein. /. Gen. Viral. 70, 20872095. ROOSSINCK,M. J., JAMEEL, S., LOUKIN, S. H., and SIDDIQUI, A. (1986). Expression of hepatitis B viral core region in mammalian cells. Mol. Celi. Biol. 6, 1393-l 400. SALFELD.J., PFAFF, E., NOAH, M., and SCHALLER,H. (1989). Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus. /. Viral. 63, 798-808. SCHLICHT, H. J., SALFELD.J., and SCHALLER,H. (1987). The duck hepatitis B virus pre-C region encodes a stgnal which is essential for synthesis and secretton of processed core proteins but not for virus formatlon. /. VYrol. 61, 370 l-3709. SERRANO,M. A., and HIRSCHMAN, S. Z. (1984). Properties of hepatitis Be antigen synthesized by rat cells transfected with circular viral DNA. J. Gen. Viral. 65, 1373-l 383. STAHL, S., MACKAY, P., MAGALIN, M., BRUCE, S. A., and MURRAY, K. (1982). Hepatitis B virus core antigen: synthesis in fscher;cichia co/i and application in diagnosis. Proc. Nat/. Acad. Sci. USA 79, 16061610. STANDRING,D. N., Ou, J.-H., MASIARZ. F. R., and RUT~ER.W. J. (1988). A signal peptide encoded within the precore region of hepatitis B virus directs the secretion of a heterogeneous population of e
233
antigens in Xenopus oocytes. Proc. Nat/. Acad. Sci. USA 85, 8405-8409. SUMMERS, M. D., and SMITH, G. E. (1987). A manual of methods for baculovirus vectors and insect cell culture procedures. Tex. Agric. Exp. Stn. (Bull.) No. 1555. SUREAU. C., ROMET-LEMONNE, J.-L., MULLINS, J. I., and ESSEX, M. (1986). Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. Ce//47,37-47. TAKEHARA,K., IRELAND,D., and BISHOP,D. H. L. (1988). Co-expression of the Hepatitis B surface and core antigens using baculovirus multiple expression vectors. J. Gen. Viral. 69, 2763-2777. TIOLLAIS, P.. POURCEL,C., and DEJEAN,A. (1985). The hepatitis B virus. Nature (London) 317,489-495. TOWBIN, H., STAEHELIN. T., and GORDON, J. (1979). Electrophoretic Transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Nafl. Acad. Sci. USA 76,4350-4354. VOLKMAN, L. E., and SUMMERS, M. D. (1975). Nuclear polyhedrosis virus detection: Relative capabilities of clones developed from Trichoplusia ni ovarian cell line TN-368 to serve as indicator cells in a plaque assay. /. Viral. 16, 1630-l 637. VOLKMAN, L. E., SUMMERS, M. D., and HSIEH, C.-H. (1976). Occluded and nonoccluded nuclear polyhedrosis virus grown in Trichoplusia n;; Comparative neutralization, comparative infectivity, and in vitro growth studies. 1. Viral. 19.820-832. WATERS,J. A., JOWET, T. P., and THOMAS, H. C. (1986). Identification of a dominant immunogenic epitope of the nucleocapsid (HBc) of the hepatitis B virus. /. Med. Viol. 19,79-86. WEIMER,T., SALFELD,I., and WILL, H. (1987). Expression of the hepatitis B virus core gene in vitro and in viva. 1. Viral. 61, 3109-31 13. WILL, H., REISER,W., WEIMER, T., PFAFF, E., BUSCHER,M., SPRENGEL, R., CATTENEO, R., and SCHALLER,H. (1987). Replication strategy of human hepatitis B virus. /. Viral. 61, 904-91 1.
176,222-233
(1990)
Expression
of Hepatitis 6 Virus Core and Precore Antigens in Insect Cells and Characterization of a Core-Associated Kinase Activity’ ROBERT E. LANFORD’ AND LENA NOTVALL
Department
of Virology and Immunology,
Southwest
Foundation
Received November
for Biomedical Research, P.O. Box 28 147, San Antonio,
Texas 78284
15, 1989; accepted January f9, 1990
The hepatitis B virus core open reading frame with and without the precore domain was expressed in insect cells using a baculovirus expression system. Precore antigen was not properly processed in insect cells and was present in highly insoluble cytoplasmic aggregates. Core antigen without the precore domain formed core particles with a diameter of 28 nm that were secreted into the medium. Both core and precore antigens were phosphorylated in insect cells. The immune response in mice to both antigens yielded antibodies with a high degree of preferential reactivity for the homologous immunizing polypeptide. A kinase activity that phosphorylated core antigen was associated with highly purified core particles. The kinase activity resembled that previously demonstrated for core particles purified from the cytoplasm of infected hepatocytes and detergent-treated Dane particles. Partial resistance of the phosphate-label to phosphatase treatment suggested that some of the phosphorylated sites are in the interior of the particle. The presence of kinase activity in recombinant core particles demonstrated that this activity is not derived from another hepatitis B virus-encoded polvpeptide. and the lack of a kinase consensus sequence in the core open reading frame suggests _. 0 1990Academic PBS. IIIC that the kinase is of cellular origin.
INTRODUCTION
1987; Moroy et a/., 1985; Will et al., 1987). Initiation of translation at the internal AUG yields the 22,000 molecular weight core protein (~22) which assembles to form the nucleocapsid; whereas, use of the upstream AUG results in the synthesis of the 25,000 molecular weight precore protein (~25) (Weimer eta/., 1987; Standring eZ al., 1988; Jean-Jean et al., 1989; Ou et al., 1986; Roossinck et a/., 1986; Chang et al., 1987; Schlicht et al., 1987; McLachlan et al., 1987; Bruss and Gerlich, 1988). The highly hydrophobic amino-terminal domain of precore directs the protein to the endoplasmic reticulum. Proteolytic cleavages remove the signal sequence and the carboxyl-terminal domain, and a polypeptide, the HBeAg, with a molecularweight of approximately 15,000 is secreted. A function for this polypeptide has not been determined, and in duck hepatitis B virus this protein is not essential for replication (Chang et al., 1987; Schlicht et al., 1987). The immunology of the two polypeptides of the core lineage are quite distinct as well. The nucleocapsid is a very powerful immunogen that is capable of functioning as a T cell dependent and independent antigen (Milich and McLachlan, 1986). The use of monoclonal antibodies has defined a single immunodominant epitope on the core particle, while two distinct epitopes have been detected on HBeAg and denatured core particles (Ferns and Tedder, 1984; lmai er al., 1982; Waters et al., 1986; Salfeld eta/., 1989). The sequence specificity of the dominant T cell epitope varies with the H-2 haplotype when different strains of mice are immunized
The hepatitis B virion (HBV) possesses an outer lipoprotein coat containing the surface antigen (HBsAg) and an inner icosahedral capsid assembled from the core antigen (HBcAg). The core particle contains a partially double-stranded DNA genome with a protein covalently bound to the 5’terminus of the minus strand of DNA and a viral polymerase (for review see Ganem and Varmus, 1987; Tiollais et a/., 1985). In addition, core particles contain a kinase activity of undetermined origin that phosphorylates the core protein in an in vitro kinase assay (Albin and Robinson, 1980; Gerlich el a/., 1982; Feitelson e2 al,, 1982; Serrano and Hirschman, 1984; Petit and Pillot, 1985). The viral polymerase reverse transcribes a pregenomic RNA after encapsidation by core protein (Ganem and Varmus, 1987; Tiollais e2 al., 1985). The exact protein-protein and proteinnucleic acid interactions required for morphogenesis of the core particle remain to be elucidated. The 5’ terminus of the core gene contains two inframe AUG initiation codons such that two proteins with a common carboxy terminus and different amino termini are synthesized. Separate mRNAs with 5’ termini upstream of each AUG permit the use of both initiation condons (Buscher et a/., 1985; Enders et a/., ’ A preliminary report on the isolation of the recombinant baculoviruses used in this study has been published previously in a symposium proceedings (Lanford et a/., 1988). ‘To whom reprint requests should be addressed, 0042.6822/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproducton I” any form resewed.
222
223
HEW CORE AND PRECORE ANTIGENS
with synthetic peptides (Milich et al., 1987). The importance of the immunological response to the core polypeptides is emphasized by the finding that HBcAg represents an effective vaccine against viral infection (Murray et a/., 1987; Roos et al., 1989). The protection is presumably due to a cytotoxic T lymphocyte response to infected hepatocytes that bear an undetermined core determinant on the cell surface. Recombinant HBV core proteins have been expressed using a variety of cell systems including bacteria (Salfeld et a/., 1989; Stahl eta/., 1982; Lanford et a/., 1987), yeast (Miyanohara et al., 1986; Kniskern et al., 1986), Xenopus oocytes (Standring et a/., 1988), insect cells (Lanford et a/., 1988; Takehara era/., 1988), and mammalian cells (Weimer et al., 1987; Jean-Jean et al., 1989; Ou et a/., 1986; Roossinck et a/., 1986). In this investigation, the baculovirus Autographa californica was used to express the core and precore polypeptides. The baculovirus insect ceil expression system (Summers and Smith, 1987) provides the advantage of high levels of synthesis, coupled with post-translational modifications similar to that obtained in mammalian cells (Luckow and Summers, 1988). The core polypeptide was assembled into core particles which were secreted into the medium. Highly purified core particles exhibited a kinase activity that phosphorylated the core protein. The precore polypeptide was not properly processed by proteolytic: cleavage and was present in highly insoluble aggregates in the cytoplasm of insect cells. The immune response in mice to core and precore proteins was quantitatively and qualitatively different. MATERIALS
AND METHODS
Cells and viruses The Sf9 cell line (Summers and Smith, 1987) derived from the fall armyworm, Spodoptera frugiperda, was cultivated in TMN-FH medium (Hink, 1970) supplemented with 10% fetal bovine serum. The medium for suspension cultures was additionally supplemented with 0.1% Pluronic F-68 (BASF Corp., Parsippany, NJ). The methods for the growth, purification, and assay of AcNPV have been described (Volkman and Summers, 1975; Volkman et a/., 1976) and a detailed procedures manual for the use of AcNPV as an expression vector has been published (Summers and Smith, 1987). Construction
and isolation
of recombinant
viruses
The baculovirus transfer vector plasmid pAC373 was used in this investigation (Luckow and Summers, 1988). The plasmid consists of pUC8 with an insert of AcNPV DNA encompassing sequences both 3’ and 5’
of the polyhedrin gene. Two plasmids were constructed with HBV DNA sequences inserted into the transfer vector in such a manner as to encode the core polypeptide (p373C) and the precore polypeptide (p373PC). Recombinant, occlusion negative viruses were isolated following cotransfection of Sf9 cells with AcNPV DNA and the respective transfer vector using the published methodology (Summers and Smith, 1987). Details of the construction and isolation of these viruses have been published previously in symposium proceedings (Lanford et al., 1988). SDS-polyacrylamide gel electrophoresis and immunoblot analysis
(PAGE)
Samples for SDS-PAGE were disrupted in electrophoresis sample buffer containing 2% SDS and 2% 2mercaptoethanol and heated at 100” for 5 min. Proteins were separated by SDS-PAGE as previously described (Lanford and Butel, 1979), electrophoretically transferred (Towbin et al., 1979) to a GeneScreen Plus membrane (New England Nuclear, Boston, MA), and processed for the immunoblot procedure as previously described (Lanford et al., 1989a) using a rabbit antiserum prepared against liver-derived core particles (Lanford eta/., 1987) and ‘251-protein A (NEN). Analysis of phosphorylation Sf9 cells growing as adherent cultures (25 cm’ flask) were infected with the recombinant viruses 373C and 373PC. Cultures were labeled at 48 hr postinfection (p.i.) for 2 hr with 100 &i/ml carrier-free [32P]or-thophosphoric acid (NEN) or 50 &I/ml [35S]methionine (ICN, Irvine, CA, Trans 35 S-label; 1000 Ci/mmol). The cells were scraped from the flasks, pelleted, and disrupted with electrophoresis sample buffer. The total cell lysates were analyzed by SDS-PAGE, Coomassie blue staining, and autoradiography. Purification
of HBcAg core particles
Sf9 cells growing in 1 liter spinner cultures were harvested 5 days p.i. with 373C. The medium was clarified at 3000 g for 20 min and core particles were pelleted through a 30% sucrose cushion by ultracentrifugation for 4 hr at 26,000 rpm in an SW28 rotor (Beckman, Fullerton, CA). The pellet was resuspended by sonication and layered onto a CsCl gradient (density 1.20-l .40 gl cm3). Equilibrium sedimentation of HBcAg was performed by ultracentrifugation for 20 hr at 36,000 rpm in an SW41 rotor (Beckman). Fractions (1 ml) were diluted 1OO-fold and used to coat a microtiter plate (Corning, Corning, NY) and positive fractions were screened by ELISA using rabbit anti-core anti-serum and peroxidase-conjugated goat anti-rabbit IgG (Fisher, Pitts-
224
LANFORD
burgh, PA). Positive fractions were dialyzed against phosphate-buffered saline (PBS) and layered onto a sucrose gradient (15-60%). Equilibrium sedimentation was performed by ultracentrifugation for 14 hr at 30,000 rpm in an SW41 rotor (Beckman). HBcAg positive fractions were detected by ELISA as described above and were dialyzed against PBS. Electron microscopy Purified core particles were examined by electron microscopy using a modification of the pseudoreplication technique (Portnoy et a/., 1977). Briefly, 10 ~1of purified core particles was pipetted onto an agar disc (2% in 0.15 M NaCI, 0.01% Merthiolate), and the agar disc was coated with a film of Parlodion, 0.75% in amyl acetate (Mallinckrodt, Paris, KY). The Parlodion film was floated onto the surface of 1% phosphotungstic acid, pH 7.0, and the film was transferred to a 3-mm copper grid and dried before observation by electron microscopy. Solubilization
of precore polypeptide
(~25)
Sf9 cells growing as adherent cultures (25-cm* flasks) were infected with the 373PC recombinant virus and were harvested at 48 hr p.i. by scraping into PBS. The cell suspension was sonicated, divided into multiple aliquots, and separated into soluble and particulate fractions by centrifugation at 15,000 g for 2 min. The insoluble fraction of each aliquot was resuspended in 100 ~1 of one of the solubilizing agents and incubated for 20 min at 23”. The samples were divided into soluble and particulate fractions as above and the fractions were analyzed for the presence of p25 by the immunoblot procedure described above. Kinase assays Assays for the HBcAg-associated kinase activity were performed by a modification of a previously described procedure (Albin and Robinson, 1980; Gerlich et al., 1982) using core particles purified as described above. Briefly, purified core particles (136 ng) were diluted in 100 ~1of TNM buffer (50 mMTris-HCI, pH 7.5, 0.5% NP40, and 10 mM MgC12) and were incubated for 1 hr at 37” with 0.5 &i [Y-~~P]ATP (NEN; 3000 Ci/ mmol). Samples were disrupted with electrophoresis sample buffer and analyzed by SDS-PAGE and autoradiography. Sensitivity of the products of the kinase reaction to alkali and acid was examined by adjusting the kinase reaction to contain 0.1 A# NaOH or 0.1 n/l HCI, respectively, and incubation for 3 hr at 60” (Gerlich et a/., 1982). The samples were neutralized, disrupted in electrophoresis sample buffer, and analyzed by SDSPAGE and autoradiography. Alkaline phosphatase
AND NOTVALL
treatment of the products of the kinase reaction was performed by incubating the samples with 6 units of calf intestinal alkaline phosphatase (Boehringer-Mannheim, Indianapolis, IN) in 50 mM Tris-HCI, pH 7.5, 10 mM MgCI,, and 5 mM dithiothreitol for 18 hr at 30”. In some assays, HBcAg was isolated from culture media and cell lysates by immunoprecipitation. Sf9 cells growing as adherent cultures (75-cm’ flasks) were infected with 373C and harvested at 48-72 hr p.i. The culture medium was clarified at 1000 g for 15 min and adjusted to contain 0.5% NP40. The cell monolayer was washed three times with PBS and extracted with 1 ml of TNM. HBcAg was immunoprecipitated from the cell extracts and culture medium using 20 lg of rabbit anti-core IgG bound to protein A-agarose (BRL, Gaithersburg, MD). The immunoprecipitates were washed three times with TNM and resuspended in 100 yl of TNM containing 50 &i of [y-32P]ATP, and used directly in a kinase assay as described above. Antigenicity and immunogenicity precore antigens
of core and
Gradient-purified core particles and precore antigen partially purified by NaOH solubilization of the insoluble fraction of p25 were assayed for HBcAg/HBeAg reactivity using the HBe EIA kit (Abbott, North Chicago, IL) as described by the manufacturer. Protein concentrations were determined prior to assay using the Bio-Rad (Richmond, CA) protein assay. Denatured core and precore were obtained by treatment of the respective antigens with 2% SDS and 2% 2-mercaptoethanol at 100” for 2 min and removal of the excess detergent and reducing agent by gel filtration on Sephadex G-25 (Pharmacia, Piscataway, NJ). The immunogenicity of core and precore antigens was determined by immunization of groups of five Balb/c mice with a single dose of 5 pg of the respective antigens in complete Freund’s adjuvant administered intraperitoneally. Mice were bled from the tail vein at 28 days postinoculation and the sera from each group were pooled. The reactivity of the antisera was tested by ELISA. Briefly, microtiter plates were coated overnight at 4” with 200 ng of core or precore antigens, unoccupied protein binding sites were blocked with 10% normal goat serum (NGS) in PBS containing 0.3% Tween-20, and the microtiter wells were incubated with serial dilutions of the mouse antiserum in 10% NGS for 2 hr at 37”. Bound antibody was detected using peroxidase-conjugated goat antimouse IgG diluted in 10% NGS (1 hr at 37”) and the peroxidase substrate, 2,2’-azino-bis(3-ethylbenzthiazoline) sulfonic acid (30 min at 23”). Between each step the wells were washed three times with PBS-Tween. Optical density was measured at 4 10 nm.
225
HBV CORE AND PRECORE ANTIGENS
MEDIA
Days 11
2 3
CELLS
4
51 1 I 2 3
4
5)
-p22
FIG. 1. Time course of accumulatton of intracellular and extracellular p22 and ~25 polypepttdes. Sf9 cells infected with 373C and 373PC were harvested every day for 5 days p.i. The medium was clarified to remove cellular debris, and a total cellular extract was prepared by disruption of the cell pellet with electrophorests sample buffer. An equal fraction of the cell lysates and media were analyzed by SDSPAGE and immunoblot. The expression of p22 in 373C-infected cultures was first detected in the cells and the medium on Days 2 and 3, respectively. The p25 polypeptrde was first detected on Day 2 in the cells and was not detected In the medium.
RESULTS Expression
of core and precore polypeptides
HBV DNA fragments encoding the open reading frames for the core polypeptide and the core polypeptide including the precore domain were cloned into the baculovirus transfer vector pAC373 and the recombinant viruses 373C and 373PC were isolated by established procedures (Summers and Smith, 1987). Sf9 cells growing in 25.cm2 flasks were infected with the two recombinant viruses and one culture was harvested each day for 5 days and examined for HBcAgrelated polypeptides. An equivalent amount of the medium and a total cell lysate were analyzed by SDSPAGE and immunoblot using a rabbit antisera prepared against liver-derived HBV core particles. An immunoreactive polypeptide with an apparent molecular weight of 22,000 (~22) was detected in the cell lysate of 373Cinfected cells on Day 2 and the maximum accumulation occurred on Day 3 (Fig. 1). The core polypeptide was observed in the medium beginning on Day 3 and the level continued to increase through Day 5. The presence of p22 in the medium did not appear to be due to cell lysis, because greater than 95% of the cells excluded trypan blue on Days 3 and 4. A precore polypeptide with an apparent molecular weight of 25,000 (~25) was detected in the cell lysate of 373PC-infected cells on Day 2 and approximately equivalent amounts of p25 were detected through Day 5. The proteolytic
processing of the precore polypeptide to HBeAg-related polypeptides was not observed in insect cells. No immunoreactive material was detected in the medium by ELISA for HBeAg (data not shown), or by immunoblot with the exception of Day 4 when a very weak reaction was observed for p25 (Fig. 1). This low level of reactivity could be accounted for by the amount of cell death occurring by Day 4. The molecular weight of the p25 suggested that the precore polypeptide was not being targeted to the endoplasmic reticulum where cleavage of the signal sequence would occur. This was substantiated by the observation that p25 synthesized in Escherichia co/i (Lanford el al., 1987) comigrated by SDS-PAGE with p25 obtained from insect cells (data not shown). The solubility of p22 and p25 in the intracellular fraction was examined to better determine the fate of ~25. Sf9 cells infected with recombinant viruses 373C and 373PC were harvested at 48 hr p.i. and the cells were divided into soluble and particulate fractions by sonication in PBS and clarification at 15,000 g for 2 min. Equivalent samples of each fraction were analyzed by SDS-PAGE and immunoblot. The core polypeptide was detected almost exclusively in the soluble fraction, whereas the vast majority of p25 was in the particulate fraction (Fig. 2). Two immunoreactive polypeptides of lower molecular weight were detected in the particulate fraction of 373PC-infected cells. These polypeptides were not routinely observed in other fractionation experiments and probably represent proteolytic degradation rather than post-translational processing. The
373 PC
- p25
* p22
FIG. 2. Solubility of intracellular core and precore antigens. Sf9 cells infected with 373C and 373PC were harvested at 48 hr p.i. and were divided into cellular soluble (S) and particulate (P) fractions by sonication in PBS and centnfugation. Equivalent samples of each fraction were analyzed by SDS-PAGE and immunoblot. Core antigen (~22) was detected almost exclusively in the soluble fraction, while the precore antigen (~25) was predominantly rn the particulate fraction.
226
LANFORD TABLE 1 SOLUBILIZATION
OF PRECORE
POLYPEPTIDE
Precore antrgen actrvrtyb Solubilizrng agent”
Pellet
Supernatant
PBS NP40 (1%) Urea (7 M) GuHCl(6 n/l)’ NP40 (1%) + DOC (1%) + SDS (0.1%) Sarc (0.1%) Sarc (0.5%) NaOH (0.01 N)
+++ ++ t + + + -
t ++ ++ ++ ++ +++ +++
a Samples of the particulate fraction from 373PC-infected Sf9 cells were resuspended in the various solubilizrng agents for 20 min at 23°C. The samples were divided into pellet and supernatant fluid by centrifugatron at 15,000 g for 2 min. ’ Fracttons were analyzed for the presence of p25 by SDS-PAGE and immunoblot. c GuHCI, guanidrne hydrochloride; DOC, sodium deoxycholate; Sarc, sodrum lauroylsarcosine.
particulate fraction of 373PCinfected cells was treated with a variety of solubilizing and denaturing agents in an attempt to determine the nature of the insoluble material and to obtain soluble p25 for other experiments. A sample of the particulate fraction was resuspended in each solubilizing agent for 20 min at 23” and the resulting soluble and particulate fractions were examined by SDS-PAGE and immunoblot. Nonionic detergents were minimally effective at solubilizing ~25, and incomplete solubilization was also observed following treatment with the chaotropic agents 7 M urea and 6 M guanidine hydrochloride (Table 1). Complete solubilization of p25 was effected by treatment with ionic detergents (SDS or sodium N-lauroylsarcosine) at concentrations of 0.5% and by 0.01 N sodium hydroxide. These results suggest that p25 is not targeted to the endoplasmic reticulum of insect cells and that the polypeptide forms highly insoluble aggregates in the cytoplasm. The ability of insect cells to modify p22 and p25 by phosphorylation was examined in 373C and 373PC infected Sf9 cells. Infected cells were labeled for 2 hr at 48 hr p.i. with carrier-free [32P]orthophosphoric acid or [35S]methionine, and total cellular extracts were examined by SDS-PAGE and autoradiography. The core polypeptide was weakly labeled with 32P in 373Cinfected cells, while p25 represented the major phosphoprotein in the 373PCinfected cells (Fig. 3). Examination of [35S]methionine-labeled cell extracts and Coomassie blue staining of the same gel indicated that the greater incorporation of 32P-label into p25 was in
AND NOTVALL
part due to the higher level of intracellular p25 in comparison to ~22. These results demonstrate that insect cells are capable of post-translational modification of both p22 and p25 by phosphorylation. Purification of core particles precore polypeptide
and soluble
The formation of intact core particles in 373C-infected cells was examined by ultracentrifugation. Sf9 cells infected with the recombinant virus 373C were harvested 5 days p.i. The medium and a soluble cell extract prepared by sonication in PBS were clarified by centrifugation at 3000 g for 20 min and subjected to sedimentation by ultracentrifugation through a 30% sucrose cushion. Essentially all of the immunoreactive p22 was recovered in the pellet fraction with little or no reactivity remaining in the supernatant. Core particles from the medium were further characterized and purified by equilibrium sedimentation first in a CsCl gradient (density 1.20-l .40 g/cm3) and then in a 15-600/o sucrose gradient. Each fraction was diluted lOO-fold and used to coat microtiterwells and the HBcAg immunoreactive material was detected by ELISA using rabbit anti-core antiserum. The density of the core particles was 1.30 g/cm3 in CsCl (Fig. 4A) and 1.19 g/cm3 in sucrose (Fig. 4B). Particles resembling the nucleocapsid of HBV with a mean diameter of 28 nm were observed
32P r&-El
35S L-J
CB L-4
FIG. 3. Phosphorylation of core and precore antigens. Sf9 cells were infected with 373C and 373PC, and cultures were labeled from 48 to 50 hr p.i. with 100 &i/ml [3’P]orthophosphate or 50 &i/ml [?S]methionine. Total cell lysates were prepared by disruption in electrophoresis sample buffer and were analyzed by SDS-PAGE, Coomassie blue staining, and autoradiography. Precore antigen (~25) was the most abundant cellular protein by Coomassie blue staining (CB) and was the major cellular phosphoprotein “P. Core antigen (~22) represented a minor cellular protein by Coomassie blue staining but was a prominent cellular phosphoprotein.
HBV CORE AND PRECORE ANTIGENS 1.5
2.0
m c z 5 n 2
1.4
1.5
i u .
1.0
u E 0
0.5
1.1
0.0 1
2
3
4
5
6
7
8
9
101112
2.0
c 3; ;5 n 2
1.25
m
1.20
'E u
1.5 !9 1.15 1.0 1.10
E g
94* 68’
g z 5 n :
0.5 1.05
g 0
433Ob - P25 20’
- p22
14 *
2 1 .oo
0.0 1
3
5
7
9
FRACTION
11
13
15
17
19
NUMBERS
FIG. 4. Purification of core particles and soluble precore polypeptide. (A) Medium from 373C-Infected Sf9 cells (5 days p.1.) was clarified at 3000 g for 20 min. and core partrcles were sedimented through 30% sucrose at 26,000 rpm for 4 hr in an SW28 rotor. Pelleted particles were layered on a CsCl gradrent (density 1.20-l .40 g/cm3) and sedimentation was performed at 36,000 rpm for 20 hr in an SW41 rotor. The densrty of each fraction was determined by refractive index and fractions were diluted 1 OO-fold prior to determination of HBcAg reactivrty by ELISA (Optical Densrty). Peak HBcAg reactivity was at a density of 1.30 g/cm3. (B) The peak fractrons from the CsCl gradient were pooled, dralyzed against PBS, and layered on a 15-60% sucrose gradient. Sedimentation was at 30,000 rpm for 14 hr In an SW41 rotor. Density and HBcAg reactivity were determined as described above. Peak HBcAg reactivity was at 1.19 g/cm3. (C)The peak fractions from the sucrose gradient were pooled, dialyzed against PBS, and examined by electron microscopy by the pseudoreplication technique. Typical core-like structures were observed with a mean diameter of 28 nm. Bar equals 100 nm. (D) Core particles (~22) purified by CsCl and sucrose densrty gradient centnfugation were analyzed by SDS-PAGE and Coomassie blue staining (lane 1). Precore antigens (~25) partrally purified by solubilization of the detergentinsoluble cellular fraction with 0.01 N NaOH (lane 2) or 0.5% N-lauroylsarcosine (lane 3) were analyzed by SDS-PAGE and Coomassie blue staining. The positions of molecularweight standards (Xl 000) are indicated at the left.
by electron microscopic analysis of the immunoreactive material recovered from the sucrose gradients (Fig. 4C). Examination of the purified particles by SDS-PAGE and Coomassie blue staining indicated that p22 had been purified to apparent homogeneity (Fig. 4D, lane 1). Several different purification schemes were examined for the partial purification of ~25. The best preparations were obtained by extracting detergent-soluble
material with 1olo NP40 and solubilizing the particulate fraction with either 0.01 N NaOH or 0.5% N-lauroylsarcosine. Examination of the solubilized fractions by SDS-PAGE and Coomassie blue staining revealed that the NaOH (Fig. 4D, lane 2) and N-lauroylsarcosine (Fig. 4D, lane 3)-solubilized materials were highly enriched for p25 but contained minor amounts of several other polypeptides.
228
LANFORD TABLE 2 ANTIGENICIT~OFCOREAND
Antigen”
Core
PRECORE POLYPEPTIDES
d Coreb
d Precore
OptIcal density
PLQ 1 0.1 0.01 0.001
Precore
>2 1.14 0.14
0.12
>2 >2 1.32
0.31
1.89
1.28
0.63
0.41
0.17 0.10
0.13 0.10
“Core particles purified on CsCl and sucrose density gradients and precore anttgen solubilized with NaOH were assayed at various protein concentrations in the Abbott HBe EIA. ’ Core particles and precore antigen were denatured (d) with SDS prior to assay.
Antigenicity and immunogenicity precore polypeptides
AND NOTVALL
from Dane particles possess a kinase activity that phosphorylates the core polypeptides (Albin and Robinson, 1980; Feitelson eta/., 1982; Gerlich eta/., 1982; Petit and Pillot, 1985). To determine whether a similar activity was present in core particles derived from insect cells, a series of kinase reactions were performed. The core particles employed in kinase reactions were purified to near homogeneity from the medium of 373Cinfected Sf9 cells by differential centrifugation through a 30% sucrose cushion, and equilibrium sedimentation in CsCl and sucrose gradients as described above (Fig. 4). Kinase reactions were pet-formed in 100 ~1 of TNM buffer containing 136 ng of core particles and 0.5 &i of [y-32P]ATP. Samples were incubated for 1 hr at 37”, disrupted in electrophoresis sample buffer, and analyzed by SDS-PAGE and autoradiography.
of core and
The core and precore polypeptides were evaluated for reactivity in a commercial ELISA for HBcAg/HBeAg reactivity (HBe EIA, Abbott) and for immunogenicity in mice. The gradient-purified core particles and the NaOH solubilized precore antigen were titrated for reactivity in the HBeAg assay before and after denaturation with SDS and 2-mercaptoethanol. Denaturation increased the reactivity of core particles by 1O-fold, whereas the reactivity of precore antigen was not affected by denaturation and was 1O-fold and 1OO-fold lower in reactivity than native and denatured core particles, respectively (Table 2). To test the immunogenicity of the two preparations, groups of five Balb/c mice were immunized with a single dose of 5 fig of the respective antigens and were bled 28 days p.i. The sera from each group were pooled and titered by ELISA using both p22 and p25 as the solid-phase antigens. Sera from mice immunized with core particles were reactive with p22 out to a dilution of 1,024,000, whereas sera from mice immunized with precore antigen were reactive only to a dilution of 1600 in the same assay (Fig. 5A). In contrast, sera from mice immunized with precore were reactive against p25 at a dilution of 256,000, while sera from mice immunized with core particles were reactive against p25 at a dilution of 1600 (Fig. 5B). These results demonstrate that the immune response to p22 containing core particles and solubilized p25-precore antigen is quantitatively and qualitatively different, with the antisera being most reactive to the homologous antigen. Kinase reactions HBV core particles derived from the cytoplasm of infected hepatocytes or by removal of the lipid envelope
2.0A
o.o\ 2
3 DILUTION
OF
-
Pre immune
w
Anti-Core
bd
Pm? ullm”ne
L
Anta-Precore
4 ANTISERA
5 (log)
6
FIG. 5. lmmunogenicity of core and precore antigens. Groups of five Balbk mice were immunized with a single dose of 5 pg of CsCl and sucrose density-purified core particles of NaOH solubilized precore antigen. The respective antigens were mixed with complete Freund’s adjuvant and administered intraperitoneally, and mice were bled 28 days post-inoculation. Sera from each group were pooled and titered by ELISA using microtiter plates coated with either core particles (A) or precore antigen (B). Values are expressed as optical density at 410 nm for the ELISA versus the log of the dilution of the antisera. In each case the sera possessed the greatest reactivity for the homologous immunizing antigen.
HBV CORE AND PRECORE ANTIGENS A.
123
4
p22-’
FIG. 6. Characterization of the products of krnase reactions with purified core partrcles. Kinase reactions were performed using gradient purified core particles in a volume of 100 ~1 containing 136 ng of core antigen and 0.5 &i of [y-32P]ATP in TNM buffer. Samples were incubated for 1 hr at 37” and either analyzed directly by SDS-PAGE and autoradrography (A, lane 1) or subjected to various treatments prior to electrophoresis. Kinase samples were incubated at 60°C for 3 hr in PBS (A, lane 2) 0.1 N HCI (A, lane 3) or 0.1 N NaOH (A, lane 4). Krnase samples were incubated at 30” for 18 hr in the absence of alkalrne phosphatase (B, lane l), or in the presence of 6 units of alkaline phosphatase (B, lane 2). Alkaline phosphatase treatment was also conducted on a kinase sample after drsruptron of the core partrcles with SDS and removal of the excess SDS (B. lane 3).
Two major phosphorylated products were detected with apparent molecular weights of 22,000 and 14,000 (Fig. 6A, lane 1). The lower molecularweight phosphorylated polypeptide probably represents a degradation product of p22 as has been suggested previously (Albin and Robinson, 1980) and as discussed below. Sedimentation of the core particles after the kinase reaction in a 15-600/o sucrose gradient revealed that both species (~22 and ~14) still migrated with intact core particles (data not shown). The specificity of the kinase activity for 373C-infected Sf9 cells was demonstrated by using media purified by the same procedure from Sf9 cells infected with the recombinant baculovirus 941T which express SV40 large T antigen (Lanford, 1988). The medium from 941T-infected cells did not possess a detectable kinase activity even when heatdenatured p22 was added to the kinase reaction as a potential phosphate acceptor (data not shown). A series of kinase reactions demonstrated that the kinase activity could utilize both Mg’+ and Mn2+ for the diva-
229
lent cation requirement, GTP could not substitute for ATP as the phosphate donor, and a variety of proteins including casein, bovine serum albumin, and immunoglobulin G were not suitable phosphate acceptors (data not shown). To determine which amino acids were phosphorylated by the kinase activity, kinase reactions were incubated at 60” for 3 hr in PBS (Fig. 6A, lane 2) 0.1 N HCI (Fig. 6A, lane 3) and 0.1 N NaOH (Fig. 6A, lane 4) before analysis by SDS-PAGE. The phosphate linkages were removed by treatment with alkali but not acid. The phosphate linkages to serine and threonine are acid labile and alkali sensitive, while the linkage to tyrosine is stable to both alkali and acid. These results are consistent with previous findings using liver-derived HBV core particles (Gerlich era/., 1982). The accessibility of the phosphate linkages was examined by treatment of the kinase reactions with alkaline phosphatase. Kinase samples incubated at 30” for 18 hr in the absence of alkaline phosphatase displayed the typical pattern of labeled products (Fig. 6B, lane 2), whereas the phosphate label associated with the lower molecularweight polypeptide was completely removed by treatment with alkaline phosphatase (Fig. 6B, lane 2). In addition, a portion of the p22-associated label was removed by the alkaline phosphatase treatment which resulted in an increased mobility of the polypeptide by SDS-PAGE (Fig. 66, lane 2). The resistance of a portion of the label to treatment with alkaline phosphatase suggested that the phosphorylated residues were on the interior of the core particle. Treatment of the core particles with SDS following the kinase reaction but prior to phosphatase treatment significantly reduced the amount of 32P-label remaining on ~22. A portion of the label was resistant to phosphatase perhaps because p22 is able to reassembled into core-like structures when the excess SDS is removed. These results suggest that the kinase activity at least in part phosphorylates residues on the interior of the core particle. The complete removal of phosphate label from the lower molecular weight polypeptide suggests that it is either present in disrupted cores that are permeable to phosphatase or is a portion of core exposed on the exterior of the particle. The relationship of the lower molecular weight product of the kinase reaction to a degradation product of p22 was further evaluated by performing kinase reactions with p22 immunoprecipitated from the medium and cell lysates of 373C-infected cells at 48 and 72 hr p.i. A definitive trend was observed in which the products of the kinase reactions from newly synthesized and rapidly purified core particles contained less of the lower molecular weight phosphorylated species. Gradient-purified core particles that were derived from the medium of 373C-infected cells 5 days p.i. and in which
230
LANFORD
1
23
4
p22 -
FIG. 7. Relatronship of low molecular werght products of the kinase reaction to ~22. Kinase reactions were performed with purified or rmmunoprecrpitated core particles still bound to the immunoglobulin and protein A agarose as described under Materrals and Methods. Kinase reactions with core particles purified from the medium of 373C-infected cells 5 days p.i. predominantly yielded lower molecular weight products (lane 1). In contrast, core particles rapidly immu nopreciprtated from the medium (lane 2) and cell lysate (lane 3) of 373~.infected cells at 3 days p.i. contained increasing amounts of phosphorylated ~22. A kinase reaction with core particles rmmunoprecipitated from a cell lysate 2 days p.i. contained almost exclusively phosphorylated p22 (lane 4).
the purification required 48 hr yielded kinase reaction products that were predominantly the lower molecular weight species (Fig. 7, lane 1). A small increase in the relative amount of phosphorylated p22 was observed with core particles rapidly immunoprecipitated from the medium 3 days p.i. (Fig. 7, lane 2) and the intensity of the phosphorylated p22 band was greater than the pl4 band when the particles were immunoprecipitated from a cell lysate of the same culture harvested at 3 days p.i. (Fig. 7, lane 3). The kinase reaction from core particles immunoprecipitated from a cell lysate prepared from 373Cinfected cells 2 days p.i. contained exclusively phosphorylated p22 (Fig. 7, lane 4). Insufficient core particles were present in the medium to examine the kinase reaction from core particles in the medium of cultures 2 days p.i. These results suggest that pl4 is derived from p22 as a result of core particles remaining in the medium at 37” for extended periods of time.
AND NOTVALL
DISCUSSION The baculovirus expression vector system has provided a useful tool for the analysis of post-translational processing and biochemical function for numerous mammalian proteins which are not normally present in sufficient quantity to facilitate such studies. In this investigation, two recombinant baculoviruses were constructed for the expression of the HBV core polypeptide with and without the precore domain. In insect cells, the precore domain was not capable of targeting the ~25 polypeptide to the ER membrane and p25 formed highly insoluble aggregates in the cytoplasm of infected cells. This is in contrast to reports using mammalian cells (Jean-Jean et a/., 1989; Ou et a/., 1986; Roossinck et al., 1986; McLachlan et al., 1987), Xenopus oocytes (Standring et a/., 1988) and in vitro translation systems (Weimer et a/., 1987; Bruss and Gerlich, 1988) for the expression of the precore polypeptide. In these systems, the precore polypeptide is targeted to the ER membrane, the amino-terminal signal sequence is cleaved, and at least a portion of the polypeptides is further processed by proteolytic cleavage and secreted as HBeAg. The precore polypeptide represents a unique instance in which a mammalian signal sequence is not recognized by insect cells. Our previous studies with the synthesis of HBsAg in insect cells demonstrated that, although the HBsAg signal sequence was recognized, HBsAg particles were not secreted into the medium (Lanford et a/., 1989b). Further investigations will be required to determine the defective step for translocation of p25 in insect cells. Modification of the precore signal sequence may circumvent the problems with p25 processing in insect cells. The recombinant baculovirus encoding the core domain induced the synthesis of large quantities of the p22 polypeptide. The core polypeptide was assembled into recognizable core structures. The density of the core particles in CsCl (1.30 g/cm3) and their appearance by electron microscopy suggested that most of the core particles do not contain nucleic acid. This is in contrast to core particles synthesized in bacteria which contain nucleic acid (Cohen and Richmond, 1982). A major portion of the core particles synthesized in insect cells were detected in the culture medium. Several lines of evidence suggest that the core particles were not released into the medium by cell lysis. Large amounts of core particles could be detected in the medium by 3 days p.i. at which time less than 1% of the cells were permeable to tt-ypan blue. By Day 4 p.i., greater than 50% of total p22 polypeptide was present in the medium yet greater than 95% of the cells still excluded trypan blue. Although the secretion of core particles was not initially anticipated, the same phe-
HEW CORE AND PRECORE ANTIGENS
nomenon has been observed in mammalian cells. Core reactivity banding in CsCl at the density of immature core particles was observed in HepG2 cells transfected with a complete HBV genome (Sureau eta/., 1986) and in HBV-infected primary chimpanzee hepatocyte cultures (Jacob et a/., 1989). Recently, a carefully controlled study indicated that the appearance of immature core particles in the culture medium of mammalian cells was not due to cell lysis. In cells expressing both core particles and /3-galactosidases only HBcAg reactivity was detected in the culture medium (Jean-Jean et a/., 1989). The secretion of a naked capsid structure is not unique to the HBV core particle. A recent electron microscopy investigation visualized the secretion of SV40 virions (a naked capsid) via large cytoplasmic vesicular structures (Clayson eta/., 1989). In addition, the use of an improved baculovirus transfer vector, pVL941 (Luckow and Summers, 1988; Lanford, 1988), which yielded increased core mRNA levels resulted in reduced accumulation of p22 in the medium due to early cell death. Although circumstantial in nature, we interpreted these findings to indicate that toxic levels of core particles were accumulating in the cells, because the rate of synthesis became greater than the rate of secretion. Again, this would imply that core is secreted from living cells, otherwise increased cell death would result in an increased level of core in the medium. The immunogenicity studies with precore and core indicated that the immune response to the two polypeptides was qualitatively different, with the reactivity of antiserum being greatest to the immunizing polypeptide. These findings are similar to those reported by others for the immunogenicity of core particles and HBeAg. Although the precore antigen was not properly processed in insect cells, the immunogenicity studies indicate that p25 may be suitable for studies comparing the immunogenicity of HBeAg and HBcAg. Core particles derived from infected hepatocytes and detergent-treated Dane particles possess an associated kinase activity that phosphorylates HBcAg (Albin and Robinson, 1980; Gerlich eT al., 1982; Feitelson et al., 1982; Petit and Pillot, 1985). In interest of determining the nature of this kinase activity and the usefulness of the baculovirus system for the analysis of this function, kinase assays were performed with highly purified core particles. Insect cell-derived core particles exhibited a kinase activity similar to the activity previously observed with core particles produced during active HBV replication. The phosphorylated products of the kinase reaction were polypeptides with molecular weights of 22,000 and 14,000. These same products were previously observed in core kinase reactions and the 14,000 molecularweight polypeptide was con-
231
sidered to be a degradation product of p22 (Albin and Robinson, 1980). Our results confirmed this relationship. Newly synthesized intracellular core antigen contained no pl4 following a kinase reaction, yet core particles accumulating in the medium have progressively increasing amounts of ~14. Following a 2-day purification scheme, pl4 was the predominant product of the kinase reaction. The events leading to the degradation of p22 are not currently apparent, but the possibility exists that the presence of a lipid envelope or encapsidation of the pregenome may stabilize the core particle. The degradation observed may resemble the process by which HBV DNA is released after penetration of the cell. Kinase reactions followed by alkaline phosphatase treatment suggested that particles possessing pl4 were permeable. All of the phosphate-label on pl4 and a portion of the p22 phosphate-label was removed by alkaline phosphatase. The accessible phosphates of p14 and p22 may be present on disrupted core particles which would be consistent with pl4 being a degradation product of ~22. An alternative hypothesis would envision a copurifying kinase that phosphory lates p14 and p22 on the exterior of the particle. The remainder of the p22 phosphate-label was completely resistant to phosphatase treatment which implies that some the phosphate moieties reside on the interior of the core particle (Gerlich et a/., 1982). An internal localization of the kinase is consistent with the observations that the activity cannot be separated from core particles after numerous purification steps and no similar activity can be purified from the medium of uninfected cells (data not shown). The presence of kinase activity in recombinant core particles demonstrates that in this system the activity is not derived from another HBVencoded protein. However, no direct evidence was obtained that would suggest that the activity or the phosphorylation sites present in Dane particles and insect cell-derived cores is the same. Comparison of the amino acid sequence of the core open reading frame with the consensus sequence for protein kinases (Hunter, 1987; Hanks et al., 1988) indicates that p22 does not possess a domain similar to known kinases. Thus, the kinase activity in insect cell-derived cores appears to be a cellular protein. Several mechanisms can be envisioned forthe encapsidation of a cellular kinase. The random entrapment of soluble cellular proteins at the site of particle morphogenesis is one such possibility. However, if a cellular kinase plays a role in HBV replication, a mechanism may have evolved for specific encapsidation. The ability to purify large quantities of core particles from insect cells should be useful in the characterization of the encapsidated kinase. In addition, the bacu-
232
LANFORD
lovirus system may be useful for the analysis of other functional characteristics potentially required of ~22 for virion morphogenesis, such as recognition of the pregenome and polymerase and association with HBsAg. Furthermore, large quantities of highly purified core particles will be useful in the preparation of a two component HBsAg-HBcAg vaccine that will provide both humoral and cellular immunity. ACKNOWLEDGMENTS We gratefully acknowledge Max D. Summers for the generous gift of the baculovirus expression system, Max D. Summers and Verne A. Luckow for helpful advtce on the use of the system, and Ana Elias for assistance In the preparation of this manuscript. This work was supported in part by the Texas Advanced Technology Program (grant 3220).
REFERENCES ALBIN, C., and ROBINSON,W. S. (1980). Protein kinase activity in hepa-
titis B virus. /. Viral. 34, 297-302. BRUSS, V., and GERLICH, W. H. (1988). Formation of transmembranous hepatitis B e-antigen by cotranslatlonal in vitro processing of the viral precore protein. \/iro/ogy 163, 268-275. BUSCHER, M., REISER, W., WILL, H., and SCHALLER, H. (1985). Transcripts and the putative RNA pregenome of duck hepatitis B virus: Implications for reverse transcription. Cell40, 717-724. CHANG, C., ENDERS,G.. SPRENGEL,R., PETERS,N., VARMUS, H. E., and GANEM, D. (1987). Expresslon of the precore region of an avian hepatitis B virus is not required for viral replication. /. viral. 61, 3322-3325. CLAYSON, E. T., BRANDO, L. V. J., and COMPANS, R. W. (1989). Release of simian virus 40 vlrlons from eplthelial cells is polarized and occurs without cell lysis. 1. Viral. 63, 2278-2288. COHEN, B. J., and RICHMOND,J. E. (1982). Electron microscopy of hepatitis B core antigen synthesized in f. co/i Nature (London) 296, 677-678. ENDERS, G. H., GANEM, D., and VARMUS, H. E. (1987). 5’.Terminal sequences Influence the segregation of ground squirrel hepatitis virus RNAs Into polynbosomes and viral core particles. /. L’irol. 61, 35-41. FEITELSON, M. A., MARION, P. L.. and ROBINSON,W. S. (1982). Core particles of hepatitis B virus and ground squirrel hepatitis virus. /. Viroi. 43, 741-748. FERNS,R. B., and TEDDER, R. S. (1984). Monoclonal antibodies to hepatitis Be antigen (HBeAg) derived from hepatitis B core antigen (HBcAg): Their use in characterization and detection of HBeAg. /. Gen. Wrol. 65, 899-908. GANEM, D., and VARMUS, H. E. (1987). The molecular biology of the hepatitis B viruses. Annu. Rev. Biochem. 56, 651-693. GERLICH, W. H., GOLDMANN, U., MULLER, R., STIBBE. W., and WOLFF, W. (1982). Specificity and localization of the hepatitis B virus-assoelated protein kinase. /. Viral. 42, 761-766. HANKS, S. K.. QUINN, A. M., and HUNTER,T. (1988). The protein kinase family: Conserved features and deduced phylogeny of the catalytic domains. Science 241,42-52. HINK, W. F. (1970). Established Insect cell line from the cabbage looper, Trxhoplusia ni. Nature (London) 226, 466-467. HUNTER, T. (1987). A thousand and one protein kinases. Cell 50, 823-829. IMAI, M., NOMURA, M., GOTANDA, T., SANO. T., TACHIBANA, K., MIYAMOTO, H., TAKAHASHI, K., TOYAMA, S.. MIYAKAWA. Y., and MAYUMI,
AND NOTVALL M. (1982). Demonstration of two distinct antlgenic determinants on hepatitis Be antigen by monoclonal antibodies. J. lmmunol. 128, 69-72. JACOB,J. R., EICHBERG.J. W., and LANFORD, R. E. (1989). In vitro repllcation and expressjon of hepatitis B virus from chronically infected primary chimpanzee hepatocytes. HepatoiogylO. 921-927. JEAN-JEAN,O., LEVRERO,M., WILL, H., PERRICAUDET,M., and ROSSIGNOL, J.-M. (1989). Expression mechanism of the hepatitis B virus (HBV) C gene and biosynthesis of HBe antigen. Virology 170, 99106. KNISKERN,P. J., HAGOPIAN,A., MONTGOMERY, D. L., BURKE,P., DUNN, N. R., HOFMANN, K. J., MILLER, W. J., and ELLIS, R. W. (1986). Unusually high-level expresslon of a foreign gene (hepatltls B virus core antigen) in Saccharomyces cerevisiae. Gene 46, 135-l 41. LANFORD, R. E. (1988). Expression of simian virus 40 T antigen In insect cells using a baculovirus expression vector. !I;ro/ogy 167, 72-81. LANFORD, R. E., and BUTEL, J. S. (1979). Antigenic relatlonshtp of SV40 early protetns to purified large T polypeptide. L/iro/ogy 97, 295-306. LANFORD, R. E., CAREY, K. D., ESTLACK, L. E.. SMITH, G. C., and HAY, R. V. (1989a). Analysts of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained In serum-free medium. ln Vitro Cell. Dev. Biol. 25, 174-l 82. LANFORD, R. E., KENNEDY, R. C., DREESMAN, G. R., EICHBERG,J. W., NOTVALL, L., LUCKOW,V. A., and SUMMERS, M. D. (1988). Expression of hepatitis B virus surface and core antigens using a baculovirus expression vector. ln “International Symposium on Viral Hepatitis and Liver Disease, London, 1987, Proceedings: Vtral Hepatltis and Liver Disease” (A. J. Zuckerman, Ed.), pp. 372-378. A. R. Liss, New York. LANFORD, R. E., LUCKOW,V., KENNEDY, R. C., DREESMAN, G. R., NOTE VALL, L., and SUMMERS, M. D. (198913). Expression and characterization of hepatitis B virus surface antigen polypeptldes In insect cells with a baculovlrus expresston system. /. Viral. 63, 15491557. LANFORD, R. E., NOT~ALL, L. M., DREESMAN, G. R., HARRISON,C. R., LOCKWOOD,D., and BURK, K. H. (1987). Expression and charactertzation of hepatitis B virus precore-core antigen in f. co/i Viral lmmunol. 1, 97-l 09. LUCKOW,V. A., and SUMMERS, M. D. (1988). Trends In the development of baculovirus expression vectors. Bioflechnology6,47P55. MCLACHLAN, A., MILICH, D. R., RANEY, A. K.. RIGGS, M. G., HUGHES, 1. L., SORGE, L., and CHISARI, F. V. (1987). Expression of hepatitis B virus surface and core antigens: Influences of pre-S and precore sequences. /. Viral. 61, 683-692. MILICH, D. R.. MCLACHLAN, A., MORIARPI, A., and THORNTON, G. B. (1987). Immune response to hepatitis b virus core antigen (HBcAg): Localization of T cell recognition sites within HBcAg/HBeAg J.lmmunol. 139, 1223-1231. MIYANOHARA,A., IMAMURA, T., ARAKI, M., SUGAWARA,K.. OHTOMO, N., and MATSUBARA,K. (1986). Expression of hepatitis B virus core antigen gene in Saccharomyces cerevisiae: Synthesis of two polypeptides translated from different initiation codons. /. Viral. 59, 176-180. MOROY, T., ETIEMBLE,J., TREPO, C., TIOLLAIS, P., and BUENDIA, M.-A. (1985). Transcription of woodchuck hepatitts virus In the chronically infected liver. fMBO/. 4, 1507-l 514. MURRAY, K., BRUCE, S. A., WINGFIELD, P., VAN EERD, P., DE REUS, A., and SCHELLEKENS,H. (1987). Protective lmmunisatlon against hepatitis B with an internal antigen of the virus. 1. Med. Viral. 23, 10 l107. Ou, J. H., LAuB, O., and RU~TER,W. J. (1986). Hepatitis B virus gene function: The precore region targets the core antigen to cellular
HBV CORE AND PRECORE ANTIGENS membranes and causes the secretion of the e antigen. Proc. Nat/. Acad. SC;. USA 83,1578-l 582. PETIT, M.-A., and PILLOT, 1. (1985). HBc and HBe antigenicity and DNA-binding activity of major core protein P22 in hepatitis B virus core particles isolated from the cytoplasm of human liver cells. J. Viral. 53, 543-551. PORTNOY, B. L., CONKLIN, R. H., MENN. M., OLARTE. J., and DUPONT, H. L. (1977). Reliable identification of reovirus-like agent in diarrhea1 stools. J. Lab. C/in. Med. 89, 560-563. Roos, S., FUCHS, K., and ROGGENDORF.M. (1989). Protection of woodchucks from InfectIon with woodchuck hepatitis virus by immunization with recombinant core protein. /. Gen. Viral. 70, 20872095. ROOSSINCK,M. J., JAMEEL, S., LOUKIN, S. H., and SIDDIQUI, A. (1986). Expression of hepatitis B viral core region in mammalian cells. Mol. Celi. Biol. 6, 1393-l 400. SALFELD.J., PFAFF, E., NOAH, M., and SCHALLER,H. (1989). Antigenic determinants and functional domains in core antigen and e antigen from hepatitis B virus. /. Viral. 63, 798-808. SCHLICHT, H. J., SALFELD.J., and SCHALLER,H. (1987). The duck hepatitis B virus pre-C region encodes a stgnal which is essential for synthesis and secretton of processed core proteins but not for virus formatlon. /. VYrol. 61, 370 l-3709. SERRANO,M. A., and HIRSCHMAN, S. Z. (1984). Properties of hepatitis Be antigen synthesized by rat cells transfected with circular viral DNA. J. Gen. Viral. 65, 1373-l 383. STAHL, S., MACKAY, P., MAGALIN, M., BRUCE, S. A., and MURRAY, K. (1982). Hepatitis B virus core antigen: synthesis in fscher;cichia co/i and application in diagnosis. Proc. Nat/. Acad. Sci. USA 79, 16061610. STANDRING,D. N., Ou, J.-H., MASIARZ. F. R., and RUT~ER.W. J. (1988). A signal peptide encoded within the precore region of hepatitis B virus directs the secretion of a heterogeneous population of e
233
antigens in Xenopus oocytes. Proc. Nat/. Acad. Sci. USA 85, 8405-8409. SUMMERS, M. D., and SMITH, G. E. (1987). A manual of methods for baculovirus vectors and insect cell culture procedures. Tex. Agric. Exp. Stn. (Bull.) No. 1555. SUREAU. C., ROMET-LEMONNE, J.-L., MULLINS, J. I., and ESSEX, M. (1986). Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA. Ce//47,37-47. TAKEHARA,K., IRELAND,D., and BISHOP,D. H. L. (1988). Co-expression of the Hepatitis B surface and core antigens using baculovirus multiple expression vectors. J. Gen. Viral. 69, 2763-2777. TIOLLAIS, P.. POURCEL,C., and DEJEAN,A. (1985). The hepatitis B virus. Nature (London) 317,489-495. TOWBIN, H., STAEHELIN. T., and GORDON, J. (1979). Electrophoretic Transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Nafl. Acad. Sci. USA 76,4350-4354. VOLKMAN, L. E., and SUMMERS, M. D. (1975). Nuclear polyhedrosis virus detection: Relative capabilities of clones developed from Trichoplusia ni ovarian cell line TN-368 to serve as indicator cells in a plaque assay. /. Viral. 16, 1630-l 637. VOLKMAN, L. E., SUMMERS, M. D., and HSIEH, C.-H. (1976). Occluded and nonoccluded nuclear polyhedrosis virus grown in Trichoplusia n;; Comparative neutralization, comparative infectivity, and in vitro growth studies. 1. Viral. 19.820-832. WATERS,J. A., JOWET, T. P., and THOMAS, H. C. (1986). Identification of a dominant immunogenic epitope of the nucleocapsid (HBc) of the hepatitis B virus. /. Med. Viol. 19,79-86. WEIMER,T., SALFELD,I., and WILL, H. (1987). Expression of the hepatitis B virus core gene in vitro and in viva. 1. Viral. 61, 3109-31 13. WILL, H., REISER,W., WEIMER, T., PFAFF, E., BUSCHER,M., SPRENGEL, R., CATTENEO, R., and SCHALLER,H. (1987). Replication strategy of human hepatitis B virus. /. Viral. 61, 904-91 1.