Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan 112, Republic of China 1
Author for correspondence: Szecheng J. Lo.Fax +886 2 2821 2880. e-mail losj{at}ym.edu.tw
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Abstract |
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Introduction |
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The HBV core protein contains either 183 or 185 aa, depending on the virus subtype, ayw, adr or adw (Tiollais et al., 1981 ). Core proteins expressed in bacteria can self-assemble into nucleocapsid-like particles (Cohen & Richmond, 1982
; Nassal, 1988
; Gallina et al., 1989
; Birnbaum & Nassal, 1990
; Kann & Gerlich, 1994
) and can encapsidate RNA nonspecifically (Pasek et al., 1979
). Studies using truncated HBV core protein have also shown that aa 10144 are responsible for self-assembly into nucleocapsids (Gallina et al., 1989
; Birnbaum & Nassal, 1990
; Zheng et al. , 1992
). In addition, the C terminus of the core protein is required for pregenome encapsidation and modulates the activity of polymerase for reverse transcription of the RNA pregenome and binding to the elongating DNA (Hatton et al., 1992
; Nassal, 1992
).
The C-terminal 33 aa of HBV core proteins contain 16 (adr and ayw) or 17 (adw) arginine (R) residues; 14 of them are clustered into four arginine repeats (IIV) (Tiollais et al., 1981 ). Previous studies have indicated that this arginine-rich domain is important for pregenome encapsidation and genomic DNA binding (Birnbaum & Nassal, 1990
; Hatton et al., 1992
; Nassal, 1992
). In the HBV core protein, the arginine-rich domain appears in three SPRRR (Ser- Pro-Arg-Arg-Arg) motifs. In the DHBV core protein, the nucleic acid- binding domain is located at aa 181228, which has ten arginine and five lysine residues but no SPRRR motif (Sprengel et al., 1985
). It is therefore of interest to know whether an HBV core protein in which the C-terminal SPRRR motif is replaced by a stretch of arginine or lysine residues, similar to that of DHBV, still retains its function for pregenome encapsidation and viral DNA maturation.
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Methods |
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Immunoprecipitation and Western blotting analysis.
Transfected HuH-7 cells were lysed in NET buffer (50 mM Tris, pH 7·5, 150 mM NaCl, 0·5 mM EDTA and 0·5% NP-40). Lysates were centrifuged to remove insoluble cell debris and were cleared by protein ASepharose (Amersham Pharmacia Biotech). The supernatant was then incubated with rabbit anti- core (Dako) or mouse anti-Flag (M2; Eastman Kodak) antiserum with protein ASepharose complexes. The beads were subsequently washed four times with NET buffer and the complexes were boiled in reducing sample buffer and resolved by 15% SDSPAGE (Laemmli, 1970 ). Western blotting was then performed as described by Towbin et al. (1979)
and a proteinantibody complex was detected by rabbit anti-core antibody and subsequently developed by enhanced chemiluminescence.
Viral nucleocapsid isolation.
To collect nucleocapsids from intracellular extract, transfected cells were collected and incubated in PBS with 1% NP-40 at 4 °C overnight (Beames & Lanford, 1995 ). After a low-speed centrifugation to remove nuclei and NP-40-insoluble cell debris, the cellular lysate was collected. To collect nucleocapsids from culture fluids, the media were incubated with NP-40 to a final concentration of 1% at room temperature for 2 h and then clarified by centrifugation at 13000 r.p.m. in a JA-20 rotor for 30 min. The viral nucleocapsids from cellular lysate and medium were then concentrated by centrifugation at 45000 r.p.m. in a Ti55.2 rotor for 2·5 h. The isolated viral nucleocapsids were resuspended in low-salt TNE buffer (10 mM TrisHCl, pH 7·5, 100 mM NaCl and 1 mM EDTA).
CsCl gradient centrifugation.
The isolated nucleocapsids were resuspended in high-salt TNE buffer (10 mM TrisHCl, pH 7·4, 150 mM NaCl, 1 mM EDTA and 0·1% sodium azide) and subjected to CsCl centrifugation (average density 1·24 g/ml, final density 1·11·5 g/ml) at 35000 r.p.m. in an SW41 rotor for 44 h. The gradients were fractionated into 0·5 ml samples from the top and each fraction was subjected to ELISA for detection of core antigen (General Biologicals). The density of individual fractions was determined by the refractive index using a refractometer.
Endogenous DNA polymerase assay.
Endogenous DNA polymerase activity assay was performed as described previously (Junker et al., 1987 ) with modification (Chiang et al., 1990
; Lin & Lo, 1992
). Briefly, one-fifth of the partially purified virus particle samples was incubated with pol-mix buffer (50 mM TrisHCl, pH 7·4, 40 mM NH4Cl, 5 mM MgCl2 , 0·5% NP-40, 0·2% 2-mercaptoethanol, and 25 µM each of dATP, dGTP and dTTP) at 37 °C for 2 h in the presence of [
-32P]dCTP (5000 mCi/mmol; Amersham). Subsequently, a chase was performed for 2 h by adding unlabelled dCTP (25 µM final concentration) at 37 °C. Contaminated nucleic acids were digested by micrococcal nuclease (5 U) for 1 h at 37 °C. Proteins were digested by 50 µg/ml proteinase K treatment in the presence of 1% SDS for 1 h at 37 °C. If necessary, 32 P-labelled 3/3097 HindIII-digested plasmid (6·4 kb) was added as a control for technical error. Glycogen was added (0·8 mg/ml final concentration) and samples were then extracted with an equal volume of phenol/chloroform. An aqueous portion was then collected and desalted, and unincorporated dNTPs were removed by a 1 ml Sephadex G-50 gel filtration column in TE buffer (10 mM TrisHCl, pH 7·5 and 0·1 mM EDTA). The labelled DNA species were adjusted to 0·3 M sodium acetate (pH 4·8) and then precipitated with 2·5 vol. ethanol; DNAs were then separated on 1% agarose gels by electrophoresis and autoradiographed.
In vitro kinase assay.
The immunoprecipitated nucleocapsids were washed three times and then incubated in kinase reaction buffer (50 mM TrisHCl, pH 7·4, 10 mM MgCl2 and 0·4% NP-40) and 10 pmol [-32P]ATP (7000 Ci/mmol; Amersham) as described previously (Jeng et al., 1991
; Lin & Lo, 1992
). After incubation at 37 °C for 1 h, the complex was washed five times with NET buffer and then subjected to SDSPAGE (15%) separation and autoradiographed.
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Results |
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After transfection with each plasmid expressing wild-type or mutant core proteins into HuH-7 cells, immunoprecipitation by anti-core antibodies followed by Western blot analysis showed that the migration of the mutated core proteins (C144Arg and C144Lys) was faster than that of the wild-type C183 (Fig. 2 , lanes 3 and 4 vs lane 1). This was expected because the mutated core proteins possess 25 aa residues less at the C terminus. Nevertheless, the expression efficiency of mutated core proteins was similar to that of the wild-type core in HuH-7 cells, which were transfected with an equal amount of corresponding plasmids (Fig. 2
).
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Discussion |
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Although the assembly of mosaic nucleocapsids from different types of core proteins has been demonstrated (Chang et al., 1994 ; Ulrich et al., 1998
), it is unclear whether co-expression of mutant with wild-type core protein will reduce or inhibit wild-type viral DNA maturation (Scaglioni et al., 1994
, 1996
; von Weizsacker et al., 1996
; Wands et al., 1997
). A decrease in HBV DNA maturation may be due to the formation of less stable mosaic capsids as postulated above. Recently, the influence of C-terminal truncation on capsid assembly has been well characterized. It is now known that the segment between aa 129149 forms an arm-like structure that plays a critical role in capsid assembly (Metzger & Bringas, 1998)
. Residue 150 has been localized on the inner side of the capsid around the 5-fold and 6-fold symmetry axes (Zlotnick et al., 1997
). Capsids formed by C-terminally truncated core proteins ending at residue 144 present a hole around the 6-fold symmetry axes that is not present in wild-type capsids (Crowther et al., 1994
). Therefore, any truncation or introduction of a stretch of positively charged amino acids at the C terminus of the core protein could generate instability of mosaic capsids. In this study, we have observed that the effect of mosaic capsids on the retention of pregenome and kinase activity is different. There is an 8-fold reduction in DNA repairing signal in the presence of FlagC151 (Fig. 5
, lane 5 vs lane 2) whereas no significant change in phosphorylated HisC183 was observed in the presence or absence of FlagC151 (Fig. 6a
, lane 4 vs lane 2).
Trans-complementation between two defective pregenomes of HBV DNA has been largely used to define the elements required for pregenome packaging and genome maturation (Schlicht et al., 1989 ; Chiang et al., 1990
, 1992
; Yu & Summers, 1991
; von Weizsacker et al., 1995
). In this study, we have employed triple-plasmid transfection and demonstrated that three different less than full- length pregenomic RNAs (3·0, 1·9 and 1·0 kb) could be successfully encapsidated from a single transfection ( Fig. 7
). To our knowledge, the 1·0 kb HBV DNA may be derived from the smallest pregenome of HBV being encapsidated and reported so far. Since the 1·0 kb band signal results from the filled-in gap of HBV DNA, it may provide insight into the mechanism of immature termination of the plus strand, which is the hallmark of hepadnaviral DNA (Tiollais et al., 1981
).
The finding of encapsidation of three less than full-length pregenomes raised a question regarding the mechanism of pregenome encapsidation. In the two-plasmid transfection studies, the higher intensity of the repaired 3·0 kb band as compared with that of the 1·9 kb band (2:1) is probably due to the fact that 3·0 kb RNA can direct polymerase synthesis and can act as cis-preference packaging (Hirsch et al., 1990 ). In contrast, such cis-preference packaging of polymerase-producing RNA apparently did not happen in the triple- plasmid transfection, since the 3·0 kb DNA is the weakest signal among three repaired HBV DNAs (Fig. 7
, lane 4). Although the mutated core proteins and various forms of less than full- length pregenomes used in this study do not occur in vivo, competition for pregenome packaging between core internal deletion and wild-type full-length pregenomes was reported in chronic hepatitis patients (Yuan et al., 1998a
, b
). Furthermore, competition between full-length and spliced pregenomes for encapsidation was also commonly observed, since only a minute amount of defective particles containing spliced RNA was detected in the sera of HBV carriers (Terré et al., 1991
; R. L. Kuo & T.-S. Su, personal communication). Therefore, the triple- plasmid transfection system can be a tool for exploring the limiting factors involved in the competition of differently sized pregenome encapsidation.
At present, how core protein, polymerase and pregenome interact to assemble into replication-competent nucleocapsids is still largely unknown. Jeng et al. (1993) have demonstrated that the corepolymerase interaction occurs in HBV nucleocapsids but the exact contact sites for these two proteins have not yet been determined. Results from this study suggest that the C terminus of wild- type core protein, in addition to interacting with a pregenome, may also be a site for direct interaction with a kinase or cellular factor. Several lines of evidence indicate that the core protein can be phosphorylated in vivo (Roossinck & Siddiqui, 1987
) and in vitro (Albin & Robinson, 1980
; Feitelson et al., 1982
; Gerlich et al., 1982
; Duclos-Vallée et al., 1998
) and that kinases can be incorporated into nucleocapsids (Albin & Robinson, 1980
; Kann & Gerlich, 1994
; Kau & Ting, 1998
). Additionally, two cellular factors, including the heat shock protein 90 complex (Hu & Seeger, 1996
; Hu et al., 1997
) and terminal-protein-associated kinase (Kau & Ting, 1997
, 1998
), have been found to interact with polymerase and are present in virus particles. Therefore, it will be interesting to know whether core mutants generated in this study either lose the ability to bind cellular kinases or factors or/and induce the instability of nucleocapsids.
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Acknowledgments |
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References |
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Beames, B. & Lanford, R. E. (1995). Insertions within the hepatitis B virus capsid protein influence capsid formation and RNA encapsidation. Journal of Virology 69, 6833-6838 .[Abstract]
Birnbaum, F. & Nassal, M. (1990). Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein. Journal of Virology 64, 3319-3330 .[Medline]
Chang, C. , Zhou, S. , Ganem, D. & Standring, D. N. (1994). Phenotypic mixing between different hepadnavirus nucleocapsid protein reveals C protein dimerization to be cis preferential. Journal of Virology 68, 5225-5231 .[Abstract]
Chiang, P.-W. , Hu, C.-P. , Su, T.-S. , Lo, S. J. , Chu, M.-H. , Schaller, H. & Chang, C. (1990). Encapsidation of truncated human hepatitis B virus genomes through trans-complementation of the core protein and polymerase. Virology 176, 355-361.[Medline]
Chiang, P.-W. , Jeng, K.-S. , Hu, C.-P. & Chang, C. (1992). Characterization of a cis element required for packaging and replication of the human hepatitis B virus. Virology 186, 701-711.[Medline]
Cohen, B. J. & Richmond, J. E. (1982). Electron microscopy of hepatitis B core antigen synthesized in E. coli. Nature 296, 677-678.[Medline]
Crowther, R. A. , Kiselev, N. A. , Bottcher, B. , Berriman, J. A. , Borisova, G. P. , Ose, V. & Pumpens, P. (1994). Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy. Cell 77, 943-950.[Medline]
Duclos-Vallée, J.-C. , Capel, F. , Mabit, H. & Petit, M.-A. (1998). Phosphorylation of the hepatitis B virus core protein by glyceraldehyde-3-phosphate dehydrogenase protein kinase activity. Journal of General Virology 79, 1665-1670 .[Abstract]
Feitelson, M. A. , Mavion, P. L. & Robinson, W. S. (1982). Core particles of hepatitis B virus and ground squirrel hepatitis virus. II. Characterization of the protein kinase reaction associated with ground squirrel hepatitis virus and hepatitis B virus. Journal of Virology 43, 741-748.[Medline]
Gallina, A. , Bonelli, F. , Zentilin, L. , Rindi, G. , Muttini, M. & Milanesi, G. (1989). A recombinant hepatitis B core antigen polypeptide with the protamine-like domain deleted self- assembles into capsid particles but fails to bind nucleic acids. Journal of Virology 63, 4645-4652 .[Medline]
Ganem, D. & Varmus, H. E. (1987). The molecular biology of hepatitis B viruses. Annual Review of Biochemistry 56, 651-693.[Medline]
Gerlich, W. H. , Goldmann, U. , Muller, R. , Stibbe, W. & Wolff, W. (1982). Specificity and localization of the hepatitis B virus-associated protein kinase. Journal of Virology 42, 761-766.[Medline]
Graham, F. & van der Eb, A. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456-457.[Medline]
Gust, I. D. , Burrell, C. J. , Coulepis, A. G. , Robinson, W. S. & Zuckerman, A. J. (1986). Taxonomic classification of human hepatitis B virus. Intervirology 25, 14-29.[Medline]
Hatton, T. , Zhou, S. & Standring, D. N. (1992). RNA- and DNA- binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication. Journal of Virology 66, 5232-5241 .[Abstract]
Hirsch, R. , Lavine, J. , Chang, L. , Varmus, H. & Ganem, D. (1990). Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as well as for reverse transcription. Nature 344, 552-555.[Medline]
Hu, J. & Seeger, C. (1996). Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase. Proceedings of the National Academy of Sciences, USA 93, 1060-1064 .
Hu, J. , Toft, D. O. & Seeger, C. (1997). Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids. EMBO Journal 10, 59-68.[Abstract]
Jeng, K.-S. , Hu, C.-P. & Chang, C. (1991). Differential formation of disulfide linkages in the core antigen of extracellular and intracellular hepatitis B virus core particles. Journal of Virology 65, 3924-3927 .[Medline]
Jeng, K.-S. , Hu, C.-P. & Chang, C. (1993). Hepatitis B core antigen forms oligomers and complexes with the p gene product in hepatitis B virus core particles. Journal of Gastroenterology and Hepatology 8, S114-S118.
Junker, M. , Galle, P. & Schaller, H. (1987). Expression and replication of the hepatitis B virus genome under foreign promoter control. Nucleic Acids Research 15, 10117-10132 .[Abstract]
Junker-Niepmann, M. , Bartenschlager, R. & Schaller, H. (1990). A short cis-acting sequence is required for hepatitis B virus pregenome encapsidation and sufficient for packaging of foreign RNA. EMBO Journal 9, 3389-3396.[Abstract]
Kann, M. & Gerlich, W. H. (1994). Effect of core protein phosphorylation by protein kinase C on encapsidation of RNA within core particles of hepatitis B virus. Journal of Virology 68, 7993-8000 .[Abstract]
Kau, J.-H. & Ting, L.-P. (1997). A serine-kinase-containing protein complex interacts with the terminal protein domain of polymerase of hepatitis B virus. Journal of Biomedical Science 4, 155-161.[Medline]
Kau, J.-H. & Ting, L.-P. (1998). Phosphorylation of the core protein of hepatitis B virus by a 46-kilodalton serine kinase. Journal of Virology 72, 3796-3803 .
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[Medline]
Liao, W. & Ou, J.-H. (1995). Phosphorylation and nuclear localization of the hepatitis B virus core protein: significance of serine in the three repeated SPRRR motifs. Journal of Virology 69, 1025-1029 .[Abstract]
Lin, C.-G. & Lo, S. J. (1992). Evidence for involvement of a ribosomal leaky scanning mechanism in the translation of the hepatitis B virus pol gene from the viral pregenome RNA. Virology 188, 342-352.[Medline]
Lin, C.-G. , Yang, S.-J. , Hwang, W.-L. , Su, T.-S. & Lo, S. J. (1995). Demonstration of the presence of protease-cutting site in the spacer of hepatitis B viral Pol protein. Journal of Virological Methods 51, 61-74.[Medline]
Metzger, K. & Bringas, R. (1998). Proline-138 is essential for the assembly of hepatitis B virus core protein. Journal of General Virology 79, 587-590.[Abstract]
Nassal, M. (1988). Total chemical synthesis of a gene for hepatitis B core protein and its functional characterization. Gene 66, 279-294.[Medline]
Nassal, M. (1992). The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly. Journal of Virology 66, 4107-4116 .[Abstract]
Nassal, M. (1996). Hepatitis B virus morphogenesis. Current Topics in Microbiology and Immunology 214, 297-337.[Medline]
Nassal, M. & Schaller, H. (1993). Hepatitis B virus replication. Trends in Microbiology 1, 221-228.[Medline]
Pasek, M. , Goto, T. , Gilbert, W. , Zink, B. , Schaller, H. , MacKay, P. , Leadbetter, G. & Murray, K. (1979). Hepatitis B virus genes and their expression in E. coli. Nature 282, 575-579.[Medline]
Roossinck, M. J. & Siddiqui, A. (1987). In vivo phosphorylation and protein analysis of hepatitis B virus core protein. Journal of Virology 61, 955-961.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press .
Sanger, F. , Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitor. Proceedings of the National Academy of Sciences, USA 74, 5463-5467 .[Abstract]
Scaglioni, P. P. , Melegari, M. & Wands, J. R. (1994). Characterization of hepatitis B virus core mutants that inhibit viral replication. Virology 205, 112-120.[Medline]
Scaglioni, P. , Melegari, M. , Takahashi, M. , Chowdhury, J. R. & Wands, J. (1996). Use of dominant negative mutants of the hepadnaviral core protein as antiviral agents. Hepatology 24, 1010-1017 .[Medline]
Schlicht, H. J. , Bartenschlager, R. & Schaller, H. (1989). The duck hepatitis B virus core protein contains a highly phosphorylated C terminus that is essential for replication but not for RNA packaging. Journal of Virology 63, 2995-3000 .[Medline]
Seifer, M. & Standring, D. N. (1993). Recombinant human hepatitis B virus reverse transcriptase is active in the absence of the nucleocapsid of the viral replication origin, DR1. Journal of Virology 67, 4513-4520 .[Abstract]
Sprengel, R. , Kuhn, C. , Will, H. & Schaller, H. (1985). Comparative sequence analysis of duck and human hepatitis B virus genomes. Journal of Medical Virology 15, 323-333.[Medline]
Summers, J. & Mason, W. S. (1982). Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 29, 403-415.[Medline]
Tavis, J. E. & Ganem, D. (1993). Expression of functional hepatitis B virus polymerase in yeast reveals it to be the sole viral protein required for correct initiation of reverse transcription. Proceedings of the National Academy of Sciences, USA 90, 4107-4111 .[Abstract]
Terré, S. , Petit, M.-A. & Brechot, C. (1991). Defective hepatitis B virus particles are generated by packaging and reverse transcription of spliced viral RNAs in vivo. Journal of Virology 65, 5539-5543 .[Medline]
Tiollais, P. , Charnay, P. & Vyas, G. N. (1981). Biology of hepatitis B virus. Science 213, 406-411.[Medline]
Towbin, H. , Stachelin, T. & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy Sciences, USA 76, 4350-4354 .[Abstract]
Ulrich, R. , Nassal, M. , Meisel, H. & Kruger, D. H. (1998). Core particles of hepatitis B virus as carrier for foreign epitopes. Advances in Virus Research 50, 141-182.[Medline]
von Weizsacker, F. , Wieland, S. & Blum, H. E. (1995). Identification of two separable modules in the duck hepatitis B virus core protein. Journal of Virology 69, 2704-2707 .[Abstract]
von Weizsacker, F. , Wieland, S. & Blum, H. E. (1996). Inhibition of viral replication by genetically engineered mutants of the duck hepatitis B virus core protein. Hepatology 24, 294-299.[Medline]
Wands, J. R. , Geissler, M. , Putlitz, J. , Blum, H. , von Weizsacker, F. , Mohr, L. , Yoon, S. K. , Melegari, M. & Scaglioni, P. P. (1997). Nucleic acid-based antiviral and gene therapy of chronic hepatitis B infection. Journal of Gastroenterology and Hepatology 12, S354-S369 .[Medline]
Wang, G. H. & Seeger, C. (1992). The reverse transcriptase of hepatitis B virus acts as a protein primer for viral DNA synthesis. Cell 71, 663-670.[Medline]
Yu, M. & Summers, J. (1991). A domain of the hepadnavirus capsid protein is specifically required for DNA maturation and virus assembly. Journal of Virology 65, 2511-2517 .[Medline]
Yuan, T. T.-T. , Lin, M.-H. , Chen, D.-S. & Shih, C. (1998a). A defective interference-like phenomenon of human hepatitis B virus in chronic carriers. Journal of Virology 72, 578 -584.
Yuan, T. T.-T. , Lin, M.-H. , Qiu, S. M. & Shih, C. (1998b). Functional characterization of naturally occurring variants of human hepatitis B virus containing the core internal deletion mutation. Journal of Virology 72, 2168 -2176.
Zheng, J. , Schodel, F. & Peterson, D. L. (1992). The structure of hepadnaviral core antigen: identification of free thiols and determination of the disulfide bonding pattern. Journal of Biological Chemistry 267, 9422-9429 .
Zlotnick, A. , Cheng, N. , Conway, J. F. , Booy, F. P. , Steven, A. C. , Stahl, S. J. & Wingfield, P. T. (1996). Dimorphism of hepatitis B virus capsids is strongly influenced by the C-terminus of the capsid protein. Biochemistry 35, 7412-7421 .[Medline]
Zlotnick, A. , Cheng, N. , Stahl, S. J. , Conway, J. F. , Steven, A. C. & Wingfield, P. T. (1997). Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein: implications for morphogenesis and organization of encapsidated RNA. Proceedings of the National Academy of Sciences, USA 94, 9556-9561 .
Received 8 June 1999;
accepted 24 June 1999.