Hepatitis B Virus DNA-negative Dane Particles Lack Core Protein but Contain a 22-kDa Precore Protein without C-terminal Arginine-rich Domain
2005; Elsevier BV; Volume: 280; Issue: 23 Linguagem: Inglês
10.1074/jbc.m501564200
ISSN1083-351X
AutoresTatsuji Kimura, Nobuhiko Ohno, Nobuo Terada, Akinori Rokuhara, Akihiro Matsumoto, Shintaro Yagi, Eiji Tanaka, Kendo Kiyosawa, Shinichi Ohno, Noboru Maki,
Tópico(s)Bacteriophages and microbial interactions
ResumoDNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera. The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles. DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera. The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles. Hepatitis B virus (HBV) 1The abbreviations used are: HBV, hepatitis B virus; HBcrAg, HBV core-related antigens; HBcAg, hepatitis B core antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; EIA, enzyme immunoassay; aa, amino acid; ER, endoplasmic reticulum; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; HBeAb, hepatitis B e antibody; rHBcAg, recombinant HBcAg; rHBeAg, recombinant HBeAg; LC, liquid chromatography; MS/MS, tandem mass spectrometry. infects more than 300 million people and is a major cause of liver diseases. The HBV belongs to the Hepadnavirus family and is a small (42 nm) enveloped DNA virus, which possesses a 27-nm icosahedral nucleocapsid composed of core protein and a 3.2-kb partially double-stranded, circular genome (1Seeger C. Mason W.S. Microbiol. Mol. Biol. Rev. 2000; 64: 51-68Crossref PubMed Scopus (1223) Google Scholar). Although the term "Dane particles" refers to the 42-nm HBV particles (2Dane D.S. Cameron C.H. Briggs M. Lancet. 1970; 1: 695-698Abstract PubMed Scopus (658) Google Scholar) and is often used in reference to the complete HBV particles, electron microscopic studies have suggested that the DNA-negative "empty" Dane particles are predominant in sera (3Gerin J.L. Ford E.C. Purcell R.H. Am. J. Pathol. 1975; 81: 651-668PubMed Google Scholar, 4Alberti A. Diana S. Scullard G.H. Eddleston W.F. Williams R. Gastroenterology. 1978; 75: 869-874Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 5Takahashi T. Kaga K. Akahane Y. Yamashita T. Miyakawa Y. Mayumi M. J. Med. Microbiol. 1980; 13: 163-166Crossref PubMed Scopus (5) Google Scholar, 6Sakamoto Y. Yamada G. Mizuno M. Nishihara T. Kinoyama S. Kobayashi T. Takahashi T. Nagashima H. Lab. Investig. 1983; 48: 678-682PubMed Google Scholar). The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. The HBV genome encodes two core-related open reading frames, precore and core genes (Fig. 1). These are expressed because of two in-frame ATG initiation codons located at the 5′ end of the genes. The first ATG encodes a 25-kDa protein (p25) containing the 29-amino acid (aa) precore sequence fused to the N terminus of the HBV core antigen (HBcAg). The p25 is directed toward the secretory pathway by a 19-aa signal sequence that is cleaved during translocation into the lumen of the endoplasmic reticulum (ER), producing a 22-kDa protein. Subsequent proteolytic cleavages within the arginine-rich C-terminal region (34 aa) generate a 17-kDa protein that is secreted as hepatitis B e antigen (HBeAg) (7Ou J.-H. Laub O. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1578-1582Crossref PubMed Scopus (242) Google Scholar, 8Garcia P.D. Ou J.-H. Rutter W.J. Walter P. J. Cell Biol. 1988; 106: 1093-1104Crossref PubMed Scopus (154) Google Scholar, 9Standring D.N. Ou J.-H. Masiarz F.R. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8405-8409Crossref PubMed Scopus (110) Google Scholar, 10Messageot F. Salhi S. Eon P. Rossignol J-M. J. Biol. Chem. 2003; 278: 891-895Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). A heterogeneous population of these precore derivatives has been observed in the sera of patients and is serologically defined as HBeAg (9Standring D.N. Ou J.-H. Masiarz F.R. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8405-8409Crossref PubMed Scopus (110) Google Scholar, 11Takahashi K. Machida A. Funatsu G. Nomura M. Usuda S. Aoyagi S. Tachibana K. Miyamoto H. Imai M. Nakamura T. Miyakawa Y. Mayumi M. J. Immunol. 1983; 130: 2903-2907PubMed Google Scholar, 12Schlicht H-J. J. Virol. 1991; 65: 3489-3495Crossref PubMed Google Scholar). Conversely, the second ATG specifies the 21.5-kDa HBcAg, which assembles into dimers that form the virus capsid (7Ou J.-H. Laub O. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1578-1582Crossref PubMed Scopus (242) Google Scholar, 9Standring D.N. Ou J.-H. Masiarz F.R. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8405-8409Crossref PubMed Scopus (110) Google Scholar, 13Weimer T. Salfeld J. Will H. J. Virol. 1987; 61: 3109-3113Crossref PubMed Google Scholar, 14Bottcher B. Wynne S.A. Crowther R.A. Nature. 1997; 386: 88-91Crossref PubMed Scopus (696) Google Scholar, 15Conway J.F. Cheng N. Zlotnick A. Wingfield P.T. Stahl S.J. Steven A.C. Nature. 1997; 386: 91-94Crossref PubMed Scopus (398) Google Scholar). HBcAg is a 183-residue protein with two domains, the assembly domain that forms the capsid and the C-terminal arginine-rich domain that is responsible for RNA packaging (Fig. 1). The assembly domain, lacking the C-terminal domain, is sufficient for self-assembly into capsid particles. The arginine-rich C-terminal domain is involved in binding to the HBV RNA pregenome or the HBV DNA genome but is dispensable for HBV capsid assembly in Escherichia coli (16Gallina A. Bonelli F. Zentilin L. Rindi G. Muttini M. Milanesi G. J. Virol. 1989; 63: 4645-4652Crossref PubMed Google Scholar, 17Birnbaum F. Nassal M. J. Virol. 1990; 64: 3319-3330Crossref PubMed Google Scholar, 18Crowther R.A. Kiselev N.A. Bottcher B. Berriman J.A. Borisova G.P. Ose V. Pumpens P. Cell. 1994; 77: 943-950Abstract Full Text PDF PubMed Scopus (433) Google Scholar, 19Wingfield P.T. Stahl S.J. Williams R.W. Steven A.C. Biochemistry. 1995; 34: 4919-4932Crossref PubMed Scopus (194) Google Scholar) and insect cells (20Beames B. Lanford R.E. Virology. 1993; 194: 597-607Crossref PubMed Scopus (56) Google Scholar). The capsid is enclosed within an envelope containing the viral glycoprotein surface antigen (HBsAg) and released to the circulation as Dane particles. We previously developed enzyme immunoassays (EIAs) for HBcAg (21Kimura T. Rokuhara A. Matsumoto A. Yagi S. Tanaka E. Kiyosawa K. Maki N. J. Clin. Microbiol. 2003; 41: 1901-1906Crossref PubMed Scopus (53) Google Scholar) and HBV core-related antigens (HBcrAg) (22Kimura T. Rokuhara A. Sakamoto Y. Yagi S. Tanaka E. Kiyosawa K. Maki N. J. Clin. Microbiol. 2002; 40: 439-445Crossref PubMed Scopus (176) Google Scholar, 23Rokuhara A. Tanaka E. Matsumoto A. Kimura T. Yamaura T. Orii K. Sun X. Yagi S Maki N Kiyosawa K. J. Viral Hepat. 2003; 10: 324-330Crossref PubMed Scopus (82) Google Scholar). Serum specimens were pretreated with SDS to release and denature antigens and to inactivate antibodies. The HBcAg assay specifically measures core protein (21Kimura T. Rokuhara A. Matsumoto A. Yagi S. Tanaka E. Kiyosawa K. Maki N. J. Clin. Microbiol. 2003; 41: 1901-1906Crossref PubMed Scopus (53) Google Scholar), and the HBcrAg assay measures precore/core proteins, including core protein and HBeAg (22Kimura T. Rokuhara A. Sakamoto Y. Yagi S. Tanaka E. Kiyosawa K. Maki N. J. Clin. Microbiol. 2002; 40: 439-445Crossref PubMed Scopus (176) Google Scholar, 23Rokuhara A. Tanaka E. Matsumoto A. Kimura T. Yamaura T. Orii K. Sun X. Yagi S Maki N Kiyosawa K. J. Viral Hepat. 2003; 10: 324-330Crossref PubMed Scopus (82) Google Scholar). The present study investigated precore/core proteins in HBV-infected human sera using the new assays. The results suggest that most of the Dane particles were DNA-negative and were composed of a 22-kDa precore protein containing the uncleaved signal peptide and lacking the C-terminal arginine-rich domain. We present a new model for the formation of HBV DNA-negative particles. Serum/Plasma Samples—Hepatitis B plasma panels were purchased from Boston Biomedica, Inc. (BBI, West Bridgewater, MA), or ProMedDx (Norton, MA). Clinical sera were collected between 1997 and 2001 at the Shinshu University Hospital (Matsumoto, Japan) from patients with persistent HBV infection. Thirteen of these serum samples containing ≥0.05 ng/ml HBcAg were immunoprecipitated to examine HBcAg/HBcrAg ratios. Of the 30 total serum samples (from 23 males and 7 females), 22 were HBeAg-positive, and 7 were HBeAb-positive. The remaining sample was positive for both HBeAg and HBeAb. None of the 30 patients was treated with anti-viral agents such as interferon or lamivudine. All sera were stored at –30 °C or below until testing. The study design conformed to the 1975 Declaration of Helsinki and was approved by the Ethics Committees of the institutions involved in this study. A written informed consent was obtained from each patient. Recombinant HBV Core-related Antigens—Recombinant HBcAg (rH-BcAg, aa 1–183) and HBeAg (rHBeAg, aa –10 –149) were expressed and purified as described (21Kimura T. Rokuhara A. Matsumoto A. Yagi S. Tanaka E. Kiyosawa K. Maki N. J. Clin. Microbiol. 2003; 41: 1901-1906Crossref PubMed Scopus (53) Google Scholar, 22Kimura T. Rokuhara A. Sakamoto Y. Yagi S. Tanaka E. Kiyosawa K. Maki N. J. Clin. Microbiol. 2002; 40: 439-445Crossref PubMed Scopus (176) Google Scholar). The concentration of these antigens was determined using the BCA protein assay kit (Pierce) and bovine serum albumin standards according to the manufacturer's instructions. Monoclonal Antibodies and EIAs for HBcAg or HBcrAg—Anti-HB-cAg and anti-HBcrAg monoclonal antibodies were established as reported previously (21Kimura T. Rokuhara A. Matsumoto A. Yagi S. Tanaka E. Kiyosawa K. Maki N. J. Clin. Microbiol. 2003; 41: 1901-1906Crossref PubMed Scopus (53) Google Scholar, 22Kimura T. Rokuhara A. Sakamoto Y. Yagi S. Tanaka E. Kiyosawa K. Maki N. J. Clin. Microbiol. 2002; 40: 439-445Crossref PubMed Scopus (176) Google Scholar). The HBcAg-specific monoclonal antibody, HB50, recognizes SPRRR repeats in the arginine-rich domain of HBcAg (21Kimura T. Rokuhara A. Matsumoto A. Yagi S. Tanaka E. Kiyosawa K. Maki N. J. Clin. Microbiol. 2003; 41: 1901-1906Crossref PubMed Scopus (53) Google Scholar), whereas the anti-HBcrAg monoclonal antibody, HB91, recognizes aa 1–19 of HBcAg and thus reacts to denatured HBcAg, HBeAg, and other precore/core proteins (22Kimura T. Rokuhara A. Sakamoto Y. Yagi S. Tanaka E. Kiyosawa K. Maki N. J. Clin. Microbiol. 2002; 40: 439-445Crossref PubMed Scopus (176) Google Scholar). HBcAg and HBcrAg were measured by EIA as described previously (21Kimura T. Rokuhara A. Matsumoto A. Yagi S. Tanaka E. Kiyosawa K. Maki N. J. Clin. Microbiol. 2003; 41: 1901-1906Crossref PubMed Scopus (53) Google Scholar, 22Kimura T. Rokuhara A. Sakamoto Y. Yagi S. Tanaka E. Kiyosawa K. Maki N. J. Clin. Microbiol. 2002; 40: 439-445Crossref PubMed Scopus (176) Google Scholar, 23Rokuhara A. Tanaka E. Matsumoto A. Kimura T. Yamaura T. Orii K. Sun X. Yagi S Maki N Kiyosawa K. J. Viral Hepat. 2003; 10: 324-330Crossref PubMed Scopus (82) Google Scholar). The assays contain a sample pretreatment step that inactivates antibodies and dissociates antigens in samples. The assays can thus detect antigens within the viral envelope or complexed with antibodies in addition to free antigens. HBV Markers and HBV DNA Measurement—HBeAg and HBsAg were measured by radioimmunoassay or by chemiluminescent immunoassay (Abbott, Tokyo), respectively. HBV DNA was detected by PCR using the Amplicor HBV monitor test (Roche Applied Science). Samples showing values over the detection range were remeasured after dilution to obtain quantitative results. Sucrose Density Gradient Ultracentrifugation—Aliquots (1.7 ml) of 10, 20, 30, 40, 50, and 60% (w/w) sucrose in a solution containing 10 mm Tris-HCl, 150 mm NaCl, and 1 mm EDTA (pH 7.5) were carefully layered in a 12-ml Ultracentrifuge tube and left at room temperature for 6 h. HBeAg-positive plasma (0.1–1.0 ml) was layered on this sucrose gradient, and ultracentrifugation was performed at 200,000 × g for 15 h at 4 °C in a Beckman Sw40Ti rotor. Fractions were collected from the top to the bottom of the gradient. The density of each fraction was calculated from the weight and volume. Each fraction was diluted 10-fold and tested for HBcAg and HBcrAg as well as for HBsAg, HBeAg, and HBV DNA. Immunoprecipitation—Immunoprecipitation was carried out using magnetic beads coated with polyclonal anti-HBsAg from the "HBV-Direct Mag kit" (JSR Corp., Tokyo) (24Mukaide M. Tanaka Y. Katayose S. Tano H. Murata M. Hikata M. Fujise K. Sakugawa H. Suzuki K. Zaunders J. Nagasawa Y. Toda G. Mizokami M. J. Gastroenterol. Hepatol. 2003; 18: 1264-1271Crossref PubMed Scopus (19) Google Scholar). A 200-μl aliquot of sample was mixed with 50 μl of reaction buffer from the kit and 50 μl of a magnetic bead suspension. The mixture was incubated for 30 min at room temperature with gentle agitation and then magnetically separated. HBcAg and HBcrAg in supernatant and precipitate were measured by EIA. Because some samples contain a large amount of HBsAg, which exceeds the capacity of anti-HBsAg beads, if the precipitated HBcAg ratio was less than 90%, the sample was diluted 10- or 100-fold and then reimmunoprecipitated. Electron Microscopy—A 500-μl aliquot of HBV-positive plasma was subjected to ultracentrifugation on linear 10–50% (w/w) sucrose density gradients. The high density HBcrAg peak fractions (corresponding to Fig. 3A, fractions 23 and 24) and HBcAg peak fractions (corresponding to Fig. 3A, fractions 25 and 26) were separated by the second ultracentrifugation through linear 35–50% (w/w) sucrose density gradients. The fractions were fixed by adding paraformaldehyde solution to a final concentration of 4%. A 4-μl aliquot of each fraction was diluted in 90 μl of distilled water in 5-mm diameter polyallomer centrifugation tubes (Beckman Instruments), and copper grids filmed with Formvar membranes and treated additionally with poly-l-lysine were placed on the bottom of the tubes in the solution. Ultracentrifugation (200,000 × g, 4 °C, 2 h) was performed in a Beckman TLS-55 swinging bucket rotor to concentrate the virus particles and allow them to attach to the Formvar membranes on the copper grids. Afterward, the attached virus particles were negatively stained with 4% uranyl acetate and observed at an accelerating voltage of 80 kV in an electron microscope (H-7500, Hitachi, Tokyo). Fifteen electron micrographs of the virus particles from each fraction were taken randomly at a magnification of ×80,000. The number of virus particles in the 3.76 μm2 area was then counted on each electron micrograph. The diameters of the virus particles in each fraction were also measured. Western Blot Analysis—Samples were subjected to SDS-PAGE through a 15–25% polyacrylamide gel under reducing conditions. Proteins in the gel were electroblotted onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore) at 15 V for 45 min. The membrane was blocked and probed using alkaline phosphatase-conjugated HB50 (for HBcAg) or HB91 (for HBcrAg) monoclonal antibody at room temperature for 1 h. The membrane was washed and incubated with 5-bro-mo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate solution (KPL, Gaithersburg, MD) for 15 min (for HBcrAg) or 90 min (for HBcAg), respectively. N-terminal Amino Acid Sequence Analysis—A 6-ml aliquot of HBV-positive plasma was subjected to ultracentrifugation on linear 10–60% (w/w) sucrose density gradients, and subsequently the high density HBcrAg peak fractions (Fig. 3A, fractions 23 and 24) were separated by gel filtration through Superose 6 HR (Amersham Biosciences). Void fractions were collected and ultracentrifuged at 200,000 × g for 15 h at 4 °C using a Beckman SW 50.1 rotor. The precipitate was separated by SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore) at 15 V for 45 min. Proteins on the membrane were stained using Coomassie Brilliant Blue-R250. The N-terminal amino acid sequence of the 22-kDa band was analyzed using the Procise 494 cLC protein sequencing system at the Apro Life Science Institute, Inc. (Tokushima, Japan). Mass Spectrometry Analysis—The 22-kDa protein was purified as described above. The 22-kDa band was cut from the SDS-polyacrylamide gel and digested in-gel by trypsin at 35 °C for 20 h. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) of the digested sample was performed on a Voyager-DE STR (Applied Biosystems) in positive ion reflection mode. External mass calibration was performed using four points bracketing the mass range of interest. Results were analyzed using the NCBI non-redundant data base (molecular mass range 15–30 kDa) by the MS-Fit 3.1.1 ProteinProspector 3.2.1 program (University of California), taking into account probable post-translational modifications. LC-MS/MS was performed using a Q-Tof2 (Micromass, Manchester, UK) quadrupole time-of-flight electrospray ionization mass spectrometer in nanoflow LC ionization mode. The analyses were performed at the Apro Life Science Institute, Inc. Statistical Analyses—The virus particle numbers on each electron micrograph were statistically compared by Welch's t test. The diameters of the virus particles were statistically compared by Student's t test. Paired t tests were used to analyze differences between log concentrations of HBcrAg and those of HBcAg. Differences were considered significant at p < 0.05. Specificity of HBcAg and HBcrAg EIAs—The specificity of the HBcAg and HBcrAg assays was confirmed by using rHB-cAg and rHBeAg. The HBcAg assay specifically reacted to rHBcAg but not to rHBeAg (Fig. 2A). The HBcrAg assay reacted equally to rHBcAg and rHBeAg (Fig. 2B). Density Distribution of HBV Precore/Core Proteins—HBV DNA-positive plasma (ProMedDx 9990776, HBsAg-positive, HBeAg-positive, HBV DNA 9.1 log copies/ml) was subjected to ultracentrifugation through a 10–60% (w/w) sucrose density gradient. Fractions were tested for HBcAg, HBcrAg, HBsAg, HBeAg, and HBV DNA (Fig. 3A). HBcAg appeared in the high density fractions and peaked in the same fraction (fraction 25) as HBV DNA. HBsAg was distributed in fractions of lower density, and HBeAg was dispersed widely in fractions of much lower density. HBcrAg peaked in fraction 24, slightly lower in density than the HBV DNA and HBcAg peaks in addition to a peak corresponding to HBeAg at much lower density. The concentration of HBcrAg in fraction 24 was 13-fold higher than the concentration of HBcAg in fraction 25. The high density HBcrAg peak was therefore predominantly composed of precore proteins other than core protein. High density HBcrAg fractions (Fig. 3A, fractions 23–26) were reanalyzed under gentler (30–50%) sucrose density gradient sedimentation (Fig. 3B). HBcrAg concentration peaked in lower density fractions than HBcAg and HBV DNA, indicating that high density HBcrAg clearly differs from HBcAg. HBsAg concentration exhibited a shoulder at the HBcrAg peak fraction. Immunoprecipitation by Anti-HBsAg—Sucrose density fractions (Fig. 3A) were immunoprecipitated by the anti-envelope protein HBsAg. Most of the HBcAg (97.5, 97.8, 96.2, and 95.1% from fractions 23–26) was precipitated by anti-HBsAg. Although more than 94% (94.5, 94.1, and 94.3% from fractions 7, 10, 13) of low density HBcrAg was observed in the supernatant, more than 96% (96.2, 96.8, 96.9, and 96.5% from fractions 23–26) of high density HBcrAg was in the precipitate. These data suggest that similar to the core protein, the high density HBcrAg exists in enveloped particles. Stability of HBcrAg Particles—The HBV core forms very stable capsid particles resistant to denaturing pH, temperature, or detergents (25Newman M. Suk F-M. Cajimat M. Chua P.K. Shih C. J. Virol. 2003; 77: 12950-12960Crossref PubMed Scopus (61) Google Scholar). Particle fractions of HBV-positive plasma were treated with or without 3% Nonidet P-40 detergent at 37 °C for 30 min and then subjected to gel filtration through Superose 6 HR (exclusion limit = 4 × 107 Da). Fractions were tested for HBcrAg and HBcAg. Regardless of Nonidet P-40 treatment, HBcrAg and HBcAg appeared in the void fractions (Fig. 4), indicating that HBcrAg formed high molecular mass (> 107 Da) particles resistant to 3% Nonidet P-40 treatment, as did the HBcAg. Electron Microscopy—HBcAg and HBcrAg in plasma 9990776 were separated by sequential sucrose density ultracentrifugation. The resultant HBcrAg-rich fraction (fraction A) contained 6.06-fold more HBcrAg than the HBcAg-rich fraction (fraction B) but contained only 3 and 38% of the HBV DNA and HBcAg, respectively, found in fraction B (Table I). Virus particles in the two fractions were concentrated and attached to the copper grids by ultracentrifugation and then negatively stained and observed under the electron microscope. Although virus particles appearing similar to Dane particles were observed in fraction B, more such Dane-like particles were seen in fraction A (Fig. 5), which contained HBV DNA at only 3% of that in fraction B. Fraction A contained 17.9 ± 11.6/3.76 μm2 Dane-like particles, which was significantly more than in fraction B (5.6 ± 3.8/3.76 μm2) (n = 15, p < 0.001) (Table I). The Dane-like particles in fractions A and B were not morphologically distinguishable (Fig. 5) but were quite similar to those reported previously (2Dane D.S. Cameron C.H. Briggs M. Lancet. 1970; 1: 695-698Abstract PubMed Scopus (658) Google Scholar, 3Gerin J.L. Ford E.C. Purcell R.H. Am. J. Pathol. 1975; 81: 651-668PubMed Google Scholar, 4Alberti A. Diana S. Scullard G.H. Eddleston W.F. Williams R. Gastroenterology. 1978; 75: 869-874Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 6Sakamoto Y. Yamada G. Mizuno M. Nishihara T. Kinoyama S. Kobayashi T. Takahashi T. Nagashima H. Lab. Investig. 1983; 48: 678-682PubMed Google Scholar). The mean diameters of the measured particles were 41.5 ± 2.2 nm in fraction A and 42.0 ± 2.2 nm in fraction B (Table I). The mean diameters were not significantly different from one another (n = 60, p = 0.27) and were similar to the sizes reported previously (2Dane D.S. Cameron C.H. Briggs M. Lancet. 1970; 1: 695-698Abstract PubMed Scopus (658) Google Scholar).Table IHBcAg, HBcrAg, HBV DNA and Dane-like particles in fractions A and BHBV DNAHBcAgHBcrAgDane-like particlesNumberDiameter×107 copies/mlng/mlng/mlin 3.76 μm2nmFraction A13812,82317.9 ± 11.6aData are presented as mean ± S.D. n = 15; p < 0.001.41.5 ± 2.2bData are presented as mean ± S.D. n = 60; p = 0.27.Fraction B3982104665.6 ± 3.8aData are presented as mean ± S.D. n = 15; p < 0.001.42.0 ± 2.2bData are presented as mean ± S.D. n = 60; p = 0.27.Ratios (A/B)0.030.386.063.20a Data are presented as mean ± S.D. n = 15; p < 0.001.b Data are presented as mean ± S.D. n = 60; p = 0.27. Open table in a new tab Identification of Particle HBcrAg as a 22-kDa Precore Protein (p22cr) Lacking the C-terminal Domain—HBV DNA-positive plasma (BBI PHM935A-14) was subjected to a 10–60% sucrose density gradient and fractionated into 15 fractions. The fractions were then analyzed by Western blotting using monoclonal antibodies for HBcAg and HBcrAg (Fig. 6A). HBcAg was detected only in fraction 8 and the original plasma. Conversely, four bands were detected by anti-HBcrAg in plasma. HBeAg and two additional proteins, which were considered HBeAg precursors, were detected in low density fractions by anti-HBcrAg. A 22-kDa protein, which was termed p22cr, was also detected in fraction 8. To confirm whether p22cr was identical to HBcAg, the p22cr band was compared with the neighboring HBcAg band (Fig. 6B). The p22cr protein exhibited slightly higher molecular weight than HBcAg. A fainter HBcAg band was also detected by anti-HBcrAg. Because p22cr did not react with the HB50 anti-HBcAg antibody, SPRRR sequences (positioned at aa 155–174 as three repeats) were presumed absent. Furthermore, p22cr maintained its 22-kDa molecular mass without the N-glycosylation consensus site. These data suggest that p22cr contains a complete or nearly complete precore region, including the signal sequence. The p22cr protein was purified, and the N-terminal amino acid sequence was analyzed. p22cr showed no significant amino acid signal (data not shown), suggesting that the N terminus of p22cr might be blocked. We then applied mass spectrum analysis. Data from MALDI-TOF MS were analyzed by MS-Fit search using the NCBI non-redundant data base. The search selected 117 of 87,559 entries for the molecular mass range 15–30 kDa. The top 20 matches were all HBV core or precore proteins. Six of 50 input peptide masses matched five precore/core peptides (Table II) that spanned 40% (86 of 212 aa) of the sequence. The N-terminal precore tryptic peptide (peptide 1, aa –28 to aa –9) was found to be N-terminally acetylated and was, therefore, not directly accessible to Edman sequencing. p22cr lacked the first N-terminal methionine of the precore protein. Another peptide, peptide 5, was identified as a precore/core peptide comprising aa 128–150. LC-MS/MS analysis was also applied. Two peptide fractions corresponding to peptides 2 and 5 of Table II were recognized as HBV precore/core proteins. Thus, the p22cr protein was confirmed to be a precore protein from N-terminally acetylated aa –28 to at least aa 150.Table IIMALDI-TOF MS analysis of p22crPeptidem/z observed[M+H]+ matchedΔPeptide sequenceModificationsAmino acidsStartEndppm12233.14382233.118311.4048QLFHLCLIISCSCPTVQASKN-terminally acetylated-28-921237.63881237.6428-3.2413DLLDTASALYR293931913.91671913.892812.4530EALESPEHCSPHHTALR405631984.91531984.9299-7.1477EALESPEHCSPHHTALRAcrylamide-modified Cys405641552.77981552.8045-15.8855DLVVSYVNTNMGLK839652490.36632490.3720-2.2840TPPAYRPPNAPILSTLPETTVVR128150 Open table in a new tab HBcAg and HBcrAg Levels in HBsAg-positive Particles from Chronic Hepatitis B Sera—The levels of precore/core proteins were investigated in HBV particles of chronic hepatitis B sera. Sera were immunoprecipitated by anti-HBsAg, and then levels of HBcAg and HBcrAg in the supernatant and precipitate were measured. More than 91% of the HBcAg was detected in precipitate fractions. HBcrAg in precipitate fractions included p22cr and HBcAg. In the precipitate fractions, HBcAg concentration ranged from 0.08 to 165 ng/ml, whereas HBcrAg ranged from 0.59 to 1,079 ng/ml (Fig. 7A). Log concentrations of HBcrAg were significantly higher than those of HBcAg (p < 0.001). HBcrAg predominated over HBcAg in precipitates from both HBeAg-positive and -negative sera. HBcAg represented only 3.1–37.4% (median 10.5%) of HBcrAg (Fig. 7B), indicating that the remaining p22cr was the dominant precore/core protein in HBsAg-positive particles. Similar results were also obtained from high density fractions of the sucrose gradient in six tested samples. In the present study, we demonstrated that HBV DNA-negative Dane particles are dominant in serum and are composed of a precore protein p22cr, which contains an uncleaved signal sequence and lacks a C-terminal arginine-rich domain. Early electron microscopic and radiolabeling studies have suggested that less than 10% of Dane particles include full cores with viral DNA (3Gerin J.L. Ford E.C. Purcell R.H. Am. J. Pathol. 1975; 81: 651-668PubMed Google Scholar, 4Alberti A. Diana S. Scullard G.H. Eddleston W.F. Williams R. Gastroenterology. 1978; 75: 869-874Abstract Full Text PDF PubMed Scopus (58) Google Scholar, 5Takahashi T. Kaga K. Akahane Y. Yamashita T. Miyakawa Y. Mayumi M. J. Med. Microbiol. 1980; 13: 163-166Crossref PubMed Scopus (5) Goo
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