Protein Kinase Activity in Hepatitis B Virus

Transcription

1 JOURNAL OF VIROLOGY, Apr. 1980, p Vol. 34, No X/80/ /06$02.00/0 Protein Kinase Activity in Hepatitis B Virus CATHERINE ALBIN AND WILLIAM S. ROBINSON* Department ofmedicine, Stanford University School ofmedicine, Stanford, California Protein kinase activity was found in hepatitis B virions (Dane particles) purified from the plasma of hepatitis B virus-infected patients, in virion cores, and in hepatitis B core antigen particles purified from hepatitis B virus-infected hepatic tissue and was not found in purified hepatitis B surface antigen particle preparations free of Dane particles. Only a fraction of the major polypeptide (apparent size, 19,700 daltons) in Dane particle cores and hepatitis B core antigen particles from infected liver appeared to be phosphorylated, and phosphorylation changed the electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels to that expected for a polypeptide of 20,600 daltons. Five minor polypeptides with apparent sizes between 38,000 and 63,000 daltons were phosphorylated in Dane particles and Dane particle core preparations but were not detected in hepatitis B core antigen particles from infected liver. None of these had electrophoretic mobilities corresponding to those of known hepatitis B surface antigen polypeptides. Prolonged storage of purified hepatitis B core antigen particles or incubation with human immunoglobulin G preparations containing antibody to the hepatitis B core antigen with or without antibody to the hepatitis B e antigen resulted in the conversion of the polypeptide with an apparent size of 20,600 daltons to ones with apparent sizes of 14,700 and approximately 6,000 daltons, suggesting proteolytic cleavage of the 20,600-dalton polypeptide under these conditions. The hepatitis B virion (also known as the Dane particle) is a spherical particle which is approximately 42 nm in diameter (6) and has a complex structure. It consists of an outer lipidcontaining envelope with hepatitis B surface antigen (HB8Ag) on its surface (1, 6) and an inner spherical core, or nucleocapsid, which is 28 mm in diameter and contains hepatitis B core antigen (HBcAg) (1), the viral DNA (12), DNA polymerase activity (8, 13), and probably hepatitis B e antigen (HBeAg) in a cryptic form (15). Nonionic detergents such as Nonidet P-40 (NP- 40) remove the HB8Ag-containing envelope of Dane particles (1), and the remaining core particles can be purified (7). Other HB.Ag forms found in the blood are small spherical and filamentous particles (1, 4, 6) containing HB5Ag but no HB,Ag, HBeAg, DNA, or DNA polymerase, and these are considered to be incomplete virus consisting of viral envelope material. HBeAg is a third hepatitis B virus (HBV)-associated antigen which was first found in the blood in a soluble form (9). HBcAg particles resembling Dane particle cores can be purified from HBVinfected liver (3, 7), and particles with similar morphology have been observed in electron micrographs of infected hepatocyte nuclei (2). Analyses of the polypeptides of purified Dane particle cores and HBcAg particles from infected liver have demonstrated consistently a major 297 polypeptide with an apparent size of between 17,000 and 19,000 daltons in different studies and sometimes minor components of a larger apparent size but variable in size and amount (5, 7). None of these polypeptides has been shown to have HBcAg reactivity (7), but the major polypeptide has been reported to react with antibody to the hepatitis B e antigen (anti-hbe) (15). All preparations of Dane particles, Dane particle cores, and HBcAg particles from HBV-infected liver that we have examined have been found to contain protein kinase activity, and this activity has followed the particles during their purification. Liver obtained at autopsy from an HBV-infected patient immunosuppressed to maintain an organ transplant was homogenized in a Waring blendor with TNE buffer (0.01 M Tris-hydrochloride [ph 7.5], 0.15 M NaCl, M EDTA). NP-40 and 2-mercaptoethanol were added to final concentrations of 0.1 and 0.05% (vol/vol), respectively, and the mixture was centrifuged in a Sorvall SS34 rotor for 15 min at 10,000 rpm. CsCl was added to the supernatant to a final density of 1.20 g/ml, and the solution was layered over a discontinuous CsCl gradient consisting of four density steps from 1.25 to 1.40 g/ml and centrifuged for 24 h in a Spinco SW27 rotor at 24,000 rpm. Peak fractions of HBcAg detected by a solid-phase micro-radioimmunoas-

2 298 NOTES say (7) were pooled, diluted, and pelleted in an SW40 rotor for 10 h at 20,000 rpm. The pellet was suspended in 200 pl of TNE buffer and layered over a 15 to 50% Renografin (E. R. Squibb & Sons) gradient containing TNE buffer and centrifuged at 60,000 rpm for 4 h in a Spinco SW60 rotor. Peak fractions of HB,Ag activity were diluted and pelleted as before. The pellet was suspended in CsCl with a final density of 1.20 g/ml and centrifuged in a discontinuous CsCl density gradient as described above. Figure 1A shows that protein kinase activity (incorporation of 32P from [y-32p]atp into an acid-insoluble form) closely followed HBcAg reactivity during the final equilibrium CsCl density gradient centrifugation step in the purification of HBcAg particles from infected liver. The same result was obtained with Dane particle cores after treating purified Dane particle preparations (7) with NP-40 and mercaptoethanol and banding the cores in a Renografin gradient as described above. The HBcAg particles from liver appeared to be highly purified because the preparations contained no detectable HB.Ag reactivity tested by radioimmunoassay (Ausria II, Abbott Laboratories), and only the 19,700-dalton polypeptide (designated P-19.7) previously shown to be the major polypeptide component of Dane particle cores (7) could be detected in such preparations by sodium dodecyl sulfate (SDS)-gel electrophoresis and staining with Coomassie brilliant blue (7, 10). Figure 1B shows that protein kinase activity closely followed Dane particle DNA polymerase activity when Dane particles purified from the plasma of infected patients (7) were centrifuged in a Renografm density gradient. Very highly purified HB8Ag particles (10) free of Dane particles were found to have little or no protein kinase activity. High levels of soluble protein kinase activity were found in serum, but the soluble activity was separated from HB,Ag particles during particle purification. The incorporation of 32p from [_y-32p]atp into an acid-insoluble form by HBcAg preparations required divalent cations. Concentrations of MgCl2 between 5 and 50 mm resulted in optimum incorporation and a fivefold-greater rate of incorporation than with MnCl2 at the same concentrations. The addition of EDTA inhibited all activity. Protein kinase activity appeared optimal at ph 7.5 and was significantly lower at ph values of 6.5 and 8.5. The addition of the nonionic detergent NP-40 resulted in approximately a threefold increase in Dane particle and liver HBCAg particle protein kinase activity. The addition of the reducing agent dithiothreitol had no effect on the activity associated with HB,Ag particles derived from infected liver and only marginal or no enhancement of Dane particle core activity. The phosphorylating activity remained associated with the detergent-treated core particles even after several subsequent purification steps, including discontinuous equilibrium CsCl density gradients and column chromatography on Sepharose 2B. High levels of incorporation occurred at temperatures between 15 and 540C, with a broad plateau between 30 and 450C. Maximum incorporation was usually achieved at 1 h, although further incorporation beyond 1 h was occasionally observed. Cyclic AMP had no effect on the protein kinase activity. After termination of the protein kinase reaction carried out with [y- 32P]ATP by the addition of EDTA and centrifugation of the cores through a sucrose solution, the acid-precipitable 32P appeared to be rapidly converted to, an acid-soluble form in several experiments, indicating the removal of phosphate from the particles. After the termination of the reaction with SDS, the 32P was made acid soluble by proteinase K digestion, indicating the association of 3P with protein. After protein kinase reactions with highly purified preparations of Dane particles, Dane particle cores, and infected-liver HBcAg particles in the presence of [y-32p]atp, radiolabeled polypeptides were identified by SDS-gel electrophoresis and autoradiography (10). Figure 2B shows that the major phosphorylated polypeptide in purified liver HBcAg particle preparations had an electrophoretic mobility corresponding to an apparent molecular weight of approximately 20,600 (designated P-20.6). In addition, variable amounts of a radioactive J. VIROL. component with an apparent size of 14,700 daltons (P-14.7) were sometimes but not always observed. These phosphorylated polypeptides had electrophoretic mobilities identical to those of the only polypeptides found in such preparations by Coomassie brilliant blue staining, and no larger radioactive components were detected. P-20.6 was also the major phosphorylated component in purified Dane particle core preparations (Fig. 2A, track 2). In addition, five other radioactive components containing smaller amounts of radioactivity and with apparent sizes of 38,000, 43,000, 48,000, 52,000, and 63,000 daltons were detected regularly in Dane particle core preparations. The 38,000-dalton component often contained a greater relative amount of 32P than shown in Fig. 2. All of the minor components, with the possible exception of the 63,000-dalton component, appeared to be electrophoretically distinct from HB8Ag particle polypeptides (10). The same spectrum of 32P-polypeptides was observed after the protein kinase reaction with purified Dane particle preparations (Fig. 2A, track 1). The

3 VOL. 34, 1980 NOTES S 3 I 5 0~~~~~~~~~~~~~ 00~~~~~~~~~~~~~ 2 A2.A x Fraction Number FIG. 1. Density gradient centrifugation for purification of HB,Ag particles from HBV-infected liver (A) and Dane particles from plasma (B). (A) Gradient fractions were collected after the third density gradient (CsCl) centrifugation step in purification ofhbcagparticles from a liver homogenate as described in the text. Each fraction was assayed for HB,Ag by solid-phase radioimmunoassay (7) (0) and for protein kinase activity (A) by incubating a sample with 0.75 mm [y-_32patp (3,000 Ci/mmol; ICN)-7 mm dithiothreitol-10 mm MgC1-50 mm Tris-hydrochloride (ph 7.5)-0.5% NP-40 in 100 il at 37 C. After 1 h, the reaction mixture was spotted on a 3MM Whatman paper disk, washed, and assayed for acid-precipitable 32P as previously described (11). (B) A Dane particle P2 preparation (7) was incubated in a DNA polymerase reaction mixture with [3H]dATP (11), and [y-]32patp was added as a substrate for the protein kinase reaction as described above. After 1 h at 37 C, the reaction mixture was layered over a 25 to 50%o Renografin gradient and centrifuged under conditions described in the text. Fractions collected from the gradient were assayed for 3H (0) and 32p (A---A) as described above. D K - A B origin of the five minor polypeptides phosphorylated in Dane particles and Dane particle cores 1 2 but not in HB,Ag particles purified from infected liver is not clear. Most of the latter particles appeared to contain no DNA and did not manifest DNA polymerase activity, as did a large fraction of the cores isolated from Dane particles, and they were not associated with HB5Ag, - -ESK as are Dane particles. Whether the minor components were HBsAg-containing polypeptides not completely removed from the cores when the Dane particles were treated with detergent and were electrophoretically different from known HB8Ag-containing polypeptides because -27 K they were phosphorylated, whether they repre- 23K sented minor polypeptide constituents of the g fh7-k- -20 f K core and may be related to the core DNA polym- 23 K erase activity or other core functions, or whether.6obk-s g _ they have some other origin remains to be determined. - 4 K Figure 3 shows a densitometer scan of Coomassie brilliant blue-stained polypeptides after SDS-polyacrylamide gel electrophoresis of HBcAg particles purified from infected liver be- FIG. 2. Autoradiograms of phosphorylated polypeptides ofpurified Dane particles (A, track 1), Dane carried out in the presence of [y-"p]atp as departicle cores (A, track 2), and HBcAg particles from scribed in the legend to Fig. 1, and radioactive poly- HBV-infected liver (B). Particles were purified as peptides were analyzed by SDS-gel electrophoresis described in the text, protein kinase reactions were and autoradiography (10).

4 300 NOTES J. VIROL. Zc B 19.7K K 19.?K Relative Migration FIG. 3. Densitometer tracings of Coomassie brilliant blue-stained polypeptides ofpurified preparations of HB.Ag particles from plasma and HBCAg particles from infected liver after electrophoresis in a 13% SDSpolyacrylamide gel. Numbers refer to the molecular weights of the indicated polypeptides. (A) HB.Ag; (B) HBcAg before phosphorylation; (C) HBcAg after in vitro phosphorylation for 2 h at 37 C with a solution containing 1 mm ATP, 10 mm MgCI2, and the remainder of the reaction mixture as described in the legend to Fig. 1. fore (Fig. 3B) and after (Fig. 3C) an in vitro phosphorylation reaction. Before phosphorylation, only one polypeptide (P-19.7) was apparent, but after phosphorylation some P-19.7 remained and two new polypeptides with sizes of approximately 20,600 (P-20.6) and 14,700 (P- 14.7) daltons were detected. P-20.6 and P-14.7 had electrophoretic mobilities identical to those of the 32P-polypeptides detected in autoradiographs such as that shown in Fig. 2B. None of the polypeptides corresponded to those of HB.Ag particles (10) shown in Fig. 3A. This result suggests that only a fraction of the 19,700- dalton polypeptide in HBcAg particle preparations was phosphorylated during this reaction, and the electrophoretic mobility of the phosphorylated fraction was changed to that expected for a polypeptide of 20,600 daltons. Even after extensive in vitro phosphorylation, not all of the 19,700-dalton polypeptide was converted to the slower-moving electrophoretic form. This could be due to a loss or absence of protein kinase activity in a fraction of the HBcAg particles or to the availability of only a fraction of the 19,700- dalton polypeptide molecules in core particles to the enzyme. Purified preparations of HBcAg particles from infected liver were sometimes found to contain the minor polypeptide described above with an electrophoretic mobility corresponding to that expected for a polypeptide of 14,700 daltons (P- 14.7) and shown in Fig. 2B and 3C. In preparations with relatively high amounts of 32P-P-14.7, a 32P-component with an electrophoretic mobility corresponding to a polypeptide of approximately 6,000 daltons (P-6) was also detected (Fig. 4, track 3). The relative amounts of 32p in P-20.6 and P-14.7 were variable in different experiments, and two conditions were found to be associated with the appearance of P-14.7 and the disappearance of P First, storage of purified HBcAg particle preparations at -70 C resulted in increasing relative amounts of 32P-P-14.7 over several weeks. Second, incubation of purified HBcAg particles with preparations of human immunoglobulin containing antibody to the hepatitis B core antigen (anti-hbc), with and without anti-hbe, appeared to result in the rapid disappearance of 32P-P-20.6 and the appearance of 32P-P-14.7 and 32P-P-6, as shown in Fig. 4. In this experiment, a preparation of purified HBcAg particles from liver was phosphorylated in the presence of [y-32p]atp, and one sample was analyzed by SDS-gel electrophoresis without further treatment (track 1) and two other samples were analyzed after incubation with different human immunoglobulin G (IgG) preparations containing anti-hbc or anti-hbe or both and then rabbit anti-human IgG (tracks 2 and 3). Very little 32P-P-14.7 was found in the untreated sample (track 1); incubation with an IgG preparation containing anti-hbc and then with rabbit anti-human IgG resulted in approximately equal amounts of the radioactivity in P and P-14.7 (track 2), and incubation with an IgG preparation containing anti-hbe and anti- HBc, and then with rabbit anti-human IgG resulted in mostly 32P-P-14.7 plus a small amount of 32p in P-6 and almost no 32p in P-20.6 (track 3). These results indicate that the 32P-polypeptide with an apparent size of 20,600 daltons

5 VOL. 34, K- _ :, 1~~~~~~~~~~~ 147sf K FIG. 4. Autoradiogram of phosphorylated polypeptides of HB,Ag particles from HBV-infected liver before and after immunoprecipitation. HB,Ag particles were purified as described in the text; a protein kinase reaction was carried out in the presence of [y-32p]atp as described in the legend to Fig. 1, and one sample (track 1) was not treated further; a second sample (track 2) was incubated for 1 h at 37 C with a human IgG preparation containing anti-hbc, followed by rabbit anti-human IgG for 1 h at 37 C, and the precipitate was collected by centrifugation and washed by suspension in TNE buffer and centrifugation three times; and a third sample (track 3) was incubated for 1 h at 37 C with a human IgG preparation containing anti-hbe and anti-hbc, followed by rabbit anti-human IgG for 1 h at 37 C, and the precipitate was collected by centrifugation and washed by suspension in TNE buffer and centrifugation three times. The three samples were then analyzed by SDS-gel electrophoresis and autoradiography. was converted to a form migrating in SDS-gel electrophoresis as expected for a 14,700-dalton polypeptide under the specific conditions described above. The mechanism of this conversion could be a cleavage of the 20,600-dalton polypeptide to a 14,700-dalton fragment containing a significant amount of 32P and a 6,000-dalton fragment with relatively little 32p. No conversion was apparent after HB,Ag 32P-particles were incubated with normal human serum, IgG preparations without anti-hbc, or bovine serum albumin, suggesting that an enzyme or other factor occurring frequently in serum was not responsible. The conversion under the conditions described above proceeded whether rabbit antihuman IgG or staphylococcal protein A was used to precipitate the HB,Ag-anti-HBc complexes, implicating the human IgG preparations with anti-hb, in conversion. If the conversion does..: NOTES 301 represent a proteolytic cleavage of P-20.6, the enzyme responsible for the cleavage may reside in the HBcAg particles since P-20.6 conversion occurred slowly with time during storage of purified particles. The mechanism of the rapid conversion, when HBcAg particles were incubated with the human IgG preparations with anti-hbc, is unclear but could have been due to an acceleration of the cleavage by the activity apparently present in purified HBcAg particle preparations triggered by specific antibody, to cleavage by protease activity in the IgG preparations, or to some other mechanism. Thus, highly purified preparations of Dane particles, Dane particle cores, and HBcAg particles from HBV-infected liver have been shown to contain protein kinase activity. In all instances, the activity copurified with the particles and was preferentially associated with the internal core of the Dane particle and HBcAg particles from liver. Core polypeptides were selectively phosphorylated, and other proteins added to the reaction mixture were phosphorylated only to a limited degree. The activity found here was similar to the protein kinase activity found in numerous other enveloped viruses in which the proteins phosphorylated are frequently associated with the viral nucleic acid and the virion-associated enzymes are not dependent on cyclic nucleotides, as are most such enzymes in cells (14). The protein kinase activity described here provides a specific and gentle method for radiolabeling Dane particles, Dane particle cores, and HBcAg particles from infected liver. This research was supported by Public Health Service grant AI from the National Institutes of Health. LITERATURE CITED 1. Almeida, J. D., D. Rubenstein, and E. J. Stott New antigen antibody system in Australia antigen positive hepatitis. Lancet ii: Almeida, J. D., A. P. Watterson, J. M. Trowell, and G. Neale The finding of virus-like particles in two Australia-antigen-positive human livers. Microbios 2: Barker, L. F., J. D. Almeida, J. H. Hoofnagle, R. J. Gerety, D. R. Jackson, and P. P. McGrath Hepatitis B core antigen: immunology and electron microscopy. J. Virol. 14: Bayer, M. E., B. S. Blumberg, and B. Werner Particles associated with Australia antigen in the sera of patients with leukemia, Down's syndrome and hepatitis. Nature (London) 218: Budkowska, A., J. W. Shin, and J. L. Gerin Immnunochemistry and polypeptide composition of hepatitis B core antigen (HB,Ag). J. Immunol. 118: Dane, D. S., C. H. Cameron, and M. Briggs Virus-like particles in serum of patients with Australia antigen associated hepatitis. Lancet ii: Hruska, J. F., and W. S. Robinson The proteins of hepatitis B Dane particle cores. J.,Med. Virol. 1:

6 302 NOTES 8. Kaplan, P. M., R. L. Greenman, J. L. Gerin, R. H. Purcell, and W. S. Robinson DNA polymerase associated with human hepatitis B antigen. J. Virol. 12: Magnius, L. O., and J. A. Espmark New specificities in Australia antigen positive sera distinct from Le Bouvier determinants. J. Immunol. 109: Marion, P. L., F. H. Salazar, J. Alexander, and W. S. Robinson Polypeptides of hepatitis B virus surface antigen produced by a hepatoma cell line. J. Virol. 32: Robinson, W. S DNA and DNA polymerase in the core of the Dane particle of hepatitis B. Am. J. Med. J. VIROL. Sci. 270: Robinson, W. S., D. A. Clayton, and R. L. Greenman DNA of a human hepatitis B virus candidate. J. Virol. 14: Robinson, W. S., and R. L. Greenman DNA polymerase in the core of the human hepatitis B virus candidate. J. Virol. 13: Rubin, C. S., and 0. M. Rosen Protein phosphorylation. Annu. Rev. Biochem. 44: Takahashi, K., Y. Akahane, T. Gotanda, T. Mishiro, M. Imai, Y. Miyakawa, and M. Mayumi Demonstration of hepatitis e antigen in the core of Dane particles. J. Immunol. 122: