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The Large Surface Protein of Duck Hepatitis B Virus Is Phosphorylated in the Pre-S Domain ELIZABETH V

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The Large Surface Protein of Duck Hepatitis B Virus Is Phosphorylated in the Pre-S Domain ELIZABETH V JOURNAL OF VIROLOGY, Nov. 1994, p. 7344-7350 Vol. 68, No. 11 0022-538X/94/$04.00+0 Copyright C 1994, American Society for Microbiology The Large Surface Protein of Duck Hepatitis B Virus Is Phosphorylated in the Pre-S Domain ELIZABETH V. L. GRGACIC AND DAVID A. ANDERSON* Macfarlane Bumet Centre for Medical Research, Melboume, Australia Received 19 July 1994/Accepted 15 August 1994 The two major envelope proteins (large [L] and small [S]) of duck hepatitis B virus are encoded by the pre-S/S open reading frame. The L protein is initiated from the AUG at position 801 in the pre-S region of the pre-S/S coding sequence, yielding an N-terminal consensus sequence for myristylation. Western immunoblots of the L protein often reveal a doublet at 36 and 35 kDa, with the latter attributed to the use of one of the three internal initiation codons. However, metabolic labelling with [3H]myristic acid results in labelling of both P35 and P36, indicating that both species must be initiated from the same start codon. Using metabolic labelling with 32P and digestion with residue-specific phosphatases, we demonstrate that L protein heterogeneity is due to phosphorylation of threonine and/or serine residues within the pre-S domain. We propose that at least one possible phosphorylation site is located at a novel (S/T)PPL motif which is conserved near the carboxyl end of the pre-SI domain in all hepadnavirus sequences. Two to three additional (S/T)P motifs are also present in the carboxyl half of the pre-Sl (but not pre-S2 or S) domain of all hepadnaviruses. L protein in serum-derived particles is resistant to phosphatase digestion in the absence of detergents, reflecting an internal disposition of the phosphorylated pre-S domain and suggesting a role for dephosphorylation in the topological shift within L during morphogenesis (P. Ostapchuk, P. Hearing, and D. Ganem, EMBO J. 13:1048-1057, 1994). Further- more, we observe that the relative amount of the phosphorylated form of L increases with time in the viral growth cycle. These findings imply that phosphorylation-dephosphorylation of the L protein is an important, regulated mechanism necessary for correct virion morphogenesis. The hepadnavirus envelope proteins display a complex N terminus (20), and metabolic labelling with [3H]myristic acid pattern of functional roles in which the large surface protein is results in both species of the doublet being labelled (reference associated with receptor binding, core interaction, and control 17 and this study). Hence, not all the forms can arise from of supercoiled DNA amplification, while the ratio of all three utilization of internal AUGs. We therefore surmised that the forms of the envelope protein, the large (L), middle (M; in heterogeneity observed may be due to further posttranslational mammalian hepadnaviruses), and small (S), plays a crucial role modification(s) of the myristylated L protein. Our results in virion morphogenesis and secretion (12). indicate that both species in the doublet are myristylated and The duck hepatitis B virus (DHBV) envelope proteins are that a proportion of this protein is phosphorylated on serine translated from a single open reading frame (pre-S/S). Two and/or threonine residues in the pre-Sl domain leading to mRNAs (2.35 and 2.13 kb) act as templates for the synthesis of slightly reduced migration in sodium dodecyl sulfate (SDS) a large surface protein of approximately 36 kDa (L or pre-S) gels. and a major, small protein of 17 kDa (S) (5). The pre-S region The significance of DHBV L protein phosphorylation is of the pre-S/S coding sequence contains start codons at discussed in light of the recent evidence on the transmembrane nucleotides 801, 825, 882, and 957 (18). The L protein, P36, is topology of the HBV L protein (3, 19). Our results provide a initiated from the AUG at position 801, yielding an N-terminal possible explanation of how L protein topology is controlled in consensus sequence for myristylation (20); however, it is hepadnaviruses. unclear whether any or all of the additional methionine codons are utilized to produce the various minor pre-S proteins frequently observed in Western immunoblots. The other pre-S MATERIALS AND METHODS protein consistently found in DHBV-infected liver and occa- Primary duck hepatocyte (PDH) cultures. Four- to five- sionally in serum is a protein of 28 kDa which has commonly day-old Pekin-Aylesbury ducklings known to be negative for been identified as pre-S2 or M by analogy with human hepatitis DHBV (14) were used for primary hepatocyte cultures. Hepa- B virus (HBV) (28). However, there is now strong evidence to tocytes were obtained according to the methods of Tuttleman suggest that this is a proteolytic product of the large surface et al. (27) as modified by Bishop et al. (1). Cells were seeded protein and that it is the minor 30-kDa protein, not always in six-well multiplates (Greiner, Solingen, Germany) at 3 x 106 detected by Western blotting, which is initiated from the AUG cells per well and infected on day 0 with a pool of sera from at position 957 (8). ducklings infected with virus from a single congenitally in- Western blots of L proteins often reveal a doublet at 35 and fected duckling (9), containing 1.23 x 109 viral genome 36 kDa (8, 17, 22, 25) which has been attributed to the use of equivalents per ml to give an approximate multiplicity of internal initiation codons. However, P36 is myristylated at the infection of 8 viral genome equivalents per cell. Biosynthetic labelling with [3H]myristic acid was carried out on PDH * Corresponding author. Mailing address: Hepatitis Research Unit, cultures prepared from congenitally infected ducklings, under Macfarlane Burnet Centre for Medical Research, P.O. Box 254, which conditions 100% of cells are infected (1). Fairfield 3078, Victoria, Australia. Phone: 61 3 280 2239. Fax: 61 3 280 Preparation of anti-L protein antisera. Antisera to the large 2561. surface protein of DHBV were prepared by immunization of 7344 VOL. 68, 1994 DHBV ENVELOPE PROTEIN PHOSPHORYLATION 7345 guinea pigs with gel-purified L protein. Virions were purified The membrane was first probed with monoclonal antibody to from DHBV-positive serum by sucrose density gradient sedi- pre-S to detect L protein as described above and then washed mentation. Briefly, 9 ml of congenitally infected duck serum thoroughly to remove ECL reagents prior to fluorography. For was layered onto a 3-ml cushion of 20% (wt/vol) sucrose in NT fluorography, the membrane was briefly dried at 80°C, im- (100 mM NaCl-10 mM Tris, pH 7.4), and virus was pelleted by mersed in 20% (wt/vol) PPO (2,5-diphenyloxazole) in diethyl centrifugation for 4 h at 35,000 rpm in an SW41 rotor at 20°C. ether, air dried, and exposed to preflashed Fuji XR film at The pellet was resuspended in 2 ml of NT, layered onto a 15 to -700C. 40% sucrose gradient, and centrifuged for 2 h at 35,000 rpm in Labelling of DHBV-infected PDH cultures with 32Pi. In- an SW41 rotor. Ten 1-ml fractions were collected from the fected and mock-infected PDHs were labelled with 1 mCi of bottom of the gradient, and proteins were precipitated with 32p; (Bresatec, Adelaide, Australia) per 3 x 106 cells on day 13 methanol at -20°C. The pre-S-containing fractions were iden- postinfection. The medium was exchanged for phosphate-free tified by SDS-polyacrylamide gel electrophoresis (SDS- Eagle's medium (ICN, Costa Mesa, Calif.) for 2 h prior to PAGE), pooled, and run on a preparative gel. The L protein labelling. Cells were then labelled for 18 h in the presence of band was cut out of the stained gel (Coomassie brilliant blue 10 nM okadaic acid (ICN), an inhibitor of serine/threonine R-250), ground in a tissue grinder with a small volume of phosphatases (PP1 and 2A). The cell monolayer was washed saline, and then emulsified with an equal volume of Hunter's twice with cold 0.85% saline, and cells were harvested by TitreMax adjuvant (CytRx Corp., Norcross, Ga.). Guinea pigs being scraped in 500 ,lI of 50 mM Tris-50 mM NaF-2 mM were immunized subcutaneously and given booster injections EDTA-1 mM EGTA [ethylene glycol-bis(P-aminoethyl ether)- after 3 weeks. Blood was collected 1 week after each immuni- N,N,N',N',-tetraacetic acid]. zation, and the sera were tested for reactivity to the L protein Treatment of L with acid phosphatase and residue-specific by Western blotting. phosphatases. L-related proteins immunoprecipitated with Immunoprecipitation of L protein from PDH cultures and guinea pig anti-L from cell lysates or sera were digested with sera. PDH cultures (10 cm2) were harvested by being scraped acid phosphatase (AP) (Boehringer-Mannheim) to test in 0.5 ml of NT. The cells were fractionated into crude whether electrophoretic heterogeneity could be removed en- cytosolic and membrane fractions by three cycles of freeze- zymatically. Both native and denatured (boiled for 5 min) thawing, followed by centrifugation at 13,000 rpm for 2 min immune complexes were treated with increasing amounts of and at 6,500 rpm for 5 min in an Eppendorf Microfuge (model AP (0.125, 0.25, and 1.0 U/ml) or mock treated for 6 h at 25°C 5415 C). The supernatant (cytosol) was removed, and the in 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane- pellet was resuspended in 450 RI of NT, to which 50 RI of 10% sulfonic acid) (pH 6.0) containing 200 mM phenylmethylsul- Nonidet P-40 (Sigma) was then added.
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