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 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 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. Nuclei were removed fonyl fluoride (Boehringer Mannheim), 10 ,ug of antipain by centrifugation as before, leaving the supernatant or mem- (Boehringer Mannheim) per ml, 10 jig of soybean trypsin brane fraction. Aliquots (500 RI) of fractionated PDH samples inhibitor (Boehringer Mannheim) per ml, 10 mM EDTA, and or a 1/10 dilution of duck sera were incubated with 5 pI of 1% Triton X-100. The reaction was halted by the addition of either guinea pig anti-L or rabbit anti-core antibody (a gift of 5 x sample buffer. Ming Qiao, Institute of Medical and Veterinary Science, Two residue-specific phosphatases were used to further Adelaide, Australia) for 4 h at 4°C and then with 40 pul of 10% examine L protein heterogeneity in day 12 PDH lysate samples (wt/vol) preswollen protein A-Sepharose (Pharmacia, Uppsala, and DHBV-positive sera (1/20 dilution in 50 mM Tris, pH 7.8), Sweden) for 15 min at 20°C with gentle agitation. The immune the latter examined either with or without the presence of 1% complex was washed 3 times with 10 mM Tris-1 mM EDTA. Nonidet P-40. Samples (20 ,u) were incubated either with 0, Detection of L proteins by Western blotting. The immune 100, or 200 U of Lambda phosphatase, a serine/threonine- complexes (40 RI) were mixed with 10 ,u of 5 x sample buffer specific phosphatase (New England Biolabs, Beverly, Mass.) or (for a final concentration of 2% SDS, 2% 2-mercaptoethanol, with 0 or 200 U of YOP phosphatase, a tyrosine-specific 25 mM Tris-HCl [pH 6.8]) for 5 min and centrifuged for 30 s, phosphatase (New England Biolabs) for 30 min at 30°C in a and the proteins were separated by SDS-PAGE (12% acryl- final volume of 40 RI containing reaction buffers specific for amide) in a Mini Protean II apparatus (Bio-Rad, Richmond, each enzyme. The Lambda phosphatase reaction required a Calif.). The separated proteins were transferred to Hybond-C final concentration of 2 mM MnCl2, 100 ,ug of acetylated membrane (Amersham, Amersham, England) with a Trans- bovine serum albumin (BSA), 5 mM dithiothreitol, and 50 mM Blot SD semi-dry transfer cell (Bio-Rad) and either exposed to Tris-HCl, pH 7.8, while the YOP phosphatase buffer contained Fuji XR film to detect 32P-labelled proteins or probed with a 50 mM NaCl, 2.5 mM Na2-EDTA, 5 mM dithiothreitol, 100 ,ug monoclonal antibody to pre-Sl (a gift from S. Bowden, Fair- of acetylated BSA per ml, and 100 mM NaOAc, pH 5.5. field Hospital, Melbourne, Australia). An anti-mouse immu- noglobulin conjugated to horseradish peroxidase (Amersham) was used as the secondary antibody, and protein bands were RESULTS visualized by use of the ECL enhanced chemiluminescence system (Amersham). Radiolabelled proteins were identified by Analysis of L protein heterogeneity by Western blotting and sequential autoradiography and immunoblotting of the mem- labelling with [3lHlmyristic acid. Examination of L protein brane essentially as described previously for the detection of from sera, PDH lysates, and liver homogenates by Western virus-specific proteins in complex mixtures (2). blot reveals a heterogeneous mixture of up to four bands Labelling of DHBV-infected PDH cultures with [3H]myris- differing in size by only 1 or 2 kDa (Fig. 1A). In contrast, S is tic acid. At 8 days after plating of congenitally infected PDHs, always a homogeneous species (6), indicating that surface 500 pRCi of [3H]myristic acid (Amersham) in L-15 medium protein heterogeneity resides in the pre-S domain. Most (Gibco BRL, Gaithersburg, Md.) was added to approximately commonly, the majority of L protein appears as a doublet at 3 x 10' cells. Cells were harvested 2 days later and fractionated approximately 35 and 36 kDa with minor bands occasionally into membrane and cytosol as before. L and related proteins detected at 37, 33, and 30 kDa. The 28-kDa doublet appears to were immunoprecipitated as described above, separated by be a proteolytic product of L, while the 33- and 30-kDa bands SDS-12% PAGE, and transferred to Hybond-C (Amersham). appear to arise from internal initiation (8). The 37-kDa band is 7346 GRGACIC AND ANDERSON J. VIROL.

A B PDH SERA 66.2- W..I

45- II. P36 .4--P37 P35------P33 P36 31- -P30 *--P28 P35--_-35U1 21.5 1 2 3 4 FIG. 1. Heterogeneity of DHBV L proteins in immunoblots of sera 1 2 3 4 5 6 7 8 from congenitally infected ducks (A) and lysates of infected PDH cells FIG. 3. Digestion with AP removes the more-slowly migrating L (B). Samples were electrophoresed on a 12% gel, the proteins were P36. L was with anti-L from transferred to nitrocellulose and blocked with casein, and L proteins protein, immunoprecipitated guinea pig PDH cell lysates or sera as indicated (above the lanes) and digested were detected by using guinea pig anti-L and horseradish peroxidase- chemi- with AP for 6 h at 25°C before electrophoresis, transfer, and immu- conjugated anti-guinea pig immunoglobulin G and enhanced anti-L and chemiluminescence. Units of luminescence. (A) Serum samples from three separate congenitally noblotting with monoclonal infected ducks. The positions of the major L doublet of 35 and 36 kDa AP per milliliter: lanes 1 and 5, 0; lanes 2 and 6, 0.125; lanes 3 and 7, 0.25; lanes 4 and 8, 1.0. and of molecular mass markers are indicated on the left; the positions of the minor large surface proteins are indicated on the right. (B) Membrane fraction of cell lysates from PDH cells congenitally infected with DHBV, showing the increase in the relative amount of P36 with Phosphatase digestion of L protein. To examine the possi- time. Lane 1 to 4, cells harvested at 8, 12, 16 and 20 days, respectively. bility that the heterogeneity observed in the L protein is due to further modification by phosphorylation, L was immunopre- cipitated from cell lysates and sera and treated with AP as rarely visible and then only with long exposure times, which has described in Materials and Methods. Digestion with AP re- precluded further analysis at this time. sulted in the loss of the more-slowly migrating species, P36, Analysis of L in infected PDH cultures shows approximately whereas the mock treatments have clearly retained the doublet 50% less P36 than P35 at day 8 postinfection, but by day 12 to (Fig. 3). 16 they appear in equal proportions (Fig. 1B). This coincides It is not possible to conclude from this experiment whether with the accumulation of large amounts of total and super- the phosphorylated residues are on the surface of the viral coiled viral DNA (reference 1 and results not shown). particle, since the AP digestion buffer included Triton X-100. Congenitally infected PDH cultures were labelled with In addition, recent work by Bruss and Thomssen (4) has shown [3H]myristic acid to identify which of these proteins were that L undergoes conformational changes under acidic condi- initiated from the AUG at position 801. Figure 2 shows that tions. both species of the doublet are labelled with myristic acid, Despite the addition of a cocktail of protease inhibitors, indicating that both species must be translated from the same treatment with the crude AP also resulted in considerable start codon in order to be myristylated through the N-terminal protein degradation. To avoid this problem and to further glycine. characterize the putative phosphorylations occurring in L, we utilized residue-specific phosphatases which were protease free. Figure 4A shows the removal of P36 from PDH lysate samples with 200 U of the serine/threonine-specific Lambda FLUOR. | ECL | _ _ - : A LserrThr-specific Tyr-specific _E B 69- XSL: w.

,. . : '' 46- _,, *,. r. @ ,4P ' ^4 X+**vs _ _ _ -ig 5, w _.. -P36 .04 _' >''- ..-P36 ji.. P35 _~ i --P35 30-- =._L _ P28=I 1 2 3 o. ; FIG. 4. Serine/threonine- but not tyrosine-specific phosphatase re- moves L protein heterogeneity. (A) Membrane fractions of PDH cell 1 2 3 4 5 6 lysates were denatured by heating (lanes 3, 4, 7, and 8) or not FIG. 2. Both P36 and P35 are myristylated. PDH cells were la- denatured (lanes 1, 2, 5, and 6) before digestion with 0 (lanes 1 and 3) belled with [3H]myristic acid from 8 to 10 days postinfection and or 200 (lanes 2 and 4) U of serine/threonine-specific Lambda phos- fractionated into membrane (lanes 2 and 5) and cytosol (lanes 3 and 6) phatase or with 0 (lanes 5 and 7) or 200 (lanes 6 and 8) U of as described in Materials and Methods. After electrophoresis, transfer, tyrosine-specific YOP phosphatase as described in Materials and and immunoblotting (lanes 4 to 6) as described in the legend to Fig. 1, Methods. L proteins were detected by immunoblotting with guinea pig the membrane was fluorographed (lanes 1 to 3). Lanes: 1 and 4, 14C anti-L. (B) Heterogeneity of the 28-kDa proteolytic product of L is and biotinylated molecular mass markers (sizes indicated on the left in also due to phosphorylation. Lanes: 1 to 3, membrane fractions of kilodaltons); 2 and 5, membrane fraction; 3 and 6, cytosol fraction. The PDH cell lysates digested with 0, 100, or 200 U of Lambda phos- positions of P36 and_F P35-<-¢c:are indicateddlSKi on....the right. phatase, respectively. VOL. 68, 1994 DHBV ENVELOPE PROTEIN PHOSPHORYLATION 7347

32p anti-L detergent + + phosph atasel - + + 60- 45.- ---- P36 46- -P35 31-It C-P36 30-F . 21.5- 1 2 3 4 5 6 FIG. 5. Phosphorylation of P36 in vivo and analysis of fractionated 1 2 3 4 cell lysates by immunoprecipitation, SDS-PAGE, autoradiography FIG. 6. Phosphorylated domains in L are not exposed on the (lanes 1 to 4), and immunoblotting with monoclonal anti-L (lanes 5 surface of virions. Serum (diluted 1/20 in the presence [lanes 3 and 4] and 6). Infected (odd-numbered lanes) and mock-infected PDH cells or absence [lanes 1 and 2] of 1% Nonidet P-40) was digested with (even-numbered lanes) were labelled with 32P as described in Materi- either 0 (lanes 1 and 3) or 200 (lanes 2 and 4) U of Lambda als and Methods, fractionated into membrane and cytosol, and immu- phosphatase as indicated above the lanes. The positions of molecular noprecipitated with either guinea pig anti-L (membrane, lanes 3 to 6) mass markers are shown at left (in kilodaltons). Note that P36 is or rabbit anti-core (cytosol, lanes 1 and 2). The sizes of molecular mass sensitive to phosphatase only in the presence of detergent. markers (in kilodaltons) are indicated at left, and the positions of P35 and P36 and of 32P-labelled P36 and core (32 to 34 kDa) are indicated with arrows. The asterisk indicates the presence of a phosphoprotein at 28 kDa (lane 3) which may be the L-derived proteolytic product, P28. be present at various stages of maturation and cytosolic fractions would include fragments of L-containing endoplas- mic reticulum. In order to assess the topological disposition of phosphatase at pH 7.8, indicating that the phosphate group(s) the phosphorylated fraction of L, pooled sera from congeni- is located on the threonine or the more-common serine tally infected ducks were digested with Lambda phosphatase in residues. Figure 4B shows that L protein heterogeneity is also the presence or absence of 1% Nonidet P-40 (Fig. 6). In the present in the 28-kDa proteolytic product (lane 1) and that this absence of detergent, P36 is resistant to phosphatase digestion can also be removed by Lambda phosphatase digestion. The (Fig. 6, lane 2), but addition of detergent renders the phos- tyrosine-specific YOP phosphatase failed to remove the phos- phate group sensitive (lane 4), indicating an internal disposi- phorylated protein. tion of the phosphorylated fraction of L protein. In vivo phosphorylation of L in DHBV-infected PDH cul- tures. To confirm that a proportion of the envelope protein DISCUSSION was modified by phosphorylation, infected and mock-infected PDH cultures were labelled with 32p on day 13 postinfection The data presented here clearly demonstrate that a propor- and immunoprecipitated with guinea pig anti-L. As a control, tion of the large envelope protein of DHBV is modified by labelled cells were also immunoprecipitated with a rabbit phosphorylation of serine and/or threonine residues within the anti-core antibody since it is known that the core protein is pre-S domain, in addition to N myristylation in both forms. phosphorylated (21, 23, 24). Figure 5 shows the separated, This finding clarifies the variable electrophoretic mobility of labelled proteins (lanes 1 to 4) and an immunoblot of the same the L species reported in the literature, demonstrating that the membrane probed with monoclonal anti-pre-S (lanes S and 6). heterogeneous L protein detected after electrophoresis con- Phosphorylated core protein was detected as two bands at sists of a doublet with the P36 species generated by phosphor- approximately 34 kDa (Fig. 5, lane 1) as expected from the ylation of P35, rather than the smaller species arising from heterogeneous phosphorylation pattern in the serine-rich car- initiation at the ATG at position 825 as previously predicted (8, boxy terminus of this protein (24, 30). A DHBV-specific 26). Most importantly, this study reveals two features which phosphoprotein was detected after immunoprecipitation with suggest a significant role for this additional protein modifica- anti-L (Fig. 5, lane 3), migrating at 36 kDa. The 36-kDa tion in DHBV replication. (i) Early in the infection of PDH protein was identified as the upper band of the L doublet by cultures, the nonphosphorylated form of L is the more abun- alignment of the autoradiograph with the ECL Western of the dant species, with levels of the phosphoprotein increasing to same blot (Fig. 5, lanes 5 and 6). A virus-specific phosphopro- equal proportions by day 12 to 16, suggesting that the level of tein of approximately 28 kDa was also detected (Fig. 5, lane 3), phosphorylation is controlled by some mechanism. (ii) Phos- which may represent the most-slowly migrating species of the phorylated L in serum-derived viral particles is resistant to degradation product of L (Fig. 1 and 4B). However, the phosphatase digestion, reflecting an internal disposition of the intensity of this band contrasts with the relative abundance of pre-S domain and suggesting a role for phosphorylation- L and P28 in most samples, and it was not possible to confirm dephosphorylation in the topological shift within the L protein its identity by probing of the Western blot for P28 as this during morphogenesis (3, 19). In addition, the L protein of antiserum was used for the immunoprecipitation. human HBV often shows similar electrophoretic heterogeneity Topological disposition of phosphorylated L protein. Our (13) and several putative phosphorylation motifs appear to be preparations of membrane-associated protein contained non- conserved in both the duck pre-S and mammalian HBV pre-Sl ionic detergent, and therefore it was not surprising that the sequences (see below). phosphate residues were accessible for digestion in both native Digestion with residue-specific phosphatases showed that and denatured proteins. However, the intracellular pattern of the modified residue(s) are either serine or threonine (Fig. L distribution is probably more complex, since particles would 4A). While the precise location of phosphorylated residue(s) 7348 GRGACIC AND ANDERSON J. VIROL.

AR outside myr _O*'r OOH ER lumen virion enveope ER membrane Inside cytosol

tDephosphorylation outside S ~~ A~~0%-.COOHH ElueER lumen virlon envelope ER membrane Inside myr cytosol

FIG. 7. A model for the role of phosphorylation-dephosphorylation in determining the transmembrane topology of DHBV L protein. Following of the pre-Sl domain, phosphorylation at one or more sites prevents translocation of this domain through the endoplasmic reticulum membrane, resulting in the initially cytosolic distribution of the pre-Sl domain (and pre-S2 in mammalian hepadnaviruses) (3, 19). After dephosphorylation, the pre-Sl (and pre-S2) domains can be translocated, resulting in a luminal and ultimately exterior disposition of these domains. within the protein is still to be determined, in DHBV they are nal disposition (and hence external disposition in the excreted likely to lie between the residue encoded by the last conserved virion) requiring a topological shift in the fully synthesized AUG (957) and the start of the S protein, since the 28-kDa molecule. This new model of L protein topology is consistent proteolytic product of L (8) also appears as a phosphatase- with evidence that the site of L protein-core interaction is near sensitive doublet (Fig. 4B) while the S protein is always a single the start of pre-S2 (4) and that approximately half the L band (6, 16). In addition, a labelled phosphoprotein was also molecules in HBV virions have an internal pre-S domain while observed at 28kDa, consistent with this protein being derived the other half, which apparently undergo this topological shift, from L (Fig. 5, lane 3). Attempts to identify the phosphory- are exposed on the surface (3). The observation that P36 in lated residues and the kinase involved should be enhanced by serum-derived viral particles can be dephosphorylated only in the ability to detect L after metabolic labelling with 32P as the presence of detergent (Fig. 5) suggests that the luminal shown here. pre-S domains are phosphorylated while the external pre-S Recent studies by Ostapchuk et al. (19) and Bruss et al. (3) domains are not. We therefore propose a model mechanism have shown that the entire pre-S region of the L protein is for the control of L protein topology in hepadnaviruses (Fig. initially located in the cytosol following translation, with lumi- 7). Immediately after translation of the pre-Sl domain and

A *23 39 *245 *257*259 DHBV CORE T1PQRAGSPLPRSSSSHHRSPBPRK-COOH 157 162 164 172 HBV CORE RSPRRRTPSPRRRRSPSPRRRRSQSRESQC-COOH

B 79 89 118 155 DHBV ENV PTPQEIPQPQWTPEE... SSPPQ... TPPL KKK MSGT 78 106 109 HBV ENV WSPQAQGILTTVSTI ..... GRQPTP BPPL RDSHPQA MQWN 121 132 135 GSHV ENV QTPSNRDQRRKPTPILTPPLERDTHPHLT Q 119 130 133 VD WHV ENV OTPTDTRnD.RwPmwA!^r I M U R PPT. RDTHPHLT Q

FIG. 8. Conserved amino acid sequences and putative phosphorylation sites in the core and envelope proteins of hepadnaviruses. (A) Core proteins of DHBV and human HBV. Phosphorylation acceptor sites proven experimentally (29) are indicated with asterisks. (B) L proteins of DHBV and human HBV and of ground squirrel (GSHV) and woodchuck (WHV) hepatitis B . Conserved (S/T)P motifs are indicated in boldface type; conserved (S/T)PPL motifs are indicated by the box. Numbering above the sequences indicates the position of the S or T with respect to the first initiation codon of the protein. Arrows indicate the starts of the S (DHBV) and M proteins (mammalian hepadnaviruses). VOL. 68, 1994 DHBV ENVELOPE PROTEIN PHOSPHORYLATION 7349 prior to translation (or translocation) of the transmembrane We thank Sotirios Kolivas for help with sequences and Dick domains in S, one or more serine or threonine residues is Wettenhall for helpful discussions. phosphorylated, resulting in a significant net negative charge sufficient to retard translocation of the first transmembrane REFERENCES domain across the endoplasmic reticulum membrane. Subse- of the pre-Si domain in approxi- 1. Bishop, N., G. Civitico, Y. Wang, K. Guo, C. Birch,I. Gust, and S. quent dephosphorylation Locarnini. 1990. Antiviral strategies in chronic hepatitis B virus mately half the L molecules allows this translocation to occur, infection. I. Establishment of an in vitro system using the duck resulting in luminal (and subsequently external) disposition of hepatitis B virus model. J. Med. Virol. 31:82-89. the pre-Sl domain. We have found that lower levels of 2. Borovec, S. V., and D. A. Anderson. 1993. Synthesis and assembly 32P-labelled L are obtained when okadaic acid is omitted of hepatitis A virus-specific proteins in BS-C-1 cells. J. Virol. during labelling (10), suggesting that phosphatases PP1 or 67:3095-3102. PP2a may be responsible for dephosphorylation of L. 3. Bruss, V., X. Lu, R. Thomssen, and W. H. Gerlich. 1994. Post- Examination of the amino acid sequence of the surface translational alterations in transmembrane topology of the hepa- protein reveals some striking similarities to the core protein titis B virus large envelope protein. EMBO J. 13:2273-2279. to potential phosphorylation sites. The C terminus 4. Bruss, V., and R. Thomssen. 1994. Mapping a region of the large with respect envelope protein required for hepatitis B virion maturation. J. of L (and hence S) has many clusters of serine residues, but Virol. 68:1643-1650. unlike the core protein this region lacks the SP or TP motif 5. Buscher, M., W. Reiser, H. Will, and H. Schaller. 1985. Transcripts shown to be essential for phosphorylation of the three serine and the putative RNA pregenome of duck hepatitis B virus: residues and one threonine acceptor residue in core (29). implications for reverse . Cell 40:717-724. However, the region of the DHBV L protein between the 6. Cheung, R. C., W. S. Robinson, P. L. Marion, and H. B. Green- residues encoded by the AUG codon at position 957 and the berg. 1989. Epitope mapping of neutralizing monoclonal antibod- codon at position 1284 has several TP and SP motifs with one ies against duck hepatitis B virus. J. Virol. 63:2445-2451. at amino acid 155 appearing as part of a more extended and 7. Civitico, G., Y. Wang, C. Luscombe, N. Bishop, G. Tachedjian, I. motif present in all HBV sub- Gust, and S. Locarnini. 1990. Antiviral strategies in chronic highly conserved ([S/T]PPL), hepatitis B virus infection. II. Inhibition of duck hepatitis B virus types as well as woodchuck and ground squirrel hepatitis in vitro using conventional antiviral agents and supercoiled-DNA viruses (Fig. 8). This motif is present near the end of the pre-Sl active compounds. J. Med. Virol. 31:90-97. domain and therefore adjacent to S in DHBV but to M in the 8. Fernholz, D., P. R. Galle, M. Stemler, M. Brunetto, F. Bonino, and mammalian hepadnaviruses (12). Conservation of these se- H. Will. 1993. Infectious hepatitis B virus variant defective in quences in the otherwise highly variable pre-Sl domain, while pre-S2 protein expression in a chronic carrier. Virology 194:137- absent in the S (or pre-S2) domains, argues for an important 148. role of phosphorylation in L function. 9. Freiman, J. S., and Y. E. Cossart. 1986. Natural duck hepatitis B The observation that the relative amount of the phosphor- virus infection in Australia. Aust. J. Exp. Biol. Med. Sci. 64:477- L is initially low and increases with time in the 484. ylated form of 10. Grgacic, E. V. L., and D. Anderson. Unpublished data. viral growth cycle (Fig. 1B) suggests that the level of phosphor- 11. Grgacic, E. V. L., L. Fan, and D. Anderson. Unpublished data. ylation is controlled by virus-specific or virus-induced factors, 12. Heermann, K. H., and W. H. Gerlich. 1991. Surface proteins of for example by the core-associated cellular kinase (15, 23). hepatitis B viruses, p. 109-143. In A. McLachlan (ed.), Molecular Alternatively, the difference in phosphorylation levels may biology of the hepatitis B virus. CRC Press, Boca Raton, Fla. result from differential localization of the L protein, induction 13. Heermann, K. H., U. Goldmann, W. Schwartz, T. Seyffarth, H. of an abundant cellular kinase, or down-regulation of a cellular Baumgarten, and W. H. Gerlich. 1984. Large surface proteins of phosphatase. While the mechanism is unclear, it is noteworthy hepatitis B virus containing the pre-s sequence. J. Virol. 52:396- that at days 12 to 16, when large amounts of surface protein 402. and the two species appear in equal 14. Jilbert, A. R., J. S. Freiman, E. J. Gowans, M. Holmes, Y. E. have accumulated Cossart, and C. J. Burrell. 1987. Duck hepatitis B virus DNA in amounts, maximal levels of total and supercoiled viral DNA liver, spleen, and pancreas: analysis by in situ and Southern blot are also reached (1, 7, 11). Summers et al. (26) have shown the hybridization. Virology 158:330-338. myristylated L protein to be active in regulating supercoiled 15. Kann, M., R. Thomssen, H. G. Kochel, and W. H. Gerlich. 1993. DNA amplification, although the mechanism is not known. We Characterization of the endogenous protein kinase activity of the suggest that phosphorylation of L protein may be involved in hepatitis B virus. Arch. Virol. Suppl. 8:53-62. this regulatory activity, perhaps through association of prege- 16. Luscombe, C. Personal communication. nome-containing core particles with populations of L protein 17. Macrae, D. R., V. Bruss, and D. Ganem. 1991. Myristylation of a containing differing proportions of phosphorylated and non- duck hepatitis B virus envelope protein is essential for infectivity L proteins, resulting in different fates of the but not for virus assembly. Virology 181:359-363. phosphorylated 18. Mandart, E., A. Kay, and F. Galibert. 1984. Nucleotide sequence newly synthesized relaxed circular viral DNA. of a cloned duck hepatitis B virus genome: comparison with The multifunctional nature of the hepadnavirus L protein in woodchuck and human hepatitis B virus sequences. J. Virol. regulation of supercoiled DNA amplification, interaction with 49:782-792. cores, particle export, and receptor binding argues for one or 19. Ostapchuk, P., P. Hearing, and D. Ganem. 1994. A dramatic shift more mechanisms to control these functions: a balanced in the transmembrane topology of a glycoprotein topological distribution through phosphorylation-dephospho- accompanies hepatitis B viral morphogenesis. EMBO J. 13:1048- rylation offers an attractive possibility which needs to be 1057. 20. Persing, D. H., H. E. Varmus, and D. Ganem. 1987. The preSl examined further. protein of hepatitis B virus is acylated at its amino terminus with myristic acid. J. Virol. 61:1672-1677. ACKNOWLEDGMENTS 21. Pugh, J., A. Zweidler, and J. Summers. 1989. Characterization of This study was supported by the Victorian Health Promotion the major duck hepatitis B virus core particle protein. J. Virol. Foundation, the Research Fund of the Macfarlane Burnet Centre for 63:1371-1376. Medical Research, and the M. Florence Beever Memorial Bequest 22. Pugh, J. C., J. J. Sninsky, J. W. Summers, and E. Schaeffer. 1987. (E.V.L.G.). E.V.L.G. is the recipient of a postgraduate research Characterization of a pre-S polypeptide on the surfaces of infec- scholarship from the National Health and Medical Research Council. tious avian hepadnavirus particles. J. Virol. 61:1384-1390. 7350 GRGACIC AND ANDERSON J. VIROL.

23. Roossinck, M. J., and A. Siddiqui. 1987. In vivo phosphorylation teins of an avian hepadnavirus. J. Virol. 65:1310-1317. and protein analysis of hepatitis B virus core antigen. J. Virol. 61: 27. Tuttleman, J. S., J. C. Pugh, and J. W. Summers. 1986. In vitro 955-961. experimental infection of primary duck hepatocyte cultures with 24. Schlicht, H. J., R. Bartenschlager, and H. Schaller. 1989. The duck duck hepatitis B virus. J. Virol. 58:17-25. hepatitis B virus core protein contains a highly phosphorylated C 28. Yokosuka, O., M. Omata, and Y. Ito. 1988. Expression of pre-Sl, terminus that is essential for replication but not for RNA packag- pre-S2, and C proteins in duck hepatitis B virus infection. Virology ing. J. Virol. 63:2995-3000. 167:82-86. 25. Schlicht, H. J., C. Kuhn, B. Guhr, R. J. Mattaliano, and H. 29. Yu, M., and J. Summers. 1994. Phosphorylation of the duck Schaller. 1987. Biochemical and immunological characterization hepatitis B virus protein associated with conformational of the duck hepatitis B virus envelope proteins. J. Virol. 61:2280- changes in the C terminus. J. Virol. 68:2965-2969. 2285. 30. Yu, M., and J. Summers. 1994. Multiple functions of capsid 26. Summers, J., P. M. Smith, M. Huang, and M. Yu. 1991. Morpho- protein phosphorylation in duck hepatitis B virus replication. J. genetic and regulatory effects of mutations in the envelope pro- Virol. 68:4341-4348.