Multiple Functions of Capsid Protein Phosphorylation in Duck Hepatitis B Virus Replication

Transcription

1 JOURNAL OF VIROLOGY, JUIY 1994, p Vol. 68, No X/94/$ Copyright (C 1994, American Society for Microbiology Multiple Functions of Capsid Protein Phosphorylation in Duck Hepatitis B Virus Replication MINSHU YU AND JESSE SUMMERS* Department of Cell Biology, University of New Mexico School of Medicine, Albuquerque, New Mexico Received 15 February 1994/Accepted 12 April 1994 We have investigated the role of phosphorylation of the capsid protein of the avian hepadnavirus duck hepatitis B virus in viral replication. We found previously that three serines and one threonine in the C-terminal 24 amino acids of the capsid protein serve as phosphorylation sites and that the pattern of phosphorylation at these sites in intracellular viral capsids is complex. In this study, we present evidence that the phosphorylation state of three of these residues affects distinct steps in viral replication. By substituting these residues with alanine in order to mimic serine, or with aspartic acid in order to mimic phosphoserine, and assaying the effects of these substitutions on various steps in virus replication, we were able to make the following inferences. (i) The presence of phosphoserines at residues 245 and 259 stimulates DNA synthesis within viral nucleocapsids. (ii) The absence of phosphoserine at residue 257 and at residues 257 and 259 stimulates covalently closed circular DNA synthesis and virus production, respectively. (iii) The presence of phosphoserine at position 259 is required for initiation of infection. The results implied that both phosphorylated and nonphosphorylated capsid proteins were necessary for a nucleocapsid particle to carry out all its functions in virus replication, explaining why differential phosphorylation of the capsid protein occurs in hepadnaviruses. Whether these differentially phosphorylated proteins coexist on the same nucleocapsid, or whether the nucleocapsid acquires sequential functions through selective phosphorylation and dephosphorylation, is discussed. Hepadnaviruses are a family of small enveloped viruses with a partially double-stranded circular DNA genome that replicates through reverse transcription. During initiation of infection, virus particles deliver the DNA genome to the nucleus, where it is converted to a covalently closed circular (ccc) DNA that serves as a transcriptional template for the production of RNA genomes (pregenome) (8, 13, 15, 17). RNA pregenomes are encapsidated in the cytoplasm by viral capsid proteins to form immature nucleocapsid particles within which viral DNA synthesis occurs. Initially, a minus-strand DNA is synthesized by reverse transcription of the pregenome (7, 12). Minusstrand DNA is subsequently used as the template for plusstrand DNA synthesis. Mature intracellular nucleocapsids containing double-stranded DNA proceed along one of two alternate pathways. Early during the infectious cycle, the DNA in mature capsid particles is delivered to the nucleus, resulting in an amplification of the copy number of cccdna (15). Late during infection, production of the large viral envelope protein inhibits this amplification, redirecting viral nucleocapsids into enveloped virus particles, which are exported from the cell (13, 14). Thus, nucleocapsids are involved in a number of sequential functions, namely, RNA packaging, DNA synthesis, delivery of viral DNA to the nucleus, and recognition of viral envelope proteins. The sequential expression of some of these functions may be regulated by protein modification. The capsid protein of the duck hepatitis B virus (DHBV) consists of 262 amino acid residues. The protein can assemble into a capsid in the absence of other viral proteins. The C terminus of the capsid protein contains one threonine and three serines that serve as potential phosphorylation sites on the surface of immature nucleocapsids (10, 11, 20). In infected * Corresponding author. Phone: (505) Fax: (505) Electronic mail address: JSUMMERS@cobra.unm.edu cells, these four sites are phosphorylated in various combinations, resulting in electrophoretic heterogeneity of the protein in sodium dodecyl sulfate (SDS) gels. While the population of intracellular viral capsids contains capsid proteins phosphorylated at zero to four of these sites, capsid proteins isolated from extracellular viruses are electrophoretically homogeneous in SDS gels and comigrate with unphosphorylated capsid protein. This difference in the phosphorylation states of intracellular and mature viral capsids suggested that phosphorylation may play a role in intracellular capsid function or viral morphogenesis (10). As a first step in understanding the role of phosphorylation in virus replication, we examined the phenotypes of a series of serine or threonine amino acid substitutions in the C terminus of capsid protein. We assayed for RNA packaging, DNA synthesis, intracellular localization of the protein, production of enveloped viruses, and viral infectivity. The results suggest that each of three of these four residues participates in a distinct manner in capsid function, depending on its state of phosphorylation. MATERUILS AND METHODS Plasmid and mutant construction. Wild-type and mutant capsid proteins were expressed in permissive cells from a plasmid in which the DHBV pregenome encoding sequences lacking the precore region was cloned immediately downstream of the immediate-early cytomegalovirus (CMV) promoter, as previously described (19). Stop codons in the envelope gene (T->A at nucleotides 1327, 1346, and 1349) prevented the expression of either envelope protein. These mutations, designated collectively as 1S, were previously described (13). In order to alter the potential phosphorylation sites at the C terminus of capsid protein, a series of point mutations were

2 4342 YU AND SUMMERS introduced by oligonucleotide-directed mutagenesis into codons coding for serine and threonine in the 3' end of the capsid gene open reading frame, resulting in individual replacement of serine and threonine by alanine or aspartic acid, as we described previously. Fully sequenced restriction fragments that contained the mutation of interest were individually subcloned into the capsid protein expression plasmid. Nomenclature for the replacement mutants followed the convention of a letter designation of the wild-type amino acid, followed by the residue number, followed by the substituted amino acid (e.g., S245A). The plasmid pspdhbv RV2650, used in testing the function of altered capsid protein by complementation, consisted of psp65 containing an EcoRI dimer of a DHBV genome in which a frameshift mutation at position 2560 destroyed the production of any capsid protein (3). We used two versions of this plasmid: one in which the envelope genes were intact, and one in which stop codons in the p17 gene prevented expression of both pre-s and S proteins (13). An infectious plasmid used for testing the effect of the mutation S259A on infectivity was constructed by substitution of the serine 259 codon by an alanine codon at the 3' end of the capsid protein gene. The mutant genome was cloned downstream of the immediate-early CMV promoter in the vector pucl 19 such that transcription began at the authentic viral cap site (14). Cell culture and transfection. Transfection of plasmid DNAs was carried out by the calcium phosphate coprecipitation method (13) in the chicken hepatoma cell line LMH (1, 4), maintained in F-12-Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum. Cells were incubated at 37 C for 4 days after addition of DNAs (10,ug/60-mm dish). Cotransfection of pspdhbv RV2650 and capsid protein expression plasmids was performed with 5,ug of each DNA. Primary duck hepatocyte cultures were used for the growth of DHBV. Primary hepatocytes were prepared from 5- to 10-dayold ducklings by perfusion of the liver with collagenase as previously described (16). Duck hepatocytes were maintained in Leibowitz medium (L-15) supplemented with glucose (1 mg/ml), insulin (1 jxg/ml), hydrocortisone hemisuccinate (10,uM), and dimethyl sulfoxide (1%) (9). Aliquots of virus concentrated from the culture fluids of transfected LMH cells by polyethylene glycol precipitation were used to infect each 60-mm dish of duck hepatocytes as previously described (14). Extraction and assay of intact viral capsids containing RNA and DNA. Transfected LMH cells in 60-mm dishes were lysed by the addition of 0.5 ml of lysis buffer (50 mm Tris-HCl [ph 8.0], 1 mm EDTA, 1% Nonidet P-40). The plate was rocked gently to distribute the buffer and was kept at 37 C for 10 min. The lysate, containing capsids, was subjected to microcentrifugation to remove nuclei and cell membrane debris. To remove DNA not present in nucleocapsids, we added 6 mm Mgacetate and 100,ug of DNase I per ml to the supernatant and incubated it at 37 C for 30 min. A portion (10,ul) of the lysate was mixed with 2 [li of sample buffer (50% glycerol, 0.1% bromphenol blue) and loaded onto a 1% agarose gel prepared in 10 Mm Na-phosphate electrophoresis buffer, ph 7.5. The capsids were electrophoresed toward the anode at 50 V with recirculation of the buffer. Capsids were transferred directly to nylon or nitrocellulose filters with TNE buffer (10 mm Tris-HCl [ph 7.4], 1 mm EDTA, 150 mm NaCl) by blotting, and the filter was washed for 10 min in distilled water and dried. At this point, the filter was probed for core antigens (nitrocellulose) by immunostaining as previously described (13), or the nucleic acid was released in situ for hybridization analysis by wetting the filter (nylon) for 10 to 20 s in 0.2 N NaOH-1.5 M NaCl, followed J. VIROL. immediately by neutralization in 0.2 N Trizma-HCI-1.5 M NaCl for 5 min, and washed again with distilled water for 10 min. The dried filter was probed for either total RNA and plus-strand DNA, or for minus-strand DNA with strandspecific 32P-labelled RNAs. Extraction and analysis of DNA replicative intermediates from cultured cells. DNA replicative intermediates were extracted either from transfected LMH cells or from infected primary duck hepatocytes as previously described (13). Four days posttransfection, cells were lysed and DNA in the supernatants was removed by DNase I digestion. Nucleic acids were purified by protease digestion and phenol extraction and collected by ethanol precipitation. Viral DNA was assayed in each sample by 1% agarose gel electrophoresis and Southern blot hybridization. Analysis of cccdna in transfected cells. Transfected cells in 60-mm plates were washed once with HBS buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [ph 7.45], 150 mm NaCl), and 1 ml of cccdna isolation buffer (10 mm Tris-HCl [ph 7.5], 10 mm EDTA, 1% SDS) was added to each plate and incubated for 5 min at 37 C. A total of 0.25 ml of 2.5 M KCl was added to the lysate, and the lysate was briefly vortexed and chilled on ice for 5 min. After removal of the detergent-protein complexes by centrifugation, the supernatant was extracted with phenol, and the nucleic acids were precipitated with ethanol. The dried DNA pellet was dissolved in 20,lI of TE (10 mm Tris-HCl [ph 7.4], 1 mm EDTA). To eliminate transfected plasmids that usually contaminated the viral cccdna fraction, the nucleic acids were digested with DpnI, which carries out methylation-dependent cleavage at many sites in the plasmid. Residual fragments of plasmid DNA were further digested with exonuclease III. Briefly, 5,ul of the cccdna solution was incubated with 5 U of DpnI and 25 U of exonuclease III in restriction buffer containing 10 mm Tris (ph 7.5), 10 mm magnesium acetate, 50 mm NaCl, 1 mm dithiothreitol, and 0.01% Nonidet P-40 and incubated at 37 C for 2 h. The viral cccdna in the digest was assayed directly by agarose gel electrophoresis and Southern blot hybridization. Assay of virus particles from supernatants of transfected cells. Virus particles were precipitated from the supernatants of transfected cells by the addition of 10% (wt/vol) polyethylene glycol (molecular weight, 7,000 to 9,000) as previously described (14). After centrifugation, the tube was carefully drained and the inside was wiped free of excess polyethylene glycol. The pellet was dissolved in 1/50 volume of 2 mm HEPES (ph 7.4)-150 mm NaCl-2 mm CaCl2. A total of 5 RI of the dissolved pellet was added to 15 pal of TE containing 750,ug of pronase per ml and incubated for 60 min at 37 C. This digestion was sufficient to disrupt viral cores that were not bound in a lipid envelope, but enveloped virus was completely resistant to pronase (Sa). Free viral DNA was removed from the suspension by the addition of 10 mm Mg-acetate and 500,ug of DNase I per ml (type I, Sigma) and incubation at 37 C for 30 min. The sample was subjected to electrophoresis through a 1% agarose gel with DNA electrode buffer with buffer recirculation. The virus particles migrated with an Rf of about 0.15 with respect to the bromphenol blue tracking dye. Virus particles were transferred to a nylon filter by blotting with TNE. The filter was thoroughly dried, and the DNAcontaining particles were denatured by soaking the filter in 0.2 N NaOH containing 1.5 M NaCl. The filter was neutralized with 0.2 M Trizma-HCl containing 1.5 M NaCl, washed in TNE, and dried. Viral DNA was detected by hybridization of the filter with a 32P-riboprobe specific for detection of the viral minus strand. Immunofluorescent staining of capsid protein in cultured

3 VOL. 68, 1994 Capsid expression vector CM I)r R1 L is -I MULTIPLE FUNCIIONS OF CAPSID PROTEIN PHOSPHORYLATION 4343 R1 229-rSkSrerrapTpqragSplprSSSShhrSpSprk-262 Capsid defective genome Rl 1S/1St DHBV Rl r S/wt v IF AL v ~1~ capsid -~M pol - ~ ~ ~ envelope RV2650 I. I / FIG. 1. Expression plasmids used in the study. Capsid proteins were produced from the expression vector depicted on the top. The immediate-early CMV promoter was used to drive expression of viral RNA. The DHBV sequences containing the 5' packaging signal were deleted (I), and stop codons were introduced into the envelope open reading frame (is). The EcoRI sites delimiting the cloned monomer viral DNA (Ri) are shown. Wild-type and mutant proteins were tested for their ability to complement a capsid-defective pregenome. The pregenome was transcribed from a viral DNA dimer, shown at the bottom. The viral DNA sustained a 2-bp deletion (RV2650) in the capsid open reading frame. The viral pregenome promoter (DHBV) is indicated. wt, wild type. cells. Transfected LMH cells and infected hepatocytes were fixed and stained with rabbit antibody specific for the DHBV capsid protein as previously described (3). RESULTS We determined the phenotypes of various altered capsid proteins after their expression in the chicken hepatoma cell line LMH. To measure the effects of mutations on steps in viral DNA synthesis and virus production, we coexpressed the capsid proteins with a DHBV genome that could not produce any capsid protein and measured the ability of the altered proteins to complement the defect in the DHBV genome, as we have previously described (19). Using this assay, we were able to avoid effects of changes in the P open reading frame, which overlaps the region of the capsid gene that we mutated. The vectors we used for expression of the capsid protein and the capsid-defective genome are shown in Fig. 1. Stop codons (1S mutation) introduced into the envelope gene that was present in the capsid protein expression vector prevented the expression of any intact envelope protein from this plasmid. In experiments in which the effect of envelope protein expression was studied, we compared the complementation of a capsid-defective genome that contained an intact envelope region with that of one that contained the 1S mutation. A series of point mutations that we introduced into the capsid gene resulted in the substitution of alanine codons for nine individual serine codons and one threonine codon at the 3' end of the core gene open reading frame. Using these mutants, we previously showed that three serines, S245, S257, Rl FIG. 2. Immunostaining of DHBV capsid protein in transfected LMH cells. LMH cells were stained for DHBV capsid protein at 48 h posttransfection with the wild-type capsid protein expression vector. Capsid protein distribution was always predominantly cytoplasmic (as shown) in all substitution mutants described in this study regardless of the presence of envelope proteins or viral DNA synthesis. and S259, and one threonine, T239, function as phosphorylation sites in the wild-type protein. Since an alanine in one of these positions might functionally mimic the nonphosphorylated wild-type amino acid residue serine, we believed that replication defects associated with these mutations might reveal the requirements for phosphorylation at these sites. We also constructed a series of mutants in which the four previously identified phosphoacceptor residues were substituted with aspartic acid, which we believed might functionally mimic phosphoserine. Defects associated with these mutations might reveal any requirement for nonphosphorylated serine at a particular site. Intracellular distribution of capsid proteins in transfected cells. Immunofluorescent staining of cells transfected with either alanine or aspartic acid substitution mutants in each case showed the capsid antigen distributed in cytoplasm in a manner similar to that observed with the wild-type capsid protein (shown in Fig. 2), suggesting that cytoplasmic localization of the capsid protein of DHBV was not affected by the phosphorylation state of any individual residue. Capsid assembly and pregenome encapsidation in replacement mutants. To determine whether prevention of serine or threonine phosphorylation at specific residues exerted any effect on viral capsid assembly, we assayed the formation of capsids by our mutants by electrophoresis of particles through nondenaturing agarose gels and Western blot (immunoblot)

4 4344 YU AND SUMMERS J. VIROL. a 6<< < < <:<:< << < > NN N X X N wen N vo co N u c, N Uz 0s < a a) < U) co QD < co LO <: a) 0 0 " C" 4 NC N N 5e l-clo Qo (5 CD 0 C) oa a) U) e r U) tt " N " N N " CN s uz w toli)c>g w w w '41.4 b X,*..~~~~ FIG. 3. Effects of serine-to-alanine replacements on capsid formation, RNA packaging, and DNA synthesis. LMH cells cotransfected with plasmids expressing the indicated capsid proteins and a capsiddefective genome were lysed, and cytoplasmic capsids were analyzed by agarose gel electrophoreses and transfer to a filter for (a) immunostaining or (b) detection of total plus-strand viral nucleic acid. In panel c, total viral replicative intermediates were extracted from the capsids and assayed by agarose gel electrophoresis and blot hybridization. WT, wild type. analysis. The electrophoretic patterns produced by the altered proteins were similar to those produced by the wild-type capsid protein (Fig. 3a), indicating that replacement mutants were able to direct the production of stable capsids. We measured the viral specific nucleic acid content of intracellular capsids by transfer of the capsids to a nylon filter, denaturation, and hybridization to a riboprobe specific for detection of viral plus strands (Fig. 3b). The results showed that individual substitution of the various potential phosphorylation sites by alanine, including threonine 239, serine 257, and serine 259, failed to affect the levels of total viral plusstrand nucleic acid (a measure of total RNA plus mature viral DNA) encapsidated compared with the wild-type protein. Alanine substitution of serine 245 caused a reduction in plus-strand nucleic acid associated with nucleocapsids; however, this reduced signal could be accounted for by a specific defect in plus-strand DNA synthesis (see below) rather than reduced packaging of RNA. The results indicated that RNA packaging into nucleocapsids did not require any specific individual phosphorylation state at these four amino acid residues _ + _ t presence of envelope protein FIG. 4. Effects of amino acid replacements on viral DNA synthesis. LMH cells were cotransfected with plasmids expressing the indicated capsid protein and a capsid-defective genome in the presence or absence of envelope protein expression from the complemented viral genome. Replicative intermediates were assayed by agarose gel electrophoresis and blot hybridization. WT, wild type. Effect of amino acid substitutions on viral DNA synthesis. When individual serine and threonine mutants were used to supply capsid proteins to a capsid-defective genome in trans, replicative intermediates could be observed in all cases (Fig. 3c). However, alanine substitution of serine 245 resulted in a specific failure to accumulate mature relaxed circular DNA (S245A). The phenotype was similar to that of mutants we have previously described (class II phenotype) that resulted from truncations of 19 to 25 amino acids at the C terminus (19). The region that is missing from the C terminus of all deletion mutants having the class II phenotype would include the serine 245 phosphorylation site, and the absence of this potential phosphorylation site in these deletion mutants could account for the class II phenotype (19). We showed previously that the downstream adjacent proline of each phosphoacceptor serine and threonine was essential for the mobility shift in SDS gels associated with phosphorylation. Whether the proline acted as a signal for recognition by a protein kinase, or whether replacement of the proline with glycine rendered the mobility in SDS gels insensitive to phosphorylation, was not determined. Replacement of proline 246 with glycine also resulted in a failure to synthesize mature viral DNA (Fig. 4, P246G). To test further whether the specific S245A and P246G phenotypes were a result of a failure to phosphorylate residue 245 or were due to the conservative missense mutations themselves, we substituted serine 245 with aspartic acid. The capsid protein of this mutant was able to support normal viral DNA maturation, as shown in Fig. 4 (S245D). It was likely, therefore, that phosphoserine rather than serine itself was functional in the synthesis of mature viral DNA. We also tested whether the presence of aspartic acid at position 245 could restore function to the mutant P246G. As shown in Fig. 4, substitution of aspartic acid for serine in mutant P246G (Fig. 4, S245D/P246G) did not restore wild-type function. The result suggests that the defect caused by the proline-to-glycine substitution was not due to prevention of phosphorylation of serine 245, but that proline, along with its adjacent upstream phosphoserine or aspartic acid, may be part of a single functional unit. Substitution of serine 259 with alanine resulted in a three- to fivefold defect in total DNA synthesis (Fig. 3c and 4, S259A).

5 VOL. 68, 1994 MULTIPLE FUNCTIONS OF CAPSID PROTEIN PHOSPHORYLATION 4345 ca cn ul qt s < N N4 C%l CM j~~~~~~ 4_..l ~~~~ I1-tCl 0) U) LO LO U) LO U) U) Lo N Cl CM CN :w_.0... < < < < 0 D C 0D CM) LO 1LC CD LO LD O CO N' U' Ln L qt LO U), N C N N N CN N CN 5 w (1 w 5 wo(: J 6. so :1 cs m LO LO P rl- m O m) In qi.. LOU LO LO LO N CN CM CM N N N N 5~~~ 5 CO 0 m CJ CJ l_ FIG. 5. Effects of amino acid replacements on cccdna synthesis. LMH cells were cotransfected with plasmids expressing the indicated capsid protein and a capsid-defective genome in the presence or absence of envelope protein expression from the complemented viral genome. Viral cccdna was selectively purified and assayed by agarose gel electrophoresis and blot hybridization. WT, wild type. We interpret this defect as an inhibition of an early step in minus-strand DNA synthesis, since RNA packaging was normal, the pattern of nascent minus strands was normal, and the minus strands were used as templates for plus-strand synthesis in a seemingly normal manner. Substitution of the adjacent downstream proline produced the same defect (Fig. 4, P260G), while aspartic acid replacement resulted in normal levels of viral DNA (Fig. 4, S259D). These results suggested that the inhibition of minus-strand synthesis by alanine substitution was due to lack of phosphorylation at residue 259. Effect of alanine substitutions on synthesis and regulation of cccdna. We analyzed the levels of cccdna in cells transfected with the various alanine substitution mutants. Since the maintenance of normal levels of cccdna in the nucleus depends on the regulation by the 36-kDa pre-s envelope protein, we assayed cccdna levels in cells expressing the altered capsid proteins in the absence and presence of pre-s envelope proteins. The results showed that with one exception (S245A), cccdna synthesis reached relatively equivalent levels in the cells transfected with the alanine substitution mutants and that cccdna levels were reduced as expected in the presence of the pre-s envelope proteins (Fig. 5). The mutant S245A showed reduced production of cccdna in the absence of the envelope protein, consistent with its defect in the production of mature DNA. However, this mutant produced nearly the same cccdna level in the presence of the pre-s envelope protein. This result indicated that the cccdna level in this mutant was not regulated by pre-s envelope protein. Alteration of cccdna regulation by a mutation in the capsid protein suggests an involvement of the 4I1 FIG. 6. Effects of amino acid replacements on the production of enveloped virus particles. LMH cells were cotransfected with plasmids expressing the indicated capsid proteins and a capsid-defective genome expressing the viral envelope proteins. Virus particles concentrated from the culture fluid were assayed by selective resistance to pronase-dnase I digestion, agarose gel electrophoresis, and blot hybridization. WT, wild type. capsid protein as well as the pre-s protein in cccdna regulation, consistent with earlier models that pre-s binding of the nucleocapsid prevents its use for cccdna synthesis (13, 14). A similar result was observed with the class II C-terminal deletion mutants that we originally described (21). Effect of aspartic acid substitutions on cccdna synthesis. Aspartic acid substitution of serine 257 produced a reduction in the levels of cccdna produced in the absence of envelope proteins (Fig. 5, S257D). Smaller reductions were seen in mutants S245D and S259D. Since all of these mutants produced levels of replicative intermediates equivalent to levels produced by the wild-type protein (Fig. 4), this result suggested that phosphorylation at position 257 would be expected to inhibit cccdna DNA synthesis. This conclusion would be consistent with models that have been proposed in the literature for control of nuclear localization of the capsid protein and cccdna synthesis by phosphorylation (2, 18), provided that nuclear localization is a step in cccdna synthesis. In these experiments, however, we never observed nuclear localization of a major portion of the capsid protein. The level of cccdna in the presence of envelope protein in all aspartic acid substitution mutants was reduced below that in the absence of envelope protein. We infer from this result that phosphorylation would not be expected to interfere with control of cccdna amplification by the pre-s envelope protein. Influence of amino acid substitutions on viral assembly and infectivity. We measured the amount of enveloped virus in the culture fluids of transfected cells by nondenaturing agarose gel electrophoresis. The results showed that mutant S245A, which was defective in viral DNA maturation, failed to produce enveloped virus (Fig. 6). This result may be a consequence of the failure of this mutant to synthesize mature relaxed circular DNA (see Discussion). Levels of virus produced by the mutant S259A were depressed three- to fivefold, commensurate with the reduction in total viral DNA synthesis attributable to this mutation. On the other hand, aspartic acid substitution of serines 257 and 259 caused specific reductions in enveloped virus production, suggesting that phosphorylation at these sites would be expected to inhibit assembly of mature nucleocapsids into virus particles. These results were confirmed by using isopycnic cesium chloride gradient centrifugation to assay for enveloped virus particles (14). No alanine or aspartic acid substitution altered the pattern of viral DNA found in extracellular virus: i.e., only mature double-stranded DNA was present in the

6 4346 YU AND SUMMERS J. VIROL. Wild type LMH Duck hcdaton vtes RI TABLE 1. Requirements for specific phosphorylated states Function Phosphorylation preference Immature capsids RNA packaging... No specific requirement Minus-strand DNA... Serine 259-phosphorylated Plus-strand DNA... Serine 245-phosphorylated Mature capsids cccdna amplification... Serine 257-unphosphorylated Virus assembly... Serine 259-unphosphorylated; serine 257-unphosphorylated Viral penetration... Serine 259-phosphorylated S259A LMH I Duck henatocvtes FIG. 7. Effect of replacement of serine 259 with alanine on infectivity of enveloped virus. LMH cells were transfected with either a wild-type viral genome or a genome in which the capsid protein codon 259 had been changed from TCG to GCG. This change did not alter the coding of the overlapping polymerase gene. Culture supernatants were used to infect cultures of primary duck hepatocytes. Viral replicative intermediates present in the LMH cells at the time of harvest are shown in the left lanes (LMH). Immunofluorescent staining of the duck hepatocyte cell layer 14 days postinfection is shown (duck hepatocytes). No evidence of infection of the duck hepatocytes by mutant S259A was seen by examination of approximately 1,000 fields of the size shown. Viral replicative intermediates present in the duck hepatocyte cultures 14 days postinfection are shown in the right lanes (RI). nucleic acids extracted from cesium chloride-purified virus particles (data not shown). The results indicated that the state of phosphorylation of any individual site was not responsible for the selective assembly of mature nucleocapsids into virus particles. We tested the infectivity of enveloped virus produced by one of our alanine substitution mutants. We cloned the S259A mutation into a wild-type DHBV genome cloned downstream of the immediate-early CMV promoter (14). Transfection of LMH cells by this mutant resulted in the production of replicative intermediates at levels approximately threefold reduced from a parallel culture transfected with wild-type genome (Fig. 7, LMH lanes). Production of enveloped virus by this mutant was reduced approximately fivefold (not shown). These results were consistent with the phenotype of this mutation assayed in the complementation experiments shown in Fig. 3, 4, and 6. Supernatants of LMH cells transfected in parallel with mutant S259A or wild-type genomes were used to infect cultures of primary duck hepatocytes, and the cells were assayed at 14 days postinfection for viral replicative DNA intermediates, and by immunofluorescent staining for viral capsid antigens. The hepatocyte cultures failed to show any evidence of infection by the mutant virus (Fig. 7). Taking into account the fivefold reduction in virus yield, we estimated from the number of staining cells that the infectivity of enveloped mutant virus was reduced by at least 2 orders of magnitude compared with that of wild type. This result was consistent with the lack of any detectable viral DNA by Southern blot analysis of the mutant infected cultures (Fig. 7, RI lanes). DISCUSSION The results of our experiments provide evidence that phosphorylation at three specific serines in the C terminus of capsid protein of DHBV influences several steps in viral replication. The presumed requirements for specific phosphorylated states are summarized in Table 1. No defects could be assigned to alanine or aspartic acid substitution of threonine 239, and therefore, these experiments provided no evidence for a role for phosphorylation of this residue in virus replication. Rationale and interpretation of results. The conclusions in Table 1 were based on the assumption that alanine could functionally replace serine at the sites of replacement, except for its ability to serve as a phosphoacceptor amino acid. Similarly, we have assumed that aspartic acid could mimic the function of phosphoserine at these sites. When defects associated with replacement of serine by alanine were corrected by aspartic acid substitution, we assumed that aspartic acid was mimicking phosphoserine rather than serine as the wild-type functional residue. Likewise, when defects introduced by aspartic acid substitution were corrected by alanine, we assumed that the alanine was mimicking serine rather than phosphoserine as the functional amino acid residue. Substitution of the adjacent downstream proline with glycine produced results that were more difficult to interpret but nevertheless were included in this paper. These substitutions produced effects on virus replication that mimicked those of the adjacent serine-to-alanine substitution, but it is not known whether the proline replacements acted by inhibiting phosphorylation of the adjacent serine. In at least one case, our evidence indicated a more complicated role for proline, since even when the upstream residue was a functional aspartic acid, the downstream proline was still required for mature viral DNA synthesis. The multiple effects of phosphorylation on virus replication. Our results provide evidence that serine phosphorylation has both positive and negative effector functions in virus replication. For example, aspartic acid replacement of two serines resulted in a stimulation of viral DNA synthesis relative to alanine substitution at these sites. This result is consistent with

7 VOL. 68, 1994 MULTIPLE FUNCTIONS OF CAPSID PROTEIN PHOSPHORYLATION 4347 phosphoserine acting as the functional wild-type residue in activation of DNA synthesis. On the other hand, aspartic acid replacement of at least two serines inhibited cccdna synthesis and/or virus production relative to alanine substitution of these residues, suggesting that nonphosphorylated serine acts as the functional wild-type residue for these processes. The results imply that differential modification of at least three serines in the capsid protein is required to produce all of the polypeptide species necessary for virus replication. Thus, posttranscriptional modification of a single capsid protein may be an alternative to multiple capsid proteins encoded by separate genes. In general, phosphorylation seemed to be required for functions carried out by immature nucleocapsids, i.e., nucleocapsids that are not direct precursors of virus particles or cccdna, while phosphorylation inhibited functions carried out by mature nucleocapsids. The data can be interpreted to suggest that dephosphorylation of phosphorylated residues on the nucleocapsid would be required for the acquisition of mature capsid functions. However, our experiments do not distinguish between a requirement for phosphorylation of some, as opposed to all, polypeptides in the nucleocapsid. For example, our data indicated a requirement of phosphoserine at residue 259 for minus-strand DNA synthesis and for unphosphorylated serine at the same residue for virus production. Since the nucleocapsid contains many copies of the capsid protein, both requirements might be met at the same time in the same nucleocapsid. Therefore, the genetic data we present do not distinguish between the following models: (i) that the nucleocapsid proteins must undergo sequential modification by phosphorylation and dephosphorylation in order for the nucleocapsid to proceed through the various stages of maturation and virus assembly, or (ii) that the nucleocapsid is modified at selected residues before any DNA synthesis occurs, and thereby acquires competence for all steps in virus replication without any subsequent modifications. Our previous report showing that the phosphorylation state of intracellular capsids differs from that of mature viral capsids, however, favors the notion that dephosphorylation does accompany the acquisition of mature capsid functions. In addition to specific requirements for phosphoserine or serine in nucleocapsid maturation and assembly into virus particles, we obtained evidence that phosphoserine at residue 259 might be required in the initiation of infection. While alanine could replace serine at position 259 without disrupting enveloped virus production, alanine could not substitute for serine at this position during initiation of an infection. Presumably the defect was due to a requirement for phosphoserine at position 259 early during infection of hepatocytes. Phosphoserine 259 may be formed by de novo phosphorylation of serine 259 during the initiation of infection or may preexist in infectious virus particles. Since alanine replacement of this residue did not inhibit cccdna formation from cytoplasmic nucleocapsids, phosphoserine at residue 259 would not seem to be required for cccdna formation from the infecting viral DNA, but for an earlier step that introduces the nucleocapsid of the infecting virus into the cytoplasm of the hepatocyte. Our data suggested that phosphoserine at position 259 would be expected to inhibit virus production. Therefore, we speculate that phosphorylation of serine 259 may interfere with a physical association between the nucleocapsid and the viral envelope that is required for virus assembly. If this were the case, phosphorylation of serine 259 might be actively required for dissociation of the nucleocapsid from the envelope at the site of viral penetration. Apart from the role of phosphorylation, mutations such as S245A that generate a class II phenotype illuminate the orderly nature of viral DNA synthesis and viral assembly. Mutations that inhibited the synthesis of mature viral DNA also inhibited cccdna regulation and virus production, two processes that depend in common on interaction of the viral nucleocapsid with the pre-s envelope protein. This result suggests that the synthesis of mature viral DNA and the acquisition by the nucleocapsid of the ability to interact with the pre-s protein may be functionally linked. We and others have repeatedly observed that only nucleocapsids that contain mature viral DNA are found in extracellular virions. Considering these two observations together, we suggest that the ability of nucleocapsids to become assembled into viral envelopes is due to the acquisition of sites for recognition and binding of the pre-s protein concomitant with the synthesis of mature viral DNA. Such sites may constitute all or part of the packaging signal postulated by Summers and Mason (12). Mechanism of action of phosphorylation. Our experiments raise the question of how the state of phosphorylation in a small region of the capsid protein differentially influences multiple functions of the capsid particle. We have suggested that a role of the several distinct phosphorylation states of the C terminus may be to stabilize the conformations of different functional domains that depend on the C terminus of the capsid protein. This model was indirectly suggested by the strong influence of phosphorylation at specific sites on the conformation of the C terminus of the capsid protein (20). The existence of alternative conformational and functional domains that depend on the particular phosphorylated state of the C terminus is consistent with the data presented here. The effects of the individual mutations that we tested would have been determined by the extent to which the conformation of the functionally specific domains depended on the particular phosphorylated state that was excluded by the mutation. ACKNOWLEDGMENTS We thank Tim Powell for excellent technical assistance. This work was supported by Public Health Service grant CA REFERENCES 1. Condreay, L., C. Aldrich, L. Coates, W. Mason, and T.-T. Wu Efficient duck hepatitis B virus production by an avian tumor cell line. J. Virol. 64: Eckhardt, S. G., D. R. Milich, and A. McLachlan Hepatitis B virus core antigen has two nuclear localization sequences in the arginine-rich carboxyl terminus. J. Virol. 65: Horwich, A. L., K. Furtak, J. C. Pugh, and J. Summers Synthesis of hepadnavirus particles containing replication-defective duck hepatitis B virus genomes in cultured Huh-7 cells. J. Virol. 64: Kawaguchi, T., K. Nomura, Y. Hirayama, and T. Kitagawa Establishment and characterization of a chicken hepatocellular carcinoma cell line, LMH. Cancer Res. 47: Kunkel, T. A., J. D. Roberts, and R. A. Zakour Rapid and efficient site specific mutagenesis without phenotypic selection. Methods Enzymol. 154: a.Lenhoff, R., and J. Summers. Unpublished data. 6. Mandart, E., A. Kay, and F. Galibert Nucleotide sequence of a cloned duck hepatitis B virus genome: comparison with woodchuck and human hepatitis B virus sequences. J. Virol. 49: Mason, W., C. Aldrich, J. Summers, and J. Taylor Asymmetric replication of duck hepatitis B virus DNA in liver cells (free minus strand DNA). Proc. Natl. Acad. Sci. USA 79: Mason, W., M. Halpern, J. England, G. Seal, J. Egan, L. Coates, C. Aldrich, and J. Summers Experimental transmission of duck hepatitis B virus. Virology 131:

8 4348 YU AND SUMMERS 9. Pugh, J., and J. Summers Infection and uptake of duck hepatitis B virus by duck hepatocytes maintained in the presence of dimethyl sulfoxide. Virology 172: Pugh, J. C., A. Zweidler, and J. Summers Characterization of the major duck hepatitis B virus core particle protein. J. Virol. 63: Schlicht, H. J., R. Bartenschlager, and H. Schaller The duck hepatitis B virus core protein contains a highly phosphorylated C terminus that is essential for replication but not for RNA packaging. J. Virol. 63: Summers, J., and W. S. Mason Replication of the genome of hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 29: Summers, J., P. Smith, and A. L. Horwich Hepadnaviral envelope proteins regulate amplification of covalently closed circular DNA. J. Virol. 64: Summers, J., P. Smith, M. Huang, and M. Yu Regulatory and morphogenetic effects of mutations in the envelope proteins of an avian hepadnavirus. J. Virol. 65: Tuttleman, J., C. Pourcel, and J. Summers Formation of the J. VIROL. pool of covalently closed circular viral DNA in hepadnavirusinfected cells. Cell 47: Tuttleman, J. S., J. C. Pugh, and J. W. Summers In vitro experimental infection of primary duck hepatocyte cultures with duck hepatitis B virus. J. Virol. 58: Wu, T.-T., L. Coates, C. E. Aldrich, J. Summers, and W. S. Mason In hepatocytes infected with duck hepatitis B virus, the template for viral RNA synthesis is amplified by an intracellular pathway. Virology 175: Yeh, C. T., S. W. Wong, Y. K. Fung, and J. H. Ou Cell cycle regulation of nuclear localization of hepatitis B virus core protein. Proc. Natl. Acad. Sci. USA 90: Yu, M., and J. Summers A domain of the hepadnaviral capsid protein specifically required for DNA maturation and virus assembly. J. Virol. 65: Yu, M., and J. Summers Phosphorylation of the duck hepatitis B virus capsid protein associated with conformational changes in the C terminus. J. Virol. 68: Yu, M., and J. Summers. Unpublished observation. Downloaded from on September 14, 2018 by guest