Download replication. phosphorylation in duck hepatitis B virus Multiple

Multiple functions of capsid protein
phosphorylation in duck hepatitis B virus
replication.
M Yu and J Summers
J. Virol. 1994, 68(7):4341.
These include:
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Vol. 68, No. 7
JOURNAL OF VIROLOGY, JUIY 1994, p. 4341-4348
0022-538X/94/$04.00+0
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 87131
Received 15 February 1994/Accepted 12 April 1994
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
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.
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
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
*
Corresponding author. Phone: (505) 277-7979. Fax: (505) 2779494. Electronic mail address: [email protected].
4341
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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.
4342
YU AND SUMMERS
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
Downloaded from http://jvi.asm.org/ on April 17, 2014 by PENN STATE UNIV
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.
MULTIPLE FUNCIIONS OF CAPSID PROTEIN PHOSPHORYLATION
VOL. 68, 1994
4343
Capsid expression vector
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FIG. 1. Expression plasmids used in the study. Capsid proteins
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.
were
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,
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)
Downloaded from http://jvi.asm.org/ on April 17, 2014 by PENN STATE UNIV
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J. VIROL.
YU AND SUMMERS
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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.
presence
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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.
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).
Downloaded from http://jvi.asm.org/ on April 17, 2014 by PENN STATE UNIV
-
VOL. 68, 1994
MULTIPLE FUNCTIONS OF CAPSID PROTEIN PHOSPHORYLATION
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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
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
Downloaded from http://jvi.asm.org/ on April 17, 2014 by PENN STATE UNIV
cs
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4346
J. VIROL.
YU AND SUMMERS
LMH
Duck hCDaton vtes
RI
TABLE 1. Requirements for specific phosphorylated states
Function
Immature capsids
RNA packaging ................
Minus-strand DNA ................
Plus-strand DNA ................
Mature capsids
cccDNA amplification ................
Wild
type
Virus assembly ................
Viral penetration ................
S259A
Duck henatocvtes
I
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
preference
No specific requirement
Serine 259-phosphorylated
Serine 245-phosphorylated
Serine 257-unphosphorylated
Serine 259-unphosphorylated;
serine 257-unphosphorylated
Serine 259-phosphorylated
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
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LMH
Phosphorylation
VOL. 68, 1994
MULTIPLE FUNCTIONS OF CAPSID PROTEIN PHOSPHORYLATION
serine 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-42542.
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