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Journal of Virology, February 2001, p. 2024-2028, Vol. 75, No. 4
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.4.2024-2028.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Does a cdc2 Kinase-Like Recognition Motif on the Core Protein
of Hepadnaviruses Regulate Assembly and Disintegration
of Capsids?
M. Inmaculada
Barrasa,
Ju-Tao
Guo,
Jeffrey
Saputelli,
William
S.
Mason, and
Christoph
Seeger*
Institute for Cancer Research, Fox Chase
Cancer Center, Philadelphia, Pennsylvania 19111
Received 7 September 2000/Accepted 15 November 2000
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ABSTRACT |
Hepadnaviruses are enveloped viruses, each with a DNA genome
packaged in an icosahedral nucleocapsid, which is the site of viral DNA
synthesis. In the presence of envelope proteins, DNA-containing nucleocapsids are assembled into virions and secreted, but in the
absence of these proteins, nucleocapsids deliver viral DNA into the
cell nucleus. Presumably, this step is identical to the delivery of
viral DNA during the initiation of an infection. Unfortunately, the
mechanisms triggering the disintegration of subviral core particles and
delivery of viral DNA into the nucleus are not yet understood. We now
report the identification of a sequence motif resembling a serine- or
threonine-proline kinase recognition site in the core protein at a
location that is required for the assembly of core polypeptides into
capsids. Using duck hepatitis B virus, we demonstrated that mutations
at this sequence motif can have profound consequences for RNA
packaging, DNA replication, and core protein stability. Furthermore, we
found a mutant with a conditional phenotype that depended on the cell
type used for virus replication. Our results support the hypothesis
predicting that this motif plays a role in assembly and disassembly of
viral capsids.
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TEXT |
An apparent paradox of virus
replication is that infected cells must be permissive for both assembly
and disassembly of viral nucleocapsids. Since both pathways cannot act
on nucleocapsids at the same time, an important question concerns the
mechanisms responsible for the switch from the uncoating to the
assembly mode. To solve this problem, viruses have adopted
different strategies, among which the best-known strategy relies on the
use of different cellular compartments for assembly and disintegration
(21). For example, in adenoviruses and orthomyxoviruses,
viral disintegration occurs in acidic compartments, and assembly occurs
in the cytosol of infected cells (6).
The mechanism controlling assembly and disintegration of the
icosahedral core particles of hepadnaviruses is not well understood. Assembly occurs in the cytosol in a two-step process where core proteins form dimers and 120-dimer subunits build capsids (23, 29). Disintegration of viral particles occurs after DNA
synthesis and may be activated by a switch that is created on the
surface of core particles in response to DNA synthesis. If correct,
such a model would predict the presence of regulatory sequence motifs on the surface of viral capsids effecting assembly and disassembly. During a search for known consensus motifs that signify recognition sites for posttranslational processing, we found a threonine-proline kinase recognition site that is conserved among core polypeptides of
all known ortho-and avihepadnaviruses. Based on structural and
biochemical data, this motif is located at the end of an 
-helix that
is required for the multimerization of the core dimers to form the
icosahedral core shells (1, 2, 5, 16, 23) (Fig.
1). Notably, the motif is located close
to the fivefold and twofold icosahedral symmetry axes of the capsid,
where it forms a junction between the interior of the capsid and a
surface-exposed loop (23). Hence, this motif is in an
ideal location to relay a signal, possibly created by phosphorylation
and dephosphorylation reactions that could produce changes in the local
conformation leading to the destabilization of the critical dimer-dimer
interaction and, consequently, to the disintegration of capsids. Such a
model predicts that mutations at this site might affect RNA packaging, DNA replication, and the stability of core particles. The genetic approach described in this report yielded variants with such defects and, thus, supported a model predicting that this site plays a pivotal
role in viral DNA replication.
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FIG. 1.
Conservation of the threonine-proline kinase motif on
hepadnavirus capsid proteins. The figure shows the predicted structure
of the HBV capsid protein as described by Bottcher et al.
(2). The cylinders represent the complete carboxy-terminal
-helix and a portion of the penultimate -helix. The bar depicts
the segment which is exposed on the surface of core particles, as
determined by Pushko et al. (16). The numbering of the
amino acids refers to the core sequences of HBV (top) and DHBV
(bottom). Aligned were the sequences of HBV (ayw), woolly monkey HBV
(wmhbv), ground squirrel and woodchuck hepatitis viruses (gshv and
whv), heron virus (hhbv), and DHBV (dhbv). The proline kinase
recognition site present on the oncogene v-fms is also
shown. Conserved residues are shown in boldface. C, carboxy terminal;
N, amino terminal.
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Mutations at Thr 174 prevent capsid formation or interfere with DNA
replication.
To examine whether mutations at the cdc2 kinase motif
can destabilize cores and interfere with RNA packaging or DNA
synthesis, we replaced Thr 174 with aspartic acid (T174D) or alanine
(T174A) to mimic phosphorylated or unphosphorylated Thr, respectively. We then tested the ability of the mutants to produce virus in transfected LMH cells (7). Five days after transfection,
viral DNA was isolated as described by Summers et al. (19)
and Yang et al. (26) and analyzed by Southern blot hybridization.
The results showed that variant T174D was defective for viral DNA
synthesis and RNA packaging and exhibited a dominant negative
phenotype
(Table
1; Fig.
2A, lanes 1, 3, and 6; Fig.
3, lane
5). Furthermore, accumulation of
core polypeptides expressed with
this variant was reduced compared to
that in wild-type duck hepatitis
B virus (DHBV) (Fig.
3, lane 5). In
contrast, mutant T174A produced
the same viral DNAs as wild-type DHBV,
including covalently closed
circular DNA (cccDNA) (Fig.
2A, lanes 1 and
2). The levels of
viral DNA intermediates that accumulated in cells
transfected
with this variant exhibited a slight, twofold reduction
compared
to the wild type. As expected, cotransfection of the wild-type
construct with variant T174A or with a plasmid expressing green
fluorescent protein did not interfere with the production of viral
DNA
intermediates (Fig.
2A through C). Under the assumption that
aspartic
acid mimics phosphorylated Thr, the results obtained
with T174D were
consistent with the hypothesis predicting that
phosphorylation at Thr
174 could trigger the disintegration of
viral capsids. The observed
dominant negative effect of T174D
suggested that the mutant is
competent for the formation of multimeric
complexes and, possibly, that
only a fraction of core proteins
in a nucleocapsid need to be
phosphorylated to signal capsid disintegration.
As predicted, mutant
T174A was competent for viral DNA synthesis,
although the presence of
cccDNA suggested either that capsids
can disintegrate without a
requirement for phosphorylation at
Thr 174 or that the mutant activated
an alternate site, such as
serine 173 (Fig.
1), as a substrate for a
cdc2-like kinase.
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FIG. 2.
Viral DNA replication in transfected LMH cells. The
figure shows Southern blots of viral core DNA (panels A I, B, and C)
and cccDNA (panel AII) extracted from LMH cells (7)
transfected with equal amounts of the indicated plasmids. The cells
were maintained in Dulbecco's modified Eagle medium-F-12 medium
supplemented with 10% fetal bovine serum, kanamycin (100 µg/ml),
penicillin (50 U/ml), and streptomycin (50 µg/ml) and transfected
with plasmid DNA using a calcium phosphate cell transfection kit
(CalPhos mammalian transfection kit; Clontech, Palo Alto, Calif.).
Plasmid DHBV directs expression of the DHBV pg from the cytomegalovirus
immediate-early promoter (22). Plasmid T174A contains a
dA-to-dG substitution at nucleotide 145 (the nomenclature is according
to Mandart et al. [12]). Plasmid T174D contains a
dAC-to-dGA substitution at nucleotides 145 and 146. Plasmid T174V
contains a dAC-to-dGT substitution at nucleotides 145 and 146. Plasmid
T174N contains a dC-to-dA substitution at nucleotide 146. Plasmid
ST173/4AA contains a dT-to-dG substitution at nucleotide 142 and a
dA-to-G substitution at nucleotide 145. Plasmid ST173/4RA contains a
dTC-to-dCG substitution at nucleotides 142 and 143 and a dA-to-dG
substitution at nucleotide 145. All mutations were verified by
nucleotide sequence analysis. RC, relaxed circular, DSL,
double-stranded linear; SS; single stranded; CCC, covalently closed
circular.
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FIG. 3.
Accumulation of packaged pg RNA and core protein in
transfected LMH cells. (A) Northern blot analysis of packaged RNA from
LMH cells transfected with the indicated plasmids. Encapsidated RNA was
extracted as described by Schultz et al. (17). The blot
was hybridized with radiolabeled DHBV RNA. (B) Western blot analysis of
a total extract from LMH cells transfected with the indicated plasmids.
Cells (35-mm-diameter dishes) were lysed in 250 µl of lysis buffer
(50 mM Tris-HCl [pH 8], 1% sodium dodecyl sulfate, 1 mM
phenylmethylsulfonyl fluoride, 2 µg of leupeptin per ml, and 0.7 µg
of pepstatin per ml). Three microliters of extract was analyzed by
Western blot using antibodies obtained from a rabbit immunized with
DHBV core protein. C, core protein; pg RNA, pregenomic RNA.
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To examine whether the phenotypes of the two mutants were specific to
the selected amino acid changes, we produced two additional
mutants
with large and small side chains, changing Thr 174 to
asparagine
(T174N) and valine (T174V), respectively. Because the
DHBV core
contains a serine residue at position 173 that could
have served as an
alternate phosphorylation site in the absence
of Thr 174, we created
two additional variants, ST173/4AA and
ST173/4RA. The Ser
173-to-arginine mutation was selected based
on the corresponding amino
acid sequence motif on the hepatitis
B virus (HBV) core protein (Fig.
1).
In contrast to the observations made with T174A, none of the other four
variants was competent for viral DNA synthesis (Table
1; Fig
2B [lanes
2 and 3] and C [lanes 2 and 3]). However, two
variants, T174V and
ST173/4RA, were competent for particle assembly
and pregenome (pg)
packaging (Fig.
3). Mutant ST173/4RA appeared
to accumulate an RNA
species with a reduced size compared to the
wild type, which could
signify replication of a truncated minus-strand
DNA species. The other
two variants, T174N and ST173/4AA, did
not appear to package pg RNA. As
with T174D, reduced packaging
of pg RNA with T174N could be explained
with the results obtained
from the Western blot showing that the core
polypeptides expressed
with this variant accumulated approximately
20-fold less than
wild-type core protein (Fig.
3B). In contrast,
ST173/4AA displayed
normal levels of core protein, suggesting that this
variant lost
the capacity to form
capsids.
Cotransfection of the wild type with ST173/4RA reduced the levels of
viral DNA synthesis approximately 10-fold (Fig.
2A, lane
6, and C, lane
6). This result suggested that this variant, like
T174D, expressed core
polypeptides that were competent to interact
with wild-type core
subunits and prevent the formation of functional
capsids in a dominant
negative fashion. In contrast, T174N, T174V,
and ST173/4AA did not
inhibit wild-type replication, suggesting
either that the core
polypeptides expressed with these plasmids
were incompetent for
assembly of capsids or that hybrid capsids
supported DNA replication
(Fig.
2B, lanes 5 and 6, and C, lane
5). The latter possibility may be
relevant in the case of T174V,
which is competent for the formation of
RNA-containing capsids
(Fig.
3A).
These results indicated that replacement of Thr 174 with amino acids
carrying bulky side chains interferes with assembly of
capsids as well
as with the accumulation of core protein in transfected
cells. However,
the dominant negative inhibition appears to be
a specific property of
phosphorylated Thr as simulated with T174D.
In contrast, replacement of
Thr 174 with amino acids containing
small side chains did not block
capsid formation. In the case
of T174V, it blocked DNA synthesis,
possibly because it prevented
proper arrangement of pg RNA in capsids
or induced a steric inhibition
of the polymerase to synthesize DNA.
Finally, the results showed
that serine 173 is required for capsid
formation presumably as
a component of the helix motif and that even
conservative changes,
such as in ST173/4RA, can interfere with viral
DNA synthesis and,
presumably, with the packaging of pg
RNA.
The phenotype of DHBV mutant T174A can vary depending on the host
cell.
The results obtained so far seemed to suggest that the
replacement of Thr 174 with alanine interfered neither with the
assembly nor with the subsequent disintegration of mature core
particles. However, it is not known whether the mechanisms for the
disintegration of cores during the intracellular amplification of
cccDNA as measured in LMH cells and following de novo infection are the
same. Therefore, we used primary duck hepatocytes (PDHs) to determine
whether the virus produced from T174A can initiate an infection. PDHs
were prepared as previously described (15, 20). Virus was
obtained from the culture supernatants of LMH cells transfected with
wild-type DHBV or with T174A. The virus was precipitated with 10%
polyethylene glycol 8000, and the pellets were resuspended in culture
medium (19). The titer of the concentrated virus
suspension was determined by Southern blot analysis using plasmid DNA
as a standard and was found to be approximately 3 × 108 virus particles per ml for both the wild type and the
mutant (Fig. 4A). Infection of PDHs with
the two samples revealed an approximately 40-fold difference in viral
DNA replication between the wild-type and the mutant viruses (Fig. 4B).
This difference did not depend on the time point used for DNA analysis,
because the results obtained with samples taken either 5 or 10 days
postinfection (p.i.) yielded comparable results. The levels of nuclear
cccDNA expressed from the mutant virus were also approximately 40-fold lower than observed with wild-type virus at both time points (Fig. 4B,
panel II, lanes 1 through 6). Thus, the formation of cccDNA did not
appear to be inhibited.
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FIG. 4.
Infection of PDHs. (A) Southern blot of DNA extracted
from enveloped virions present in samples of transfected LMH cells used
for the infection of PDHs. A plasmid standard (lane 4) was used to
determine the amount of DNA present in lanes 1 and 3. The concentrated
culture supernatants were protease treated and incubated with DNase I
to remove contaminating core particles, as described by Yu and Summers
(27). A sample containing wild-type virus was incubated
with NP-40 as a control to demonstrate the efficacy of the protease to
digest nonenveloped core particles (lane 2). (B) Southern blot from
core (BI) and cccDNA (BII) extracted from PDHs infected with the
indicated virus samples. Cells were harvested 5 (d5) and 10 (d10) days
postinfection. RC, relaxed circular; DSL, double-stranded linear; SS,
single stranded; CCC, covalently closed circular.
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To determine whether the reduction in viral DNA synthesis was due to a
block in the initiation of infection, we next asked
whether the mutant
virus could convert its relaxed circular genome
into cccDNA. The levels
of cccDNA derived directly from the infecting
virus in the absence of
DNA replication were determined with PDHs
that were infected in the
presence of phosphonoformic acid (PFA),
a known inhibitor of the viral
reverse transcriptase (
13). Under
these conditions, cccDNA
was synthesized in PDHs infected with
the mutant, albeit at
approximately twofold reduced levels compared
to wild-type virus (Fig.
5A). Based on Northern blot analysis,
the
cccDNA derived from T174A was competent for the transcription
of the
three known viral mRNAs, pg RNA, pre-S-RNA, and S-RNA (Fig.
5B). These
results showed that the mutant virus was competent
to infect PDHs,
enter the uncoating pathway, convert the DNA genome
into cccDNA, and
produce all three viral RNAs.
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FIG. 5.
Formation of cccDNA from virion DNA, viral RNA
expression, and packaged RNA in PDHs. (A) Southern blot of core DNA
(input virus) (panel AI) and cccDNA (panel AII) extracted from PDHs
infected with the indicated virus samples in the presence of PFA. (B)
Northern blot analysis of poly(A)-enriched RNA isolated from PDHs
infected with the indicated virus samples. The blot was first
hybridized with radiolabeled DHBV DNA (panel BI) and subsequently with
a probe corresponding to the glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) gene (panel BII). Plates were maintained in the presence of PFA
and collected 48 and 72 h p.i. or on day 7 p.i. PDHs were
infected with 5 × 107 virions per 60-mm culture dish
for DHBV and 4 × 107 virions for T174A (panels A and
B). (C) Northern blot analysis of packaged RNA from PDHs infected with
the indicated virus samples at 3.5 × 108 virions per
dish. Encapsidated RNA was extracted as described by Schultz et al.
(17). The blot was hybridized with radiolabeled DHBV
RNA.
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Because the 40-fold reduced level of viral DNA synthesis did not result
from a deficiency in nucleocapsid breakdown and cccDNA
formation during
initiation of infection, we examined whether
RNA-containing core
particles accumulated in the PDH infected
with the mutant. The results
showed that RNA packaging occurred
at an approximately 100-fold reduced
rate compared to that in
wild-type virus (Fig.
5C). As noted above,
packaging of the same
mutant occurred at normal levels in LMH cells,
indicating that
the defect is host cell
specific.
Implications for viral replication.
Our results showed that
mutations at a conserved cdc2 kinase-like motif on the hepadnavirus
core protein can have profound effects on DNA replication, RNA
packaging, and the stability of core particles and, hence, can support
a model predicting that this site might play a role in assembly and
disintegration of capsids. These experimental observations are in
agreement with structural data obtained with the HBV core protein
revealing the presence of hydrogen bonds between the hydroxyl of Thr
128 (corresponding to Thr 174 in DHBV) and the backbone of valine 124 and tryptophan 125, respectively (Fig. 1). These interactions stabilize
the carboxyl end of the -helix and thus dimer-dimer formation of
core subunits. Hence, changes in the local conformation around Thr 128 are predicted to destabilize the core structure, essentially as
demonstrated by our results. The proposal that this motif could relay a
signal, created as a consequence of viral DNA synthesis, from the
interior of capsids to the outside is supported by the results showing that different mutations in this motif can interfere with DNA replication (ST173/4RA and T174V) and RNA packaging (ST173/4AA), as
well as with the stability of core polypeptides (T174D and T174N)
(Table 1). Additional support for a functional role of this motif in
the viral life cycle has been provided by the observation showing that
the phenotype of the variant T174A depends on the cell line selected
for virus replication. One possible explanation is that the mutant core
protein expressed with T174A is less stable in PDHs than in LMH cells.
However, the fact that cccDNA formation was not affected by the T174A
mutant indicates that phosphorylation at this site does not play a role
in virus disassembly. Nevertheless, independent support for a role of
this region in the regulation of viral replication also comes from a
recent report by Yuan and Shih (28). These authors found
that proline 130 of HBV (Fig. 1) is involved in relaying a signal from
the interior of capsids to the outside, as a consequence of DNA
synthesis, which induces the interaction of cores with envelope components.
Although the oncogene v-
fms is phosphorylated at a Thr
residue within a cdc2 sequence motif similar to the conserved motif
in
hepadnaviruses (Fig.
1) (
18), we so far have not found any
evidence to suggest that cores expressed in LMH cells are
phosphorylated
at this site. However, because our results with variant
T174D,
mimicking phosphorylated Thr, indicated that phosphorylation of
the Thr residue could induce rapid degradation of the core protein,
our
results do not exclude a role for phosphorylation in capsid
disassembly. Phosphorylation is known to regulate assembly and
disintegration of several multisubunit structures in eukaryotic
cells.
For example, phosphorylation triggers the breakdown of
the nuclear
membrane and the fragmentation of the Golgi complex
during mitosis
(
8,
14). The role of phosphorylation in disassembly
has
found major support through the identification of the proline
isomerase
PIN1, which induces isomerization of prolines adjacent
to
phosphorylated serine or Thr residues (
9,
25). Proline
isomerization may also play a major role during the assembly and
disintegration of human immunodeficiency virus type 1, which depends
on
the presence of cyclophilin in nucleocapsids (
3,
10,
11).
In addition, the life cycle of the yeast retrotransposon Ty1 appears
to
be regulated by the kinase Fus3. Fus3 could suppress transposition
by
phosphorylation of Ty1 capsids, which, in turn, could trigger
the
degradation of the viruslike particles (
4,
24). We are
in
the process of examining the possible role of core phosphorylation
in
capsid disintegration with the help of a recently developed
in vitro
system permissive for viral DNA synthesis in
vitro.
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ACKNOWLEDGMENTS |
We acknowledge services provided by the Fox Chase Cancer Center
nucleotide sequencing facility. We thank Mike Sauder for help with
structural analyses.
This work was supported by grants from the National Institutes of
Health and by an appropriation from the Commonwealth of Pennsylvania.
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FOOTNOTES |
*
Corresponding author. Mailing address: Institute for
Cancer Research, Fox Chase Cancer Center, 7701 Burholme Ave.,
Philadelphia, PA 19111. Phone: (215) 728-4312. Fax: (215)
728-4329. E-mail: c_seeger{at}fccc.edu.
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Journal of Virology, February 2001, p. 2024-2028, Vol. 75, No. 4
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.4.2024-2028.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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