From the Department of Molecular
Microbiology and Immunology and from the § Saint
Louis University Liver Center, Saint Louis University School of
Medicine, St. Louis, Missouri 63104
Received for publication, August 30, 2002, and in revised form, October 15, 2002
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ABSTRACT |
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Hepadnaviral reverse transcription occurs
in subviral capsids in which the core protein surrounds the reverse
transcriptase ("polymerase") and the pregenomic RNA. The pregenomic
RNA is the template for reverse transcription and also the bicistronic
mRNA for core and polymerase. The pregenomic RNA structure and the capsid stoichiometry imply that vastly more core would be translated than polymerase. Previously, we found that duck hepatitis B virus polymerase unexpectedly accumulates in the cytoplasm (Yao, E., Gong,
Y., Chen, N., and Tavis, J. E. (2000) J. Virol.
74, 8648-8657). The production mechanism and function of the excess
polymerase are unknown. Here, we determined the kinetics of expression
and degradation of polymerase and core in cells producing virus.
Polymerase was translated 10% as rapidly as core, the half-life of
nonencapsidated polymerase was very short, core had a very long
half-life, and very few polymerase molecules were encapsidated. The
presence of excess polymerase indicates that the translation rate of
the polymerase is not limiting for encapsidation. Therefore,
encapsidation must be regulated by other events, most likely binding of
the polymerase to the pregenomic RNA. These data support the hypothesis that polymerase may have functions beyond copying the viral genome by
demonstrating that the polymerase is a cytoplasmic protein that
is only rarely encapsidated.
Hepadnaviruses are small DNA-containing viruses that replicate by
reverse transcription. Human hepatitis B virus is a major cause of
liver disease and liver cancer world-wide (1), and other hepadnaviruses
can be found in woolly monkeys, rodents, and birds (2-4). All
hepadnaviruses share a high degree of hepatotropism, are highly
species-specific, employ the same replication cycle, and share a nearly
identical genetic organization. These viruses have an envelope studded
with viral glycoproteins that surrounds an icosahedral core particle.
The shell of the core particle is formed from the viral
C1 protein, and it contains
the viral nucleic acids and the viral reverse transcriptase, known as
the P protein.
Hepadnaviral reverse transcription occurs in cytoplasmic core
particles. Core particle formation begins with binding of P to the C accumulates in cells to easily detectable levels that rise steadily
over the first few days of expression in liver or in cultured cells.
The accumulation pattern of P has never been directly measured because
of the lack of appropriate antibodies. Expression of P is believed to
be very low for three reasons. First, P is translated from the
downstream ORF of the bicistronic pgRNA. Such ORFs are usually very
poorly translated, especially when the context of the initiation codon
(17) is suboptimal and there are multiple upstream AUGs, as is the case
for P. Second, the best estimate indicates that only one or two P
molecules are within each core particle (18), and thus little P would
be needed relative to C for assembly of core particles. Third, using
kinase epitope-tagged P, it has been proposed that P is a translational
repressor (18). These observations led to the idea that slow
translation of P is the rate-limiting step in encapsidation (18).
However, using specific antibodies for P, we found that DHBV P
accumulates in the cytoplasm to detectable levels and that the majority
of P in cells is not encapsidated (19). Therefore, much more P
accumulates in cells than was anticipated. The mechanism of the
accumulation of this excess P and its biological function are unknown.
P could be translated slowly and have a very long half-life.
Alternatively, P could be translated rapidly relative to the demands of
P for encapsidation. In any case, the kinetics of P biosynthesis and
turnover could play an important role in viral assembly and thus on
viral replication, pathology, or antigen presentation by infected cells.
To understand the kinetic mechanism for the expression of excess DHBV P
in cells, we performed a quantitative analysis of P and C expression
and degradation. These experiments are the first such
experiments to directly analyze P using specific antibodies rather than
employing surrogate reporter genes, and thus this is the first analysis
of the synthesis and degradation of P in the context of ongoing reverse transcription.
Virus and DNA Constructs--
DHBV Type 3 was employed (20).
D1.5G is an overlength DHBV3 expression construct containing a
5' duplication of nucleotides 1658-3021 in pBluescript( Cell Culture, Transfection, and Infection--
LMH cells were
maintained in DMEM/F12 supplemented with 10% fetal bovine serum. Cells
were transfected with FuGENE 6 (Roche Molecular Biochemicals) as
recommended by the manufacturer.
Lysate Preparation, Western Blotting, and
Immunoprecipitation--
LMH cells were lysed in 0.75× or 1×
radioimmune precipitation assay buffer (1× is 20 mM Tris,
pH 7.2, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 150 mM NaCl) containing 1 mM phenylmethylsulfonyl fluoride and 3 µg/ml leupeptin on ice for 10 min, and the lysates were clarified at 14,000 × g for 10 min at
4 °C.
For immunoprecipitation, antibody was bound to protein A/G-agarose
(Oncogene Research Products) and incubated with lysates at
4 °C overnight. Immunocomplexes were washed three times with 1 ml of
0.75× or 1× radioimmune precipitation assay buffer, and proteins were
released with Laemmli buffer. Samples were resolved by SDS-PAGE and
detected by PhosphorImager analysis or Western blotting.
For Western blotting, proteins were resolved by SDS-PAGE and
transferred to Immobilon-P (Millipore) membranes. P was detected with
anti-DTP3'-His monoclonal antibody mAb9. Following incubation with the
appropriate IgG-alkaline phosphatase conjugate (Promega), P was
visualized by incubation with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Promega) according to the manufacturer's instructions.
Metabolic Labeling and Pulse-chase Determination of Half-life of
P and C--
Transfected LMH cells were washed twice with DMEM lacking
methionine and cysteine (labeling medium) and pulsed with labeling medium supplemented with 120 µCi/ml EasyTag Express
(PerkinElmer Life Sciences) and 1% fetal bovine serum. For pulse-chase
experiments, cells were labeled for 15 min (for P) or 3 h (for C),
rapidly washed twice with DMEM/F12, and then fed with DMEM/F12
containing 10% fetal bovine serum. Great effort was taken to perform
these steps as rapidly as possible, and all media were equilibrated to
37 °C/5% CO2 prior to use. Cells were incubated at
37 °C/5% CO2 for varying times and then placed on an
ice-water slurry and lysed for immunoprecipitation. Following SDS-PAGE,
radioactive P or C was detected and quantitated by PhosphorImager analysis.
Calculation of the Molar Translation Ratio of P and
C--
The molar translation ratio of P and C (P/C; moles of P
min The Half-lives of P and C Are Vastly Different--
The half-life
of P was determined by transfecting LMH cells with DHBV expression
constructs and performing pulse-chase experiments at day 1 or 3 after
transfection. There are no cell lines infectable by DHBV, but
transfection of LMH chicken hepatoma cells with expression constructs
for DHBV (e.g. D1.5G) produces viral core particles competent for reverse transcription and results in release of infectious virions from the cells (22). Transfected LMH cells were
metabolically labeled with [35S]methionine/cysteine for
15 min, washed twice with non-radioactive medium, supplied with
non-radioactive medium containing 10% fetal bovine serum, and
incubated for various times. At each time point, cells were lysed, P
was immunoprecipitated, and radioactivity in P was determined by
phosphorimage analysis. This assay measures the half-life of
nonencapsidated P because encapsidated P cannot be precipitated with
anti-P antibodies (19). A representative experiment for
wild-type D1.5G-transfected cells at day 3 after transfection is shown
in Fig. 1. Table I shows that
nonencapsidated P had a very short half-life
of 25 min at day 1 and 15 min at day
3.
We were unable to measure the half-life of C because it was
exceptionally stable. A representative experiment with
D1.5G-transfected cells at 3 days after transfection is in Fig. 1.
Chase periods as long as 29 h revealed no reproducible diminution
of the radioactivity immunoprecipitated. The amount of radioactive C
immunoprecipitated in pulse-chase experiments could be reduced by
secretion of virus particles or by degradation of C. The persistence of
radioactive C indicates that C is stable in this system and that its
degradation and secretion rates can be ignored in kinetic analyses. The
negligible secretion rate is consistent with the low rate of production
of mature viruses from LMH cells at early times after transfection (data not shown).
P Is Translated at 10% the Rate of C--
P and C are translated
from the bicistronic pgRNA (11-14). The downstream position of the P
ORF on the pgRNA, the suboptimal Kozak context (17) of the DHBV P AUG
(UAUAUGG), and the presence of 15 AUG codons between the
mRNA cap and the P AUG (four of which are in identical or
equivalent Kozak contexts to the P AUG) lead to the prediction that P
would be translated a great deal less frequently than C. To test this
prediction, LMH cells were transfected with a wild-type DHBV expression
vector (D1.5G) and labeled with [35S]methionine/cysteine
for 1.5 or 4 h prior to lysis at 1 or 3 days after transfection. P
and C were immunoprecipitated from equal fractions of the lysates, and
radioactivity incorporated into the proteins was measured by
PhosphorImager analysis of the gels exposed simultaneously to the
same plate. A representative experiment is shown in Fig.
2.
The total amounts of P and C synthesized during the labeling period
were calculated from the radioactivity immunoprecipitated, the labeling
time, the half-life of P (25.3 min for day 1 or 15.2 min for day 3;
Table I), and the numbers of methionine and cysteine residues in P and
C. Dividing the total amount of P by the total amount of C yielded a
molar P/C translation ratio of 0.10 ± 0.020 at day 1 and
0.11 ± 0.001 at day 3 (Table II). A
measure of the validity of this approach is that it should be
insensitive to the length of the labeling period, and indeed, the
values for the day 1 P/C ratio in Table II reveal little difference
when the labeling time was 1.5 or 4 h. Therefore, P is translated
only 10-fold less rapidly than C, despite the downstream location of the P ORF on the bicistronic mRNA, the suboptimal Kozak context of
the P AUG, and the presence of 15 other AUGs upstream of the P
AUG.
P Is Rarely Encapsidated--
The level of nonencapsidated P in
cells exceeds the amount of encapsidated P by 3-fold at day 3 after
transfection (19). Because encapsidation greatly stabilizes P, this
implies that very few P molecules are encapsidated. We tested this
hypothesis by examining the decay kinetics of P using
mutations to DHBV that blocked encapsidation.
The disappearance of P in pulse-chase experiments measured by
immunoprecipitation could occur by either encapsidation or degradation. If the rates of encapsidation and degradation are appreciably different, and if both processes affect significant fractions of P,
then the decay curve of nonencapsidated P would be biphasic. To
determine whether disappearance of nonencapsidated P is monophasic or
biphasic, LMH cells were transfected with DHBV or core-deleted DHBV
(DHBV(dlCore)) expression constructs, the cultures were metabolically labeled with [35S]methionine/cysteine for 15 min, and
pulse-chase experiments were performed with time points as close as 4 min apart. Representative half-life experiments are shown in Fig.
3.
The decay profile for nonencapsidated P was monophasic when P was
expressed from the wild-type genome on day 1 after transfection, shortly after encapsidation begins (Fig. 3A; linear
correlation coefficient of 0.973). The decay profile for
nonencapsidated P from the wild-type genome was also linear at day 3 after transfection, when the encapsidation reaction is well established
(Fig. 3B; linear correlation coefficient 0.979). Next, we
measured the decay profile of nonencapsidated P expressed from
DHBV(dlCore), where encapsidation is blocked because C is not produced.
The decay profile at day 3 after transfection for nonencapsidated P
expressed from DHBV(dlCore) was also linear (Fig. 3C; linear
correlation coefficient 0.980). These data indicate that the decay of
nonencapsidated P is monophasic and that the turnover rates of P are
the same from wild-type and encapsidation-defective DHBV. Therefore,
the fraction of P molecules that becomes encapsidated is too small to
affect the decay kinetics of P.
These data reveal that the kinetic mechanism for the accumulation
of excess P is rapid translation coupled with inefficient encapsidation
and that the steady-state level of P in the cells is limited by the
short half-life of P. These experiments are the first to analyze P in
the context of active reverse transcription because previous
quantitative analyses of the synthesis or degradation of P relied on
in vitro systems or employed replication-deficient constructs in which P was replaced by a marker gene (11, 14, 23-28).
DHBV P was translated from the pgRNA at 10% the rate of C
(i.e. P/C = 0.1). This relatively high rate is similar
to the P/C rate of 0.25-0.33 that was determined using a P-LacZ
fusion gene initiating at the P AUG (23). The P/C translation rate for
HBV has been measured at 0.1 by employing a reporter gene for HBV P
(24), indicating that rapid synthesis of P is probably conserved between the avian and mammalian hepadnaviruses. The unusually high
translation rate of P and the overlap between the P and C ORFs raises
the possibility that translation of the P and C ORFs could interfere
with each other. Given that the P AUG is probably located by the
ribosomes through an unusual mechanism (14, 26), we suspect the pgRNA
may be segregated into two translational pools. One of these pools
would produce C, and the other would produce P. Because encapsidation
appears to occur in cis (i.e. P binds to the same mRNA
molecule from which it was translated (6)), this implies that those
pgRNA molecules that produce C would not be encapsidated.
Three observations indicate that the proportion of P molecules that
become encapsidated is very small. First, blocking encapsidation did
not affect the decay profile of P or the intracellular accumulation of
P (Fig. 3), and thus the proportion of P that was encapsidated must be
much smaller than the proportion that was not encapsidated. Although we
cannot directly determine the level of encapsidation that would be
required to leave a mark on the decay pattern of nonencapsidated P, we
estimate that the sensitivity limit of these assays would be about
10-20% of the total P synthesized. Second, we previously demonstrated
that the amount of nonencapsidated P exceeds the amount of encapsidated
P in cells by 3-fold at day 3 after transfection (19). Here, we found
that nonencapsidated P has a half-life of only 15 min, whereas
encapsidated P must be as stable as the core particle, which has a
half-life of many hours to days. This indicates that the fraction of P
molecules that are encapsidated must be considerably below the 10-20%
estimated detection limit of our kinetic assays. Third, much more P was made than the minimum needed to assemble core particles. At day 3 after
transfection in LMH cells (when encapsidation is well established), P
is synthesized 10% as rapidly as C. However, core particles contain
240 C molecules (8, 9) and 1-2 P molecules (18), so the P translation
rate was 12-24-fold higher than the minimum required to supply P for
core particles.
The kinetic mechanism for the metabolism of P is in Fig.
4. In this mechanism, newly synthesized P
(PNew) is made from amino acids. PNew can
either be encapsidated (PE) or interact with cellular components as nonencapsidated P (PNE). Secretion of capsids
containing PE results in viral P (PVirus). The
rates important to the data presented here are the translation rate of
P (KSynth), the encapsidation rate
(KEncap), the rate of production of the
cell-associated PNE (KNE), and the
degradation rate of PNE (KDeg). This
mechanism shows that at steady-state KNE = KDeg and that KNE and
KEncap are in competition with each other. Our
results indicate that KSynth is unanticipatedly
large, that
KNE
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
stem loop at the 5' end of the viral pgRNA (5-7). This complex is then
encapsidated through polymerization of 120 dimers of C around it (8,
9). Encapsidation appears to be rapid because no intermediates between
dimers of C and complete particles have been found in cells (10).
Expression of the three viral products needed for encapsidation (pgRNA,
C, and P) is closely linked because the pgRNA is also a bicistronic
mRNA that encodes C and P (11-14). C and P are both translated by
de novo initiation from their ORFs on the bicistronic pgRNA
(15, 16).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
)
(Stratagene). Mutations introduced into D1.5G were dlCore (deletion of
nucleotides 2846-2849 that truncates C and blocks encapsidation); P-OF
(deletion of nucleotide 424 that truncates P after amino acid 84 (15)); and dlBulge (deletion of nucleotides 2571-2576 that removes the
bulge of
and blocks binding of P and encapsidation (21)).
1/moles of C min
1) was calculated by
dividing the total amount of P made during the labeling period by the
total amount of C made during the same period. Because the half-life of
C in LMH cells is too long to be measured (i.e. C does not
degrade appreciably), all label that was incorporated into C was
detected by immunoprecipitation. Determining the total amount of P
synthesized presented two problems. First, P has more methionine and
cysteine residues than C, and thus it is labeled to a higher molar
specific activity. Therefore, the amount of radioactivity incorporated
by P was divided by 8.6 to correct for the differing numbers and
proportions of methionine and cysteine residues in the two proteins.
Second, the half-life of P is very short, so the large majority of P
synthesized over the labeling period was degraded and was not detected
by immunoprecipitation. We corrected for this difference by calculating
the total amount of P synthesized from its half-life and the amount of
radioactivity detected in P by immunoprecipitation. The first-order
decay equation is y(t) = y0e
t, where
y0 is the instantaneous synthesis rate,
is
the decay constant, and t is decay time. The radioactivity
detected by immunoprecipitation (A) is the area under the
decay curve for the labeling period (T), as shown in
Equation 1.
The solution to this equation is
y0 = A/[(1/
(Eq. 1)
)-(1/
)e
T].
Solving for
in terms of half-life (T1/2) yields
=
ln(0.5)/T1/2. The
instantaneous translation rate, y0, was
calculated from these equations. We then assumed that the translation
rate remained constant during the labeling period and multiplied
y0 by T to yield the total amount of
P synthesized.
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ABSTRACT
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DISCUSSION
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Fig. 1.
Representative pulse-chase experiments to
determine T1/2 for P and C. LMH cells
were transfected with D1.5G. At day 3 after transfection, the cells
were labeled for 15 min (for P) or for 3 h (for C) with
[35S]methionine/cysteine and incubated in non-radioactive
medium for the indicated times, and then the cells were lysed, and P or
C was immunoprecipitated.
Halflife of nonencapsidated P
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Fig. 2.
Measurement of 35S
incorporation for calculation of the P/C molar translation ratio.
LMH cells were transfected with D1.5G, D1.5G(P-OF), or D1.5G(dlCore)
and labeled with [35S]methionine/cysteine for 1.5 h
prior to lysis on either day 1 or 3 after transfection. P and C were
immunoprecipitated from equal aliquots of the lysate and were resolved
by SDS-PAGE. Incorporated 35S was quantitated by
PhosphorImager analysis, and the raw quantitation values for the
experiment shown are given in phosphorimaging units.
Molar P/C translation rates
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Fig. 3.
P decays with monophasic kinetics. LMH
cells were transfected with DHBV expression constructs, and 1 or 3 days
later, the stability of the nonencapsidated P was determined by
pulse-chase analysis. P was quantitated by PhosphorImager analysis.
DHBV(dlCore) is encapsidation-deficient.
DISCUSSION
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ABSTRACT
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RESULTS
DISCUSSION
REFERENCES
KEncap, and that
KDeg is large and monophasic.
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Fig. 4.
Kinetic mechanism for metabolism of P. P
is translated from amino acids (AA) to produce
PNew. PNew can then either interact with
cellular components to become nonencapsidated P (PNE) or be
encapsidated (PE). Secretion of capsids containing
PE results in virion-associated P (PVirus). The
flow of P through this mechanism is controlled by the translation rate
(KSynth), the rate of association of
PNew with cellular components (KNE),
the rate of encapsidation of PNew
(KEncap), the rate of degradation of
PNE (KDeg), the rate of
disintegration of capsids (KDisint), and the
secretion rate of core particles as virions (KSec).
The half-life of P drops from 25.3 ± 4.0 min at day 1 after transfection to 15.2 ± 1.5 min on day 3 after transfection (Table I). Because the half-life of a protein is inversely proportional to its degradation rate, this 40% reduction in the stability of P implies that degradation of P is regulated. The reduction in stability of P is independent of the encapsidation reaction (Table I), indicating that the destabilization of P is not controlled by the level of encapsidation. The mechanism for the turnover of P is unknown, but it may involve the ubiquitin/proteosome pathway because addition of the proteosome inhibitor MG-132 increases the steady-state level of P in cells 2-3-fold (data not shown).
These data disprove the accepted model that slow translation of P is
limiting for encapsidation (18). Because encapsidation requires binding
of P to on the pgRNA (5-7), our data imply either that the
intracellular concentration of P and the pgRNA combined with the P:
binding constant do not favor P:
binding or that only a small amount
of P ever binds to the cellular chaperones that are needed for P to
bind to
(29-31).
The large amount of cytoplasmic P must be the source of antigen
resulting in the strong and early immune response to P, which can be as
rapid and strong as the response to C, a major viral structural protein
(32-35). Production of this excess of P is a liability to the virus
through induction of additional immune pressure. The hepadnaviral
replication strategy is to establish a persistent infection lasting
decades, and the presence of an avoidable liability such as production
of a large excess of P implies that the nonencapsidated P may be
important for the virus. We have speculated that P may play a
regulatory role on cellular or viral functions in addition to
synthesizing the viral genome (19). These results support this
hypothesis by demonstrating that P is primarily a cytoplasmic protein
that is only occasionally encapsidated.
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ACKNOWLEDGEMENTS |
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We thank William Mason and Jesse Summers for the gifts of anti-C antibodies. We thank Abdul Waheed, Paul Fishback, Ranjan Srivastava, Heinz Schaller, and Jesse Summers for helpful discussions.
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FOOTNOTES |
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* This work was supported by Grants AI38447 and CA91327 from the National Institutes of Health (to J. E. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8441; Fax: 314-773-3403; E-mail: tavisje@slu.edu.
Published, JBC Papers in Press, November 19, 2002, DOI 10.1074/jbc.M208895200
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ABBREVIATIONS |
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The abbreviations used are:
C, core
protein;
P, polymerase protein (reverse transcriptase);
HBV, human
hepatitis B virus;
DHBV, duck hepatitis B virus;
D1.5G, plasmid
expression vector for DHBV;
DMEM, Dulbecco's modified Eagle's medium;
DMEM/F12, a 1:1 mixture of DMEM and F12 medium;
LMH, chicken hepatoma
cells;
ORF, open reading frame;
pgRNA, pregenomic RNA;
, the stem
loop structure on the pgRNA to which P binds;
T1/2, half-life.
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