From the Laboratoire de Génétique des Virus, Gif sur Yvette, France
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ABSTRACT |
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The Hepatitis B virus encodes the secreted e
antigen (HBe) whose function in the viral life cycle is unknown. HBe
derives from a 25-kDa precursor that is directed to the secretory
pathway. After cleavage of the signal sequence, the resulting 22-kDa
protein (P22) is processed in a post-endoplasmic reticulum compartment to mature HBe by removal of the 34-amino acid C-terminal domain. The
efficiency of HBe secretion is specifically decreased in cells grown in
the presence of tunicamycin, an inhibitor of
N-glycosylation. Inasmuch as HBe precursor is not
N-glycosylated, our data suggest that a cellular
tunicamycin-sensitive protein increases the intracellular transport
through the HBe secretory pathway. The study of the secretion of HBe
derived from C-terminal-truncated precursors demonstrates that the
tunicamycin-sensitive secretion absolutely requires a part of the
C-terminal region that is removed to form mature HBe, indicating that
the cellular tunicamycin-sensitive protein increases the efficiency of
the intracellular transport of P22. We have also shown that the
Escherichia coli -galactosidase can be secreted when
fused to the HBe precursor signal sequence and that the P22 C-terminal
domain renders the secretion of this reporter protein also
tunicamycin-sensitive.
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INTRODUCTION |
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The Hepatitis B virus (HBV)1 e antigen (HBe) is found into the serum of patients suffering from acute hepatitis (1). Even though a role for infectivity or viral multiplication is excluded (2-4), conservation of this antigen during evolution (all viruses from the family encode a similar e antigen) suggests that it has an important role in the life cycle of hepadnaviruses.
HBe derives from a precursor (the precore protein) encoded by the entire HBV C open reading frame, which contains two in-frame initiation codons delimiting the pre-C sequence (87 nucleotides) and the C gene. The precore protein is translated from the pre-C AUG on the pre-C RNA, whereas the core protein (the subunit of the capsid) is translated from the C AUG on the pregenomic RNA, a slightly shorter transcript that does not include the pre-C AUG. The precore protein, a 25-kDa unglycosylated protein (P25), is directed to the secretory pathway by a 19-amino acid-long signal sequence that is cleaved during translocation into the lumen of the endoplasmic reticulum (ER) (5, 6), producing a 22-kDa protein (P22) (Fig. 1A). P22 is further processed in a post-ER compartment by removal of its C-terminal extremity (7, 8), most likely through a multiple-steps process.2 The resulting mature HBe (17 kDa) is then secreted into the blood in a monomeric form (9). The cleaved C-terminal domain of P22 is 34 amino acids long and contains 16 arginine residues arranged in clusters (arginine-rich domain) (Fig. 1B). Maturation of e antigen from precore protein is similar for two other hepadnaviruses (10, 11).
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It has been shown that the cleaved C-terminal domain of P22 is crucial for the efficiency of the HBe secretion process (12, 13). Here, we provide evidence that a cellular protein contributes to the efficiency of this process by a direct or indirect interaction with the P22 C-terminal domain.
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EXPERIMENTAL PROCEDURES |
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Plasmids--
Schematic representation of proteins referred to
in this study is shown in Fig. 2.
Proteins were expressed under the control of the adenovirus major late
promoter. Plasmids pHPC, pHPC25 and pHPC
39
have been described previously (13). For plasmids pHPC
18
and pHPC
14, stop codons were introduced, respectively, at
codons 195 or 199 of the entire C open reading frame by site-directed
mutagenesis of pHPC using polymerase chain reaction (14). Plasmid
pPC-LZ was assembled from pHPC and pGH101 (15). Plasmid pPCLZE derives from pPC-LZ and contains a stop codon at codon 1003 of the
Escherichia coli lacZ gene. Plasmids pPC-LZE-C58 and
pPC-LZE-C39 were assembled from pPC-LZ and pHPC.
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Labeling of Transfected Cells and Immunoprecipitation-- Adenovirus-transformed human embryo cells (line 293-31) (16) were grown as described (13). Cells at 80% confluency were transfected by the calcium phosphate method (17) with 30 µg of DNA/100-mm dish. Forty-eight h post-transfected cells were grown for 1 h in 10 ml of methionine-free cysteine-free Eagle's minimal essential medium (ICN), then for 3 h in 6 ml of methionine-free cysteine-free Eagle's minimal essential medium containing 500 µCi of Pro-Mix protein labeling mix (Amersham Pharmacia Biotech, specific activity >1,000 Ci/mmol). After labeling, media and cell extracts were prepared, and proteins were immunoprecipitated and analyzed as described previously (13, 18).
Time Course Experiments-- Forty-eight h post-transfected cells were grown for 1 h in 10 ml of Eagle's minimal essential medium then for 2, 3, or 5 h in 6 ml of Eagle's minimal essential medium containing 500 µCi of Pro-Mix protein labeling mix. After labeling, media and cell extracts were prepared, and proteins were immunoprecipitated and analyzed as described above.
Tunicamycin Treatment-- Cells were exposed to 6 µM tunicamycin (Boehringer Mannheim) for 6 h before methionine/cysteine depletion in 10 ml of fresh Dulbecco's modified Eagle medium. Tunicamycin was also present during depletion and protein labeling.
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RESULTS |
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HBe Secretion Decreases in Cells in Which N-Glycosylation Is Abolished-- To obtain new insights into the mechanism of P22 intracellular transport, we first examined the effect of the inhibition of N-glycosylation upon HBe secretion. Cells expressing the precore protein were grown in the presence of tunicamycin, an inhibitor of N-glycosylation. As shown on Fig. 3, the addition of tunicamycin 7 h before labeling provoked a significant and reproducible decrease in the amount of secreted HBe, whereas the amount of neosynthesized P22 was not reduced. This diminution of HBe secretion cannot be explained by a direct effect of tunicamycin on the biosynthesis of HBe inasmuch as there is no N-glycosylation consensus site in the sequence of the precore protein and must be interpreted as an indirect consequence of the inhibition of N-glycosylation.
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The Action of the Cellular Tunicamycin-sensitive Protein Requires
the P22 C-terminal Region--
As the C-terminal domain of P22 is
important for HBe secretion (12, 13), it was tempting to speculate that
TSP would interact with this region. To test this hypothesis, we first
determined if the inhibition of N-glycosylation would still
adversely affect the secretion of HBe derived from precursors truncated
at different positions3
within the C-terminal part: P2514, P25
18,
P25
25, and P25
39 (Fig. 2A). As
shown in Fig. 6, tunicamycin treatment
reduced the level of secretion of HBe, HBe
14 and
HBe
18 but not that of HBe
25 or
HBe
39. In this particular experiment, HBe
18
appears to migrate slightly slower when synthesized in the absence of
tunicamycin, and HBe
25 appeared as two fuzzy bands when
tunicamycin was present. These slowest migrating bands most likely
correspond to non-fully mature HBe
25 molecules, which are
sometimes observed in pHPC
25-transfected cells, even in
the absence of tunicamycin. However, these results show that a region
located upstream of position
18 (relative to the P22 C terminus) is
crucial for tunicamycin sensitivity of HBe secretion. This strongly
suggests that the C terminus of P22 facilitates an interaction with
TSP. Furthermore, as HBe
25 and HBe
39 were
still secreted in the presence of tunicamycin, the action of TSP, even
if it is required for optimal HBe secretion, is dispensable.
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DISCUSSION |
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Our study shows that the inhibition of N-glycosylation
in cells expressing the HBe precursor led to a significant decrease in
HBe secretion, an unexpected finding since the HBe precursor contains
no glycosylation consensus sites. A general effect of tunicamycin on
the cellular secretion machinery is ruled out as the secretion of
HBe25, HBe
39, and PCLZ was not adversely
affected by the inhibitor. Thus, our findings are consistent with the
idea that a cellular protein increases specifically HBe secretion and
that the activity of this protein (TSP) depends upon the
N-glycosylation status of the cell.
The molecular mechanism by which TSP increases HBe secretion remains to
be determined, although we demonstrate that the tunicamycin sensitivity
of secretion absolutely requires a part of the P22 C-terminal domain, a
region absent in mature HBe. First, P22 truncations larger than 18 amino acids abolished tunicamycin sensitivity, suggesting that the
sequence upstream of position 18 is important for the interaction
with TSP. Second, the 39 C-terminal amino acids of P22 conferred
tunicamycin sensitivity on PCLZEC39. Taken together, these results
demonstrate that a sequence located between amino acids
19 and
39
is involved in the interaction with TSP. However, since in PCLZEC39
both the N- and C-terminal domains of P22 are present, we cannot
exclude the possibility that the P22 N terminus may also be involved in
this interaction, a possibility not supported by reports on the native
structure of HBe (7, 21) and
-galactosidase (22).
How might tunicamycin affect the activity of TSP? The simplest
explanation is that TSP is only active when N-glycosylated. Another possibility is that its activity is indirectly affected by
tunicamycin. TSP would require the involvement of a
N-glycoprotein for its correct folding, processing,
subcellular localization, or activity. The next question is how does
such a protein function. TSP could be a chaperone that favors the
folding of P22, increasing the amount of P22 that exits from the ER and
is further transported to the cell surface. The first possibility is
that the sequence 19 to
39 promotes the binding of TSP in a direct
manner. This seems unlikely since it is generally assumed that, to
recognize unfolded proteins, chaperones interact with exposed
hydrophobic residues, free exposed sulfhydryl groups, or partially
glucose-trimmed oligosaccharides (23, 24), features that are not
present in the sequence
19 to
39 (see Fig. 1B). In
particular, we can exclude a direct binding of BiP to this sequence as
it has been shown that BiP preferentially binds a heptameric consensus
motif containing a subset of aromatic and hydrophobic residues in
alternating positions (25, 26), a motif not present in the sequence
19 to
39. Similarly, binding of calnexin can also be ruled out, as
this chaperone is a lectin that recognizes specifically partially
trimmed, monoglucosylated N-linked oligosaccharides
(27-29). Involvement of the protein disulfide isomerase can also be
excluded, as this chaperone requires hydrophobic interactions and
formation of disulfide bonds (30). Alternatively, the sequence
19 to
39 may promote the binding of TSP to P22 in an indirect manner. This
sequence would slow the folding of P22 and thus would indirectly
promote the binding of TSP to a folding intermediate, allowing proper folding to proceed.
Whatever TSP binds to the sequence 19 to
39 or to another region of
P22, abolishment of the N-glycosylation could reduce its
chaperone activity as envisaged above. Another possibility is that
treatment of cells with tunicamycin for 7 h, which is known to
increase significantly the level of ER chaperones, leads to an
increased level of TSP, which could consequently slow the export of P22
from the ER as demonstrated with GRP78/BiP for the secretion of
different proteins (31-33).
Alternatively, TSP could be a "cargo receptor." Transported (or cargo) proteins are carried from one compartment to the next by small coated vesicles (34) and packaged into these vesicles by nonselective diffusion, a default pathway termed "bulk-flow" (35). Recent data have shown that, in contrast, some cargo proteins are specifically concentrated into vesicles, in particular at the exit from the ER (36-38). In this selective-transport model, sorting of soluble proteins would be mediated by specialized transmembrane cargo receptors. It is tempting to speculate that TSP would play such a role, mediating the packaging of P22 at one step of its vesicular transport, therefore increasing HBe secretion. So far, none of our data either support or argue against this hypothesis.
The role of HBe in the viral life cycle is still unknown (39 for review). However, recent data have shown that P22 down-regulates the level of HBV replication (40-43) by means of cytosolic P22 molecules (6, 42, 43) that most likely associate with core proteins in unstable capsids (43). Thus, one might speculate that the relative levels of P22 returned to the cytosol and P22 transported to the cell surface should be controlled and that TSP could be an actor in this repartition. Whether TSP intervenes in vivo during natural HBV infection remains to be determined.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr. I. B. Holland for critical review of the manuscript and helpful discussion. We thank Dr. S. Salhi for her critical reading of the manuscript. Thanks are also due to M. T. Bidoyen for performing the routine cell culture.
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FOOTNOTES |
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* This work was supported by a grant from the Association pour la Recherche sur le Cancer.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.
Supported by a training fund from the Ministère de
l'Education Nationale, de l'Enseignement Supérieur et de la
Recherche.
§ Present address: Laboratoire d'Hygiène de la Ville de Paris, 11 rue George Eastman, 75013 Paris, France.
¶ To whom correspondence should be addressed: Laboratoire de Génétique des Virus, CNRS-UPR 9053, Avenue de la Terrasse, 91198 Gif sur Yvette cedex, France. Tel.: 33 1 69 82 38 47; Fax: 33 1 69 82 43 08; E-mail: jmrossi{at}gv.cnrs-gif.fr.
1 The abbreviations used are: HBV, hepatitis B virus; ER, endoplasmic reticulum; HBe, hepatitis B virus e antigen; PAGE, polyacrylamide gel electrophoresis; TSP, tunicamycin-sensitive protein.
2 F. Messageot and J.-M. Rossignol, unpublished observations.
3
Truncated proteins are named according to the
number of removed amino acids: for example, P2514 is the
precore protein truncated of the 14 last amino acids, and
P22
14 and HBe
14 are, respectively, the
corresponding truncated P22 and mature HBe. Please note that
HBe
39 is identical to P22
39 and is 5 amino
acids shorter than mature HBe.
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REFERENCES |
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