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INTRODUCTION |
The transmembrane topology of most eukaryotic polytopic proteins
is established cotranslationally at the endoplasmic reticulum (ER)1 membrane and is
maintained during subsequent steps of folding and transport. The
biogenesis of these proteins involves a series of coordinated
translocation and membrane integration events that is directed by
topogenic determinants within the nascent chains and that ultimately
leads to a uniform topology for any given polypeptide (1, 2). In recent
years, however, it has become evident that certain cellular polytopic
proteins exhibit variations in biogenesis such that two or more
distinct topological orientations are generated (3, 4). For example,
the multidrug resistance P-glycoprotein (5), the transporter ductin
(6), and the aquaporin-1 water channel protein (7) have been observed
to be expressed in alternate topological forms with the diversity apparently generated at the time of translocation at the ER membrane. Because different orientations seem to serve different functions (6,
8), it is less surprising that viral proteins such as the hepadnavirus
large L envelope protein exploit a similar strategy of topological
heterogeneity (9-13), likely to optimize the generally limiting
information potential of virus genomes.
The hepatitis B virus (HBV) L envelope protein with its pre-S domain
plays vital roles in the viral life cycle by mediating receptor binding
during host cell attachment, by performing a matrix-like function in
nucleocapsid envelopment, and by exerting various regulatory functions
(14-16). The multifunctional nature of L depends on its dual
transmembrane topology that is established after the completion of
polypeptide synthesis via an as yet uncharacterized mechanism (9, 11,
12). Upon biogenesis, L as well as two closely related homologues, the
middle (M) and small (S) envelope proteins, are expressed from a single
open reading frame of the viral genome by means of three different
start codons that are spaced at intervals of 108 (or 119, depending on
subtype) and 55 codons. Therefore, the 226-amino acid sequence of S is
repeated at the C termini of M and L, which carry the additional pre-S2 domain or pre-S2 and pre-S1 domains, respectively (Fig. 1A)
(17). All three proteins are cotranslationally integrated into the ER membrane, likely directed by the action of a signal(anchor) and a
stop-transfer sequence encoded within the first and second
transmembrane (TM) segments (TM1 and TM2) of their S domains (18, 19).
Two further membrane-spanning segments are predicted in the C-terminal third of the S domain (Fig. 1B) (20, 21); it has not been established whether these putative TM3 and TM4 segments contain topogenic information. In contrast, the hydrophilic pre-S1 and pre-S2
domains have been shown to lack any signal sequence activity (11, 22).
Hence, M and L are translocated into the ER membrane by the proximal
signals of their S domains that also govern cotranslational translocation of the upstream pre-S2 region of M into the ER lumen (Fig. 1B) (11, 22). Conversely, the pre-S2 plus pre-S1
(pre-S) domain of L fails to be translocated and thus faces the
cytosolic side of the ER membrane in nascent chains. During maturation, approximately half of the L molecules post-translationally translocate the pre-S region into the microsomal vesicle lumen, thereby generating a dual topology that is maintained in the secreted virion envelope (Fig. 1B) (9, 11, 12). By disposing the pre-S domain either at a cytosolic (i.e. inside the virus) or luminal
(i.e. outside the virus) location, L seems to serve its
topological conflicting functions such as capsid envelopment and
receptor binding, respectively.
In search for the mechanism(s) leading to this highly unconventional
split mixed topology and the cellular factors involved, we previously
identified the pre-S1-specific amino acid (aa) sequence 70-94 of L as
a bona fide translocation control region, because deletion
of this sequence leads to cotranslational pre-S translocation and a
uniform topology (23). This sequence specifically mediates the binding
of L to the cognate heat shock protein Hsc70 (23), suggesting this
chaperone to be responsible for the repression of cotranslational pre-S
translocation while simultaneously keeping the trapped pre-S domain in
a configuration competent for post-translational reorientation. How the
hydrophilic pre-S domain traverses the lipid bilayer is still unknown,
but it is postulated to occur through the complexing of HBV-envelope
subunits into an aqueous channel (10, 20, 21, 24). The formation of
such a membrane channel has been proposed to depend on dimerization of
S, M, and/or L chains concomitant with lateral interactions between
their three amphipathic TM1, TM3, and TM4 segments, which pack together
thereby shielding their hydrophilic faces from the lipid (20, 21). According to computer modeling, such a channel, if being composed of
six TM helices, could reach a diameter of 1.5 nm (20) and hence would
allow the transmembrane passage of pre-S, thereby circumventing the
need of, for example, the host cell ER translocon.
In this study, we explored structural and compartmental requirements of
the post-translational pre-S translocation of L with special regard to
the hypothetical HBV-specific channel serving as a potential pre-S conduit.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
The mammalian expression vector
carrying the HBV L gene (pNI2.L) under the transcriptional control of
the human metallothionein IIA promoter has been described (12). For
epitope tagging, an influenza virus hemagglutinin (HA) epitope
(YPYDVPDYASL) was fused in frame to the C terminus of L by
site-directed mutagenesis using a recombinant M13mp19.HBV bacteriophage
and the antisense oligonucleotide 5'-GTTTTGTTAGGGTTTATAAGCTAGCGTAATCGGTAAATCGTATGGGTAACATATGTACCCAAAG-3'. Simultaneously, an NdeI restriction site was
introduced upstream of the HA tag to enable subsequent cloning steps
(in the primer sequence, the HA-specific sequence is underlined, and
mismatches caused by the engineered NdeI site are in
boldface). The resulting HA-tagged L gene was employed for ectopic
protein expression and further plasmid constructions.
To sequentially delete TM segments from the C terminus of L, two
mutants were constructed by excising a BamHI (nucleotide (nt) position 470, as referred to the HBV genome, subtype
ayw)-NdeI (nt 830) fragment, or a BssHII (nt
387)-NdeI (nt 830) fragment from pNI2.L. After appropriate
modification of the ends, plasmid DNAs were religated, thereby
generating pNI2.L
TM3/4 and pNI2.L
TM2/3/4, respectively, that
lacked either the last two or three TM segments. For the in-frame
deletion of TM1, a StyI (nt 180)-XbaI (nt 249) fragment was removed in mutant L
TM1. The precise deletion of TM2
(L
TM2) was achieved by M13 mutagenesis with two antisense oligonucleotides,
5'-GAAGATGATTAATCGCCGCAG-3'
and 5'-TGATAGTCGACAGGTACCAACAAG-3'
(restriction sites are underlined, and mismatches are in boldface),
that introduced an AseI (nt 395) and a HincII (nt
450) restriction site at the 5' and 3' borders of TM2, respectively,
and subsequent removal of the AseI-HincII
fragment. Mutant L
TM1/3/4 was generated by replacing an
MscI (nt 2972)-MscI (nt 305) fragment of plasmid pNI2.L
TM3/4 by the correspondingly fragment of pNI2.L
TM1. To substitute TM segments by foreign hydrophobic domains, a hybrid protein
was constructed that carried the pre-S domain fused to the N-terminal
half of the M envelope protein of coronavirus mouse hepatitis virus
strain A59 (MHV). Therefore, an NdeI site was introduced at
codon positions 81/82 of the MHV M gene, present in plasmid pTZ19R-M (a
generous gift from P. J. M. Rottier), by polymerase chain reaction
mutagenesis using the primer
5'-AAATCCAAGATACATATGATTTAGCGC-3' together with the primer 5'-ACTCTAGAGGATCCCTCGAGGAAATCT-3',
annealing to the 5'-noncoding region of the M gene. The polymerase
chain reaction product obtained was then used to replace the
XhoI (nt 127)-NdeI (nt 830) fragment of plasmid
pNI2.L, generating the construct pNI2.L::MHV that encoded the
first 155 aa of L, an 8-residue spacer (SRKSNPNI), and the N-terminal
82 aa of the MHV M protein, followed by the HA tag. To abolish
concomitant expression of the HBV M and S envelope proteins from the L
open reading frame, their translational start codons
(Met109 and Met164) (Fig. 1A) were
changed to threonine residues (pNI2.Lo) as described (25).
Subsequently, most deletion mutants described above were introduced
into this Lo background.
Cell Culture, Transfection, Trypsin Protection Assay, and Western
Blotting--
To produce viral envelope proteins, transient
transfection of COS-7 cells by electroporation was used. Two days
post-transfection, microsomes were prepared and subjected to the
trypsin protection assay essentially as described (12). Briefly, cells
were disrupted by dounce homogenization, and microsomes were recovered
by ultracentrifugation prior to proteolysis with trypsin (25 µg/ml) in the presence or absence of 0.5% Nonidet P-40.
After incubation on ice for 60 min, trypsin was inactivated by the
addition of 30 µg/ml aprotinin. Each sample was then
adjusted to 0.5% Nonidet P-40 and solubilized for 20 min on ice. After
centrifugation for 5 min at 13,000 × g and 4 °C,
proteins of the supernatants were precipitated with 10%
trichloroacetic acid (TCA) and washed twice with 5% TCA and once with
acetone. TCA precipitates were resolved by SDS-PAGE and Western blotted
to nitrocellulose membranes. Immunoblots were incubated with a mouse
monoclonal antibody against the HA epitope (Babco), diluted 1:1000 in
blotting buffer (phosphate-buffered saline with 5% skim milk) and
peroxidase-labeled goat anti-mouse secondary antibody (Dianova) (1:5000
dilution in blotting buffer), and developed with enhanced
chemiluminescence detection reagents (Amersham Pharmacia Biotech).
Metabolic Labeling and Immunoprecipitation--
For metabolic
labeling, cells were washed 48 h after transfection and starved
for 40 min in methionine-free minimal essential medium without fetal
calf serum. Cells were pulsed for 10 min with 300 µCi of
[35S]methionine/cysteine (PerkinElmer Life
Sciences) (1000 Ci/mmol) and either harvested directly or
harvested after a chase of 45 min performed by replacing the medium
with Dulbecco's modified Eagle's medium supplemented with 10% fetal
calf serum and 1.5 mg/ml unlabeled methionine and cysteine. Microsomes
were prepared and subjected to trypsin digestion as described above
except the proteins were immunoprecipitated with an L-specific
(i.e. pre-S1-specific) polyclonal antiserum (23).
Immunoprecipitates were separated by SDS-PAGE and analyzed by
PhosphorImager scanning (Molecular Dynamics). For protein synthesis in
the presence of dithiothreitol (DTT), the reducing agent was
added 5 min before as well as during the pulse (20 min) and during the
chase (20 min) labeling at a final concentration of 10 mM.
Cells were processed as described above except the phosphate-buffered
saline washing buffer and the homogenization buffer were supplemented
with 20 mM N-ethylmaleimide to block free
sulfhydryl groups.
Subcellular Fractionation--
Transfected cells were washed
twice in ice-cold homogenization buffer (10 mM
triethanolamine, 10 mM acetic acid, 250 mM
sucrose, 1 mM EDTA, and 1 mM DTT) and suspended
in the same buffer supplemented with 10 µg/ml each of
chymostatin, leupeptin, antipain, and pepstatin. The cells were
homogenized by passing 12 times through a 25-gauge needle. The
homogenate was centrifuged for 5 min at 1500 × g and 4 °C to obtain a postnuclear supernatant. The postnuclear
supernatant was separated on nycodenz (Nycomed) step gradients that
were formed by the method described by Hammond and Helenius (26).
Briefly, a discontinuous gradient of 10, 14.66, 19.33, and 24%
nycodenz in 0.75% NaCl, 10 mM Tris, pH 7.4, 3 mM KCl, and 1 mM EDTA was formed manually and
centrifuged for 4 h at 235,000 × g and 15 °C
in an SW40 rotor (Beckman). The postnuclear supernatant was then loaded
on top of the gradients and centrifuged for 1.5 h as described
above. Fifteen fractions were collected from the bottom, and proteins
from each fraction were precipitated with TCA prior to immunoblot
analysis. The assignments of ER, Golgi, and intermediate compartment
(IC) fractions were based on the marker analysis using anti-ID3 (a
generous gift from S. Fuller), anti-58K (Sigma), and anti-rab2 (Santa
Cruz) for immunoblotting, respectively, whereas the distribution of L
was analyzed with anti-HA.
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RESULTS |
Experimental Guide--
Topological analyses have demonstrated
previously that the initial topology of the hepadnaviral L envelope
protein at the ER membrane contains its entire pre-S domain oriented to
the cytosolic side, whereas the mature protein disposes this domain in
addition to the luminal side of intracellular membranes (Fig.
1B) (9-13). The topological
reorientation of L, i.e. the partial post-translational translocation of its pre-S domain across membranes, can be scored by
its protection from exogenously added trypsin in the absence but not
presence of nondenaturing detergents. While the pre-S domain of newly
synthesized L chains is almost fully sensitive to cleavage with
trypsin, it becomes increasingly protected over time, yielding up to
50-80% resistant chains at steady state (9, 12, 13). The
post-translational mode of this translocation event is evidenced
further by the absence of N-linked glycosylation of the
pre-S domain that carries two modification-competent glycosylation sites (Asn4 and Asn123) (12, 23). Based on
these observations, partial trypsin protection at steady state and the
lack of pre-S-linked N-glycans are convenient markers for
post-translational pre-S translocation and are thus used herein.

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Fig. 1.
Structure and proposed transmembrane
topologies of the HBV envelope proteins. A, the large
L, middle M, and small S envelope proteins are translated from three
separate in-phase start codons in the HBV envelope open reading frame
located at aa positions 1, 109, and 164, respectively. Accordingly, all
three proteins share the 226-aa sequence of the S domain. The 55-aa
long pre-S2 domain is common to M and L, whereas L additionally
contains the N-terminal pre-S1 domain with 108 residues. The four
hydrophobic segments in the S domain are indicated by open
ovals, designated 1-4. Partial
N-glycosylation occurring at Asn309 within the S
domain is indicated by crossed lines symbol in
parentheses, whereas the crossed lines symbol
refers to the pre-S2-specific glycosylation of M. Below the pre-S
domain of L, the positions of the potential trypsin cleavage sites
(R, arginine; K, lysine) are depicted.
B, models for S, M, and L protein topologies at the (post)ER
membrane. The predicted four membrane-spanning segments of S
(1-4) project their N and C termini into the ER lumen. M
exhibits a topology similar to S with its N-terminal pre-S2 domain
protruding into the ER lumen, whereas L displays an unconventional
split mixed topology. Upon cotranslational membrane integration, the
pre-S1 and pre-S2 domains of L are initially located on the cytosolic
side of the ER membrane (left model) with TM1 not being
inserted into the membrane. During maturation (marked by the
arrow), ~50% of L molecules post-translationally
translocate their pre-S region to the luminal space (right
model).
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Post-translational pre-S Translocation Does Not Require the Helper
Function of S and M Proteins--
Because of the genetic organization
of the HBV envelope open reading frame, L expression is accompanied by
the synthesis of S and M, which together assemble the virion envelope
(17). The complexing of envelope subunits has led to the proposal that
post-translational pre-S translocation of L may occur through a channel
created from lateral interactions between the amphipathic TM regions in
the S domains of M and/or S chains (10, 20, 21, 24). To examine this
hypothesis, we probed whether S and M are required for pre-S translocation. To prevent concomitant M and S synthesis from the L
gene, the start codons were inactivated by mutagenesis (designated L
only (Lo)), and an HA-epitope tag was engineered into the C terminus of
mutant and wild-type (wt) L to enable detection by Western blotting.
The topology of the proteins synthesized in transiently transfected
COS-7 cells was then determined by trypsin protection assays of
microsomes, prepared 2 days post-transfection, and immunoblotting with
an HA-specific monoclonal antibody. As shown in Fig.
2, L was obtained in nonglycosylated
(p39) and single-glycosylated (gp42) forms as a consequence of partial
modification at Asn309 in its S domain (lane 1)
(17). In addition, nonglycosylated and glycosylated forms of both the S
(p24 and gp27) and the M (gp33 and ggp36) proteins appeared because of
the internal initiation of translation (lane 1) (17).
Importantly, the M and S forms were absent in cells transfected with
mutant Lo (Fig. 2, lane 4), confirming the prevention of
their expression. Consistent with previous works (9, 12), proteolysis
with trypsin in the absence of detergent (Nonidet P-40) yielded two
fractions of wt L: trypsin-resistant full-length molecules and
trypsin-sensitive chains with cleavage occurring at least at a very
distal site within pre-S, most likely at Arg156 (Fig. 2,
lane 2; see also Fig. 1A). Therefore, a 25-kDa
nonglycosylated (T) and a 28-kDa glycosylated (gT) HA-reactive tryptic
fragment were generated that migrated slightly slower than the 24- and 27-kDa forms of S. Upon disruption of the microsomal membranes with
detergent, trypsin completely converted L to these two fragments (Fig.
2, lane 3), thus demonstrating that trypsin resistance is likely to be a consequence of pre-S translocation to the microsomal vesicle lumen rather than intrinsic protease resistance of the domain
itself. Importantly, however, when L was synthesized in the absence of
M and S chains, basically the same pattern of protection was obtained,
because ~67% of Lo chains were found to be inaccessible to trypsin
unless Nonidet P-40 was present, whereas the remaining chains were
cleaved to the gT and T fragments (Fig. 2, lanes 4-6). The
slightly more intense band obtained for the T fragment is likely to be
caused by comigration of trypsin (molecular mass, ~23.5 kDa) and its
unspecific staining in Western blotting. These results demonstrate that
L formed its dual topology even when synthesized without S and M
chains.

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Fig. 2.
Post-translational pre-S translocation does
not require S and M chains. COS-7 cells were transfected with the
wt L gene (left panel) or mutant Lo gene (right
panel) carrying missense mutations of the S- and M-specific start
codons. Two days after transfection, microsomal vesicles were prepared
and either left untreated or digested with trypsin in the absence ( )
or presence (+) of Nonidet P-40 (NP-40) as denoted above
each lane. After TCA precipitation, proteins were analyzed by SDS-PAGE
and HA-specific immunoblotting. Nonglycosylated and glycosylated forms
of L, M, and S are indicated on the left, and the nonglycosylated
(T) and single-glycosylated (gT) tryptic
fragments are marked on the right. Numbers to the
right refer to positions of molecular mass standards in kDa.
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Post-translational pre-S Translocation Does Not Depend on
Disulfide-linked Dimerization of L Chains--
Alternatively, we
reasoned that L on its own might create a putative pre-S-conducting
channel that has been predicted to build up during dimerization and/or
oligomerization of envelope chains concomitant with intermolecular
covalent disulfide linkage formation (20, 21, 27). Therefore, we
assessed whether the inhibition of disulfide bond formation in newly
synthesized L chains had an effect on pre-S translocation. To this aim,
Lo-transfected cells were treated with DTT during metabolic pulse-chase
labeling with [35S]methionine/cysteine, followed by
trypsin protection analysis and immunoprecipitation with a
pre-S1-specific antiserum. As shown in Fig.
3, Lo molecules, synthesized and matured
in the presence of DTT, translocated their pre-S domains to the
microsomal lumen as efficiently as Lo polypeptides expressed in
untreated cells (lanes 1-6). This is evident from the
almost identical degree of trypsin protection in intact microsomes.
Under both assay conditions, a significant fraction of Lo chains were
resistant to trypsin unless the protecting membranes were destroyed
(Fig. 3, compare lanes 2 and 5). Of note, the
L-tryptic fragments were undetectable in this experiment, because a
pre-S1-specific antiserum was used for immunoprecipitation.

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Fig. 3.
Post-translational pre-S translocation does
not depend on disulfide bond formation of L chains. COS-7 cells
transfected with the Lo gene were metabolically pulse-labeled for 20 min and chased for the same period in the absence (left
panel) or presence (right panel) of 10 mM
DTT. After alkylation, microsomes were mock-treated or proteolyzed with
trypsin with (+) or without ( ) Nonidet P-40 (NP-40) as indicated.
Proteins were then subjected to immunoprecipitation with a
pre-S1-specific antiserum prior to SDS-PAGE and PhosphorImaging.
Positions of the L forms are indicated.
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Amphipathic TM1, TM3, and TM4 Segments Are Dispensable for
Post-translational pre-S Translocation--
Although the above data
raised first doubts on the channel hypothesis, it nonetheless seemed
possible that noncovalent complexing of L chains, such as ionic
interactions between the hydrophilic faces of the TM segments, may
enable dimerization and pore formation. Because such interactions are
difficult to interrupt in living cells, we employed a mutagenesis
approach by constructing a series of mutant L proteins carrying
deletions of TM segments (Fig.
4A). According to the channel
model, we initially focused on the amphipathic
-helices TM1, TM3,
and TM4 and constructed two mutants, L
TM1 and Lo
TM3/4, devoid of
either the first or last two TM segments, respectively. In the latter
mutant, flanking residues of the upstream luminal peptide loop
including the glycan acceptor Asn309 were also deleted
(Fig. 4A). Consistent with previous reports (18, 19), the
deletion of TM1 did not affect cotranslational membrane integration as
evidenced by the membrane association of mutant L
TM1 and its partial
N-glycosylation at Asn309 (Fig. 4B,
lane 4). Presumably, the stop-transfer segment TM2 substitutes for the loss of the signal(anchor) TM1 (18, 19). After
trypsin digestion, protease-resistant chains of the 37-kDa nonglycosylated and 40-kDa single-glycosylated forms of mutant L
TM1
were identified in the absence but not the presence of detergents (Fig.
4B, lanes 5 and 6), indicating a
partial luminal pre-S location and hence post-translational pre-S
translocation. A similar degree of trypsin protection in intact
microsomes was observed with mutant Lo
TM3/4 that appeared only in
the nonglycosylated form of 32 kDa caused by the missing
Asn309 residue (Fig. 4B, lanes 7-9).
In combining these data, it seems that none of the three putative
channel-forming TM segments of L is required for post-translational
pre-S reorientation.

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Fig. 4.
Role for the TM segments in
post-translational pre-S translocation. A, schematic
representation of mutant L proteins carrying deletions in the S
domains. As in Fig. 1A, the pre-S1, pre-S2, and S domain
structures of wt L are shown in the most upper line with
open ovals indicating its four TM segments
(1-4). The positions of aa residues at the borders of these
segments are shown, and the C-terminally fused HA-epitope tag is
depicted by a hatched symbol. Partial
N-glycosylation occurring at Asn309 is indicated
by the crossed lines symbol in parentheses. The HA-tagged
mutant L proteins carrying C-terminal truncations and/or internal
deletions are aligned below. Deletions are indicated by dotted lines
with the deleted aa residues shown beneath. B, COS-7 cells
were transfected with the constructs as indicated above each panel. Two
days post-transfection, microsomal vesicles were prepared and subjected
to trypsin protection analysis and HA-specific Western blotting as
described in the Fig. 2 legend. For mutant L TM2, the crude lysate of
transfected cells was analyzed in addition (lane 10).
Because samples were run on different gels, positions of molecular mass
standards in kDa are shown on the left of each panel, and arrow
tips mark L and its derivatives.
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Hydrophobic TM2 Segment Is Essential and Sufficient for
Post-translational pre-S Translocation--
These results prompted us
to investigate next which role the most hydrophobic TM2 helix played in
this process. Therefore, we analyzed the mutants Lo
TM2 and
Lo
TM2/3/4 that lacked this segment either alone or in combination
with TM3 and TM4, respectively (Fig. 4A). The individual
deletion of TM2 (L
TM2), however, yielded an unstable polypeptide
that failed to associate with microsomal membranes and thus could be
only detected in the crude cellular lysate (Fig. 4B,
lane 10). Although this observation hints to an essential
role of TM2 in the cotranslational membrane integration of L, it
precludes definition of its precise role in post-translational pre-S
reorientation. Evidence for an important contribution of TM2 to the
latter event was obtained by the mutant Lo
TM2/3/4 that left only TM1
as a hydrophobic element (Fig. 4A). This mutant was stably
expressed as a membrane-bound 29-kDa polypeptide in accordance with the
molecular mass calculated for its nonglycosylated chain (Fig.
4B, lane 14). Its proper membrane integration was further confirmed by extraction with sodium carbonate (data not shown).
Digestion with trypsin revealed that this mutant was fully sensitive to
proteolytic attack even in intact microsomes, because no protected
chains could be detected by either HA-specific (Fig. 4B,
lanes 15 and 16) or pre-S1-specific
immunoblotting (data not shown). Because mutant Lo
TM2/3/4 no longer
supported pre-S translocation and mutant Lo
TM3/4 did, TM2 seemed to
be a critical determinant in pre-S reorientation. Addressing this
point, we finally constructed a mutant missing all but the second TM
segment (Lo
TM1/3/4) (Fig. 4A). This mutant, synthesized
in membranous nonglycosylated 28-kDa form, clearly acquired partial
protease protection by the microsomal membrane (Fig. 4B,
lanes 17-19). From these data we conclude that among the
hydrophobic domains of L, only TM2 is needed for pre-S translocation.
To unequivocally demonstrate that pre-S translocation of mutant
Lo
TM1/3/4 occurs post-translationally, the kinetics of its chain
maturation was monitored using a pulse-chase labeling protocol that we
had established previously for wt L (12). As shown in Fig.
5, after a 10-min pulse without chase,
the majority of newly synthesized Lo
TM1/3/4 polypeptides were
sensitive to trypsin, independent of whether membranes were disrupted
(lanes 1-3). By contrast, after a chase for 45 min, about
30% of these polypeptides were found to resist protease digestion
(lanes 4-6). The delayed pre-S translocation of mutant
Lo
TM1/3/4 indicates that it reoriented its pre-S domain after
polypeptide synthesis.

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Fig. 5.
TM2 is sufficient for
post-translational pre-S translocation. Analysis of the topology
of the mutant Lo TM1/3/4 using trypsin and pulse-chase labeling is
shown. Transfected cells were pulse-labeled for 10 min and either lysed
immediately (lanes 1-3) or chased for 45 min (lanes
4-6). Microsomes were mock-treated or digested with trypsin in
the absence ( ) or presence (+) of Nonidet P-40 (NP-40) as
denoted above each lane. As a control for unspecific
immunoprecipitation, nontransfected cells were pulse-chase labeled and
processed in a similar manner (lanes 7-9).
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Foreign TM Segments Are Unable To Substitute for TM2 in
Post-translational pre-S Translocation--
To unravel the critical
role of TM2 in pre-S transfer, we then evaluated whether it could be
functionally replaced by heterogeneous sequences. Because of the
structural homology between the S domain of L and the coronavirus MHV M
envelope protein, a triple-spanning protein with its first and second
TM segments likely serving as a signal anchor and a stop-transfer
signal, respectively (28), these two segments were chosen to substitute
for TM1 and TM2 of L (Fig. 6,
A and B). The chimera L::MHV was
engineered in the wt pre-S background to simultaneously monitor the
topology of the M::MHV construct, which was derived from the
internal translational initiation at the HBV M-specific start codon
within pre-S. As shown in Fig. 6C, both L::MHV and
M::MHV fusion proteins were efficiently expressed in
transfected cells (lane 1). In intact microsomes, the
M::MHV construct was clearly protected from trypsin digestion
(Fig. 6C, lane 2), which indicates that the
foreign TM segments translocated the upstream pre-S2 domain into the
vesicle lumen in a manner similar to the HBV-specific segments. It
therefore came out as a surprise that the MHV-specific TMs failed to
confer partial pre-S reorientation as evidenced by the full trypsin
sensitivity of the L::MHV construct in intact membranes (Fig.
6C, lane 2). We took these data as further proof
for the specific requirement of TM2 in L topogenesis.

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Fig. 6.
Coronavirus TM segments fail to confer
post-translational pre-S translocation. A, proposed
topology of the MHV M envelope protein at the ER membrane with its
three membrane-spanning segments indicated (28). B,
schematic representation of the L::MHV hybrid carrying the
HBV pre-S domain fused to the first 82 aa of the MHV M protein. TM1 and
TM2 of MHV M protein are depicted by black ovals with the aa
positions at their borders indicated; the C-terminal-attached HA tag is
drawn as a hatched oval. C, trypsin protection
assay of microsomes from L::MHV-transfected cells prepared at
steady state. As described in the Fig. 2 legend, microsomal vesicles
were either left untreated or digested with trypsin in the absence ( )
or presence (+) of Nonidet P-40 (NP-40) prior to SDS-PAGE
and HA-specific immunoblotting. The L::MHV construct as well
as the M::MHV construct, derived from the translational
initiation at the start codon preceding the pre-S2 domain, are
indicated on the left, and numbers to the right refer to
molecular mass standards in kDa.
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Post-translational pre-S Translocation Occurs at the ER
Membrane--
Because our results altogether do not favor the need of
a specialized HBV envelope structure for pre-S translocation, we
finally determined where in the secretory pathway pre-S translocation takes place. Because of (i) the lack of pre-S-linked
N-glycosylation, (ii) the inability of canine pancreas
microsomes to confer pre-S reorientation, and (iii) the prevailing
channel hypothesis, we and others have suggested previously that L
maturation occurs at a post-ER intermediate compartment (9-13), where
the HBV envelope assembly is thought to take place (27). Addressing
this issue, we used fractionation of Lo-transfected cells to separate
the ER, IC, and Golgi complex. As shown by the distribution of markers for the ER (protein disulfide isomerase), IC (Rab2), and the Golgi complex (58K), the organelles banded according to density, with the ER
being the heaviest and the Golgi complex being the most buoyant (Fig.
7A). The majority of
organelle-specific markers were found to peak in distinct fractions,
indicating a sufficient resolution of the three compartments (Fig.
7A). Fractions were next assayed for the distribution of Lo
that was predominantly present in the ER as expected (Fig.
7A). To our surprise, fractions of the IC, where pre-S
translocation had been assumed to occur, were almost free of Lo, while
it was also found in cis/medial Golgi fractions (Fig. 7A),
an as yet unknown intracellular location of L. Next, the Lo
polypeptides present in a peak ER fraction (no. 4) were digested with
trypsin as described above. This analysis clearly showed the typical
partial protease protection of about half of the Lo chains in the
absence of detergent (Fig. 7B, lane 2). Virtually
identical results were obtained when transport of L out of the ER was
blocked with brefeldin A (data not shown). Together these findings
demonstrate that the dual L topology is generated at the ER
membrane.

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Fig. 7.
Post-translational pre-S translocation occurs
at the ER membrane. For subcellular fractionation, Lo-transfected
COS-7 cells were homogenized and fractionated by isopycnic density
centrifugation on nycodenz gradients. A, fractions were
precipitated with TCA and probed for the distribution of marker for the
ER (protein disulfide isomerase (PDI)), the IC
(Rab2), and the Golgi complex (58K), or for L
protein by immunoblotting using specific antibodies. In the top, the
deduced gradient positions of the ER (closed triangles), IC
(open squares), and Golgi complex (closed
circles) are indicated together with the density profile of the
gradient. B, trypsin protection assay of L molecules present
in the ER fraction 4 of the nycodenz gradient was done as described in
the Fig. 2 legend, and samples were analyzed by HA-specific
immunoblotting. The positions of L and its tryptic fragments
(gT and T) are indicated.
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|
 |
DISCUSSION |
The HBV L envelope protein is the first example of a viral protein
exhibiting different transmembrane topologies for functional diversity
(9, 11, 12, 14, 16) and hence is an addition to a restricted but
growing number of membrane proteins that seem to have developed
variations on cotranslational biogenesis pathways (3-7). In contrast,
however, and in being novel, the topological heterogeneity of L is
generated in a noncotranslational manner (9, 11, 12). Accordingly, a
prevailing model predicts post-translational pre-S translocation of L
to be enabled by an HBV-specific structure created during virion
envelope assembly (10, 20, 21, 24). By analyzing the parameters
influencing L topogenesis, the results of this work raise doubts on the
validity of such a channel model.
First evidence that pre-S refolding of L may be uncoupled from envelope
assembly was obtained by the finding that neither the S nor the M
envelope protein is required for this event. The dispensability of S is
particularly surprising because this protein is the predominant
constituent of the viral envelope and forms its scaffold (17), which is
why it has been implicated as the likely candidate for providing
the pre-S conducting channel (20). Indeed, recent studies on the pre-S
translocation of the related L protein of duck HBV demonstrated an
essential role for the avian S counterpart in this process (24). We do
not yet know the reason for this discrepancy, but assume that the
envelope proteins of the two virions may have evolved different folding
pathways to acquire their final transmembrane topology, as has been
also shown recently for the two closely related aquaporin-1 and
aquaporin-4 proteins (29). In favor of this view, assuming different
folds, is the inability of the duck HBV envelope proteins to substitute for the HBV homologues in virus envelope assembly (30).
As post-translational pre-S reorientation is an intrinsic feature of
the HBV L protein, it nonetheless may proceed via a channel created by
L itself. Analysis of L deletion mutants, however, revealed that the
topological switch of L does not require any of its amphipathic TM
helices but rather the most hydropathic TM2 segment. By analogy to
other viral single-spanning membrane proteins, such as the M2 protein
from influenza A and the NB protein from influenza B, that form
homotetramer bundles of transmembrane helices surrounding a central
ion-permeable pore (31), mutant L
TM1/3/4 might also still
homo-oligomerize with the helical packaging of its single TM2 segment.
Because of its extremely hydrophobic character, we consider TM2,
however, unlikely to line a pore of sufficient hydrophilicity to enable
transmembrane pre-S passage.
Although being in seeming conflict with a current view of
post-translational pre-S translocation, our results do not rule out
that aqueous pores may exist in the secreted virion envelope. This has
been implicated by low pH treatment of hepadnaviral particles that
triggers further exposure of pre-S domains of L that were originally
hidden and even surface display of the TM1 segment, a putative fusion
sequence involved in viral entry (9, 32, 33). Such repositioning events
indeed may proceed through aqueous pores in the extracellular envelope,
the formation of which is likely facilitated by the viral lipid
structure that is devoid of a unit membrane, because hepadnaviral
envelope assembly is accompanied by a substantial reorganization of
membrane lipids (34). Therefore, the mechanisms responsible for the
pre-S translocation of L across intracellular membranes and its
rearrangement(s) in the extracellular envelope might not necessarily be coincident.
In combining these data, we consider other transmembrane transport
mechanisms more feasible at generating the dual topology of L at a time
when it is embedded in microsomal membranes. Oess and Hildt (35) have
recently identified a cell-permeable motif within the pre-S2 region of
M and L that enables pre-S, if separated from the S domain, to cross
over the plasma cell membrane. Involvement of this motif in
post-translational pre-S translocation, however, is largely excluded,
because this sequence has been shown to be dispensable for
morphogenesis and infectivity of HBV that both depend on the alternate
L structure (14, 16, 36). Moreover, the failure of mutant Lo
TM2/3/4
to refold pre-S, as shown herein, argues against a role for the
pre-S2-specific motif in this process. Alternatively, the structural
change of L might be achieved by reversible flipping of pre-S across
the lipid bilayer, as has been demonstrated for a subdomain of the
colicin Ia channel protein (37). Although such a transmembrane slipping
back and forth might well explain the partial mode of pre-S
translocation, a further puzzling feature of L maturation, the
phenotypes of distinct L derivatives, does not support this proposal.
First, mutant L proteins, in which cotranslational pre-S translocation
has been artificially enforced, maintained their uniform luminal pre-S orientation (14, 23). Second, mutant Lo
TM2/3/4 uniformly disposed
its pre-S domain to the cytosol, thus contradicting a reversible pre-S
flipping mechanism.
Rather, according to our fractionation data, it is reasonable to assume
that pre-S reorientation of L is governed by the ER translocation
machinery, with TM2 being the most crucial topogenic determinant. It
seems possible that upon cotranslational translocation, the TM domains
of nascent L chains might not be integrated into the bilayer
immediately but instead are held within the Sec61p translocon even
after their synthesis is completed. This would be consistent with the
large internal diameter of the translocon pore (20-60 Å) as well as
with recent studies suggesting that TM segments, even those of several
chains of a single protein, may accumulate within or near the
translocon before entering the membrane (38-40). Inconsistently,
however, the detachment of the ribosome after chain termination (41)
would keep the Sec61p translocational channel, occupied with L
chain(s), in an unphysiologically open state. Possibly, binding of the
Hsc70 chaperone with cytosolically disposed pre-S domains (23) somehow
might accomplish channel gating, similar to established chaperone
functions in post-translational protein translocation in yeast and
bacteria (42). The bound Hsc70 molecule might additionally serve as a
regulatory device, preventing the premature exit of L from the
translocon. Alternatively, it is also quite possible that L chains may
be released into the lipid after translation but reenter the
translocation channel for reorientation. Re-association of proteins
with the Sec61p channel after they have been integrated into the lipid
bilayer is suggested by experiments that implicate the Sec61p complex in retrograde transport of, for example, MHC class I molecules (43) or
apoprotein B100 (44). Irrespective of the path L could use, the
translocation channel would then provide the hydrophilic environment
needed for transmembrane pre-S transport. In the simplest case, this
would involve a 90° rotation of TM1, that had been originally left
out or ignored by the translocation machinery, and a concurrent
translocation of the flanking N-terminal pre-S domain to the luminal
space (see Fig. 1B). A similar topological reorientation of
internal TM segments and adjacent peptide loops has recently been
observed to occur during the maturation of aquaporin-1 that, in
contrast to L, is initiated during translation but enhanced after
synthesis is almost completed (7). Although we cannot formally exclude
that such a TM1 rotation may direct the alternate pre-S topology of
wild-type L, the results of our mutagenesis analysis indicate that TM1
is not actually involved in pre-S repositioning, at least of mutant L
proteins. Rather, our data hint to an essential and specific function
for TM2 in this process that could be fulfilled by neither TM1 nor
foreign TM segments. Considering the mandatory requirement of TM2, it
is tempting to speculate that this domain confers a putative prolonged
interaction of L with the ER translocon to promote pre-S refolding in a
subset of L chains. To do so, TM2 must reorient from a
membrane-spanning to a membrane-lining segment or to a helical hairpin
structure. To account for the absence of pre-S-linked
N-glycosylation in this scenario, initial folding of pre-S
before its post-translational delivery to the luminal ER space may
hamper recognition by the oligosaccharyl transferase. In regard to
recent suggestions that certain polytopic proteins may require
specialized components to achieve their proper topology in addition to
the basic components needed for protein translocation (3, 45), it also
seems conceivable that post-translational pre-S translocation may occur
in a specialized ER subdomain where glycosylation enzymes might be
missing. Our previous observation that canine pancreas microsomes are
unable to reconstitute pre-S reorientation (12) would fit with such a
proposal. Intriguingly, a similar result has been reported for
aquaporin-1 (noted above) that likewise failed to reorient and thus to
mature in canine rough microsomal membranes (7).
To conclude, the precise mechanism(s) by which the functionally
important dual topology of L is generated is still elusive and remains
to be defined. Our demonstration that the topological reorientation of
L is seemingly not established by a virus-specific structure but is
physically linked to the ER membrane renders the HBV L protein as a
challenging substrate to unravel the complex and dynamic nature of
events occurring during polytopic protein topogenesis.