From the Centro de Biología Molecular Severo
Ochoa (CSIC-UAM), Facultad de Ciencias, Universidad Autónoma,
Cantoblanco, 28049 Madrid, Spain and
Unidad de
Biofísica (CSIC-UPV/EHU), Departamento de
Bioquímica, Universidad del País
Vasco, 48080 Bilbao, Spain
Received for publication, July 3, 2002, and in revised form, October 18, 2002
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ABSTRACT |
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Alphavirus 6K is a short, constitutive
membrane protein involved in virus glycoprotein processing, membrane
permeabilization, and the budding of virus particles. The
amino-terminal region that immediately precedes the transmembrane
anchor contains a conserved sequence motif consisting of two
interfacial domains separated by Asn and Gln residues. The presence of
this motif confers on the 6K pretransmembrane region the tendency to
partition into the membrane interface. To study the functional
importance of the interfacial sequences, three different Sindbis
virus 6K variants were obtained with the following
modifications: 9YLW11xAAA, 18FWV20xAAA, and 9YLW11xAAA/18FWV20xAAA.
Reconstituted mutant viruses were infectious and showed no defects in
glycoprotein processing, although virus budding was hampered. Single 6K
expression in Escherichia coli cells showed interfacial
mutants to have a diminished capacity to modify membrane permeability
and to have lower toxicity. In particular, the 9YLW11xAAA/18FWV20xAAA
variant was expressed at high levels and did not enhance membrane
permeability significantly, although it retained its integral membrane
protein condition. Parallel analyses of membrane permeabilization in
baby hamster kidney cells were carried out using a Sindbis virus
replicon that synthesized both capsid protein and 6K. Transfection of
the construct with wild-type 6K strongly increased permeability
to the antibiotic hygromycin B. Replicons encoding 6K interfacial mutants induced lower membrane permeabilization. Again, the greatest impairment was observed for the 9YLW11xAAA/18FWV20xAAA variant, permeabilization activity of which was ~10% that of wild-type 6K.
These findings show the importance of the interfacial 6K sequence for
virus budding and modification of membrane permeability.
Alphaviruses are enveloped animal viruses with single-stranded
positive RNA tightly packaged by the capsid. The structural proteins
are synthesized from a subgenomic mRNA encoding a polyprotein that
is proteolytically processed (for review, see Refs.
1-3). Capsid (C)1 protein is
first synthesized and detaches from the rest of the polyprotein by
autocatalytic proteolysis. Once C protein has been liberated into the
cytoplasm, polyprotein synthesis continues to be associated with
endoplasmic reticulum (ER) membranes. The exposed amino terminus
contains a signal sequence that interacts with ER membranes and directs
the glycoprotein precursor (E3-E2-6K-E1) into the lumen of the ER. The
precursor is associated with the ER membrane, spanning the lipid
bilayer six times. Soon after synthesis, this precursor is cleaved at
both ends of the 6K protein by a cellular protease present in the ER,
generating the products PE2 (E3+E2), 6K, and E1. PE2 and E1 then
associate to form dimers that travel with 6K through the vesicular
system to the plasma membrane. As a final activation step, PE2 is
cleaved by a furin-like protease present in a post-Golgi compartment,
giving rise to glycoproteins E3 and E2. The glycoproteins transported
to the plasma membrane expose their amino terminus ectodomains to the
external medium, whereas the carboxyl domains remain facing the
cytoplasm. The virus genomes replicated in the cytoplasm interact with
the C protein to form nucleocapsids. The assembled nucleocapsids
subsequently interact with the carboxyl domain of E2. This interaction
provokes the wrapping of the capsid with the lipid envelope,
concomitant with the budding of virus particles (1-3).
6K is a small hydrophobic polypeptide acylated with fatty acids (4, 5).
Despite the association of the 6K protein with the plasma membrane and
its interaction with E1-E2, very little 6K is incorporated into the
budded virus particles (4, 5). Although 6K protein provides the
cleavage sites in the glycoprotein precursor for signalase activity, a
Semliki forest virus variant lacking the entire 6K is processed between
E2 and E1 (6, 7). This virus mutant is not defective in synthesis and
transport of glycoproteins or in nucleocapsid formation; its major
defects concern the budding process. Another function assigned to
Semliki forest virus 6K protein is to provide the signal sequence for E1 translocation to the lumen of the ER (8). Notably, E1 is properly
translocated in the 6K-deleted Semliki forest virus mutant (6, 7).
Similarly, Sindbis virus (SV) variants with single or multiple amino
acid substitutions in the 6K have defects in virion release, leading to
the formation of multinucleated virus particles (4, 9-11). Proper
proteolytic processing of the virus glycoproteins is hampered in an SV
variant bearing an insertion of 15 amino acids in the 6K protein. This
variant exhibits a transdominant phenotype, but virus particles display
a morphology similar to that of wt virus (12). An SV variant with
deleted 6K 22 amino acids shows defects in glycoprotein proteolytic
processing (13). A revertant of this mutant in which these defects were
corrected was subsequently isolated, but virus release was still
impaired. Furthermore, the in trans expression of a genuine
6K from an extra subgenomic promoter placed in the same genome did not
produce appreciable reversion. In addition, the functions of the 6K
protein cannot be rescued by the corresponding counterparts from
related virus species. Thus, the substitution of the SV 6K gene by the 6K counterpart from Ross River virus leads to the small plaque phenotype and reduced formation of infectious virus (14). This SV
variant with the 6K gene from Ross River virus was able to cleave the
glycoprotein precursors proteolytically and to transport them into the
plasma membrane, although the budding process was impaired. Together,
these observations suggest a function for 6K in the release of virions
from infected cells. However, the exact molecular mechanism by which 6K
protein enhances virion release remains unknown.
This article reports an unexplored feature of SV 6K that might be
related to the promotion of the virion-release process (i.e. the affinity of 6K for the membrane interface). It was found that the
amino-terminal 6K ectodomain contains two hydrophobic-at-interface segments that can mediate association of this sequence with the external membrane monolayer. Conservation of the interfacial 6K segments among divergent members of the Alphavirus genus
suggests a functional role for these motifs. Accordingly, SV 6K
variants containing substitutions that interfere with the capacity of
the amino terminus to partition into membranes without affecting
overall 6K hydrophobicity were obtained and characterized. The data
support the hypothesis that 6K participates in virus release through
direct interaction with membranes that compromise the permeability
barrier. A model is proposed that accounts for the involvement of 6K in this phenomenon.
Cells, Viruses, and Plasmids--
Baby hamster kidney (BHK-21)
cells were grown at 37 °C in Dulbecco's modified Eagle's medium
supplemented with 5% fetal calf serum and nonessential amino acids. SV
was derived from the cDNA clones described below after transfection
of the RNAs synthesized in vitro.
Full-length SV cDNA Clones--
A full-length cDNA clone
of Sindbis virus pT7SVwt (13) was used as the wt parental clone to
generate mutagenized SV variants. pT7SV YLW/AAA, pT7SV FWV/AAA, and
pT7SV YLW/AAA/FWV/AAA carry mutations in the 6K gene: 9YLW11xAAA,
18FWV20xAAA, and both 9YLW11x AAA and 18FWV20xAAA, respectively. They
were generated by replacing the BssHII/SplI
fragment in pT7SVwt by PCR-amplified and subsequently digested
fragments incorporating the 6K substitutions.
SV Replicons--
Several SV replicons were obtained by deleting
glycoprotein genes: C+6Kwt, C+6K YLW/AAA, C+6K FWV/AAA, and C+6K
YLW/AAA, FWV/AAA. These replicons were made in two steps. First,
pT7SVwt was digested with BssHII and StuI and the
recessed ends filled in with DNApol I, Large (Klenow fragment).
Thus, part of the sequence coding for the proteins E3 and E2 was
deleted. The open reading frame was not broken, and the remaining
sequence coded for the 45 amino-terminal amino acids of E3 fused to 31 carboxyl-terminal amino acids of E2 plus 6K and E1. In a second step,
the different 6K sequences were introduced at the same time as the E1
sequence was deleted, using the unique restriction sites
BssHII, upstream of 6K, and ApaI, downstream of
E1. Sequences with the different 6Ks were amplified by PCR using
oligonucleotides that incorporated the restriction sites
BssHII and ApaI and a stop codon placed after the
6K sequence. Digested products replaced the sequences located between
the BssHII and ApaI sites in the above plasmid.
The replicon coding only for the capsid protein was obtained by
replacing the pT7SVwt sequence between the AatII and
ApaI sites by a PCR-amplified fragment encoding the
Capsid sequence from the AatII site to the end and a
stop codon.
Bacterial Expression Plasmids--
pET11 plasmid (15) was used
for this purpose. pET11 6Kwt, pET11 6K YLWxAAA, pET11 6K FWVxAAA, and
pET11 6K YLWxAAA/FWVxAAA were constructed replacing the
NdeI/BamHI fragment in pET11 by PCR-amplified
products encoding the different 6K protein variants. Plasmids were
checked by sequencing the inserts from PCR amplification by standard
sequencing techniques.
Transfection of BHK Cells--
Subconfluent BHK cells were
harvested, washed with ice-cold phosphate-buffered saline, and
resuspended in phosphate-buffered saline at a density of about 2.5 × 106cells/ml. A 25-µl aliquot of T7 RNA polymerase
transcription mixture with about 10 µg RNA from the different
cDNA constructs was added to 0.4 ml of cells, and the mixture was
transferred to a 2-mm electroporation cuvette. Electroporation was
performed at room temperature by generating two consecutive 1.5-kV,
25-microfarad pulses using a Gene Pulser apparatus (Bio-Rad) as
described Liljeström et al. (6). The cells were then
diluted in growth medium and seeded onto culture plates.
Electron Microscopy--
Transfected cells were processed for
electron microscopy as follows. At 16 h.p.e. (hours
post-electroporation), cells were fixed with 2% glutaraldehyde in 0.2 M HEPES buffer, pH 7.4, for 1 h at room temperature
and immediately scraped off the plate. These were then washed twice and
resuspended in 0.2 M HEPES buffer, pH 7.4. After fixation,
they were dehydrated and infiltrated with Epon. Thin sections were
obtained and stained with uranyl acetate and lead citrate.
6K Expression and Membrane Permeabilization in E. coli
BL21(DE3)--
Permeability changes to the antibiotic hygromicin B
(HB) induced by the expression of 6K proteins were measured in E. coli cells as described previously (16, 17). Briefly, E. coli BL21(DE3) cells transformed with the described pET11
recombinant plasmids were grown in M-9 medium (18) and induced with 1 mM isopropyl-1-thio- Permeabilization of 6K-expressing BHK Cells--
106
BHK cells were electroporated with 10 µg of RNA synthesized in
vitro from the different plasmids and further seeded in three
wells of an L-24 plate. At 16 h.p.e., proteins were radiolabeled for 45 min with 200 µl Dulbecco's modified Eagle's medium without methionine/cysteine but supplemented with 2 µl of Trans label [35S]Met-Cys (15 mCi/ml, Amersham Biosciences) per well
in the presence or absence of 0.5 or 1 mM HB. Cells were
harvested and labeled proteins analyzed by SDS-PAGE and fluorography.
Hydrophobicity Analysis of SV 6K--
The sequence alignments for
representative 6K products derived from divergent alphaviruses (1) are
shown in Fig. 1A. Trp-11 and
Trp-19 are totally conserved at the amino terminus of these sequences
(Fig. 2A, bold characters). When average
interfacial hydrophobicity was plotted for several of these
representative sequences (Fig. 1B), the hydropathy plots detected two
consecutive peaks at the short amino-terminal 6K ectodomain,
immediately preceding the transmembrane domain (TMD). The average
hydropathy plots in Fig. 1B were calculated according to the
so-called Wimley-White(WW)hydrophobicity-at-interface scale (19, 20).
In contrast with classical hydrophobicity scales, this is a
whole-residue scale (i.e. it includes contributions from
peptide bonds as well as amino acid side-chains) based on the
water-to-membrane interface transfer-free energies for each amino acid.
Consequently, the average interfacial hydrophobicity of an arbitrary
sequence directly reflects the tendency of such a sequence to partition
from water into membrane interfaces or, in other words, to promote the
first step of the protein-membrane interaction process leading to
integration (21). The positive 6K peaks indicate that the conserved
amino-terminal stretches have a propensity to interact with and to
remain immersed in the interfacial region of the membrane. Because this
side of 6K faces the external side of the membrane, the presence of
these segments confers on the 6K ectodomain the ability to interact
differentially with the external membrane monolayer.
Generation of 6K Interfacial Mutants--
To assess the role of
the interfacial domains in 6K protein, several variants were generated
(Fig. 2). Because the goal was to explore
the functional importance of the hydrophobic-at-interface character of
the sequences, substitutions specifically affecting this parameter that
did not, however, induce appreciable changes in the overall sequence
hydrophobicity, were selected. The hydropathy plots in Fig. 2
illustrate this strategy. Kyte-Doolittle plots (22), based on relative
side-chain hydrophobicities, accurately predict transmembrane regions
in constitutive integral membrane proteins (translocated with energy
cost during biogenesis). Positive peaks are consistent with
translocated TMD regions remaining stably inserted in membranes.
Accordingly, the Kyte-Doolittle plots in Fig. 2 detect the TMD region
of 6K as a positive peak. None of the 6K mutations generated altered
this hydrophobicity pattern (Fig. 2).
In contrast with Kyte-Doolittle, the WW algorithm is based on the
water-to-membrane interface transfer-free energies for each amino acid
(19). The membrane interfaces are distinct regions of the bilayer,
characterized by their chemical heterogeneity and sharp polarity
changes with distance. Wimley and White (19) found unexpected
differences in the relative hydrophobicities of the amino acid residues
when these residues partition from water into interfaces of
phospholipid bilayers. In particular, hydrophobic sequences rich in Trp
residues show the greatest tendency to partition spontaneously from the
aqueous phase into the membrane. The different substitutions made in SV
6K in the present work resulted in abolition of the first, second, or
both amino interfacial subdomains (Fig. 2).
Analysis of Sindbis Viruses with 6K Interfacial Variants--
To
test the effect of the 6K mutations on different aspects of the virus
life cycle, the different SV variants in 6K were first reconstituted.
BHK cells were transfected with RNA transcribed from these SV variants.
The proteins synthesized at 16 h.p.e. were analyzed by SDS-PAGE.
Fig. 3A shows that the three
SV 6K mutants synthesized viral proteins as efficiently as wt SV. Thus, the levels of synthesized protein and host translation inhibition were
similar for all viruses assayed. In addition, no defects were found in
the processing of the different viral structural proteins. These
results indicate that the cleavage sites in the viral polyprotein
contributed by the 6K variants were able to undertake proteolytic
processing adequately. Moreover, no alterations were noted in the
migration of the viral glycoproteins, suggesting that their
post-translational processing was not hampered by the mutations in
6K.
Previous analyses have indicated that one of the major defects of
alphaviruses with a mutated 6K appears at a very late step in the virus
life cycle: the process of virus budding. Therefore, the exit of
viruses from the infected BHK cells was investigated by electron
microscopy. Fig. 3B shows a number of SV particles exiting
from wtSV-infected cells. The morphology of these virus particles was
normal. In contrast, a clear defect was observed with the three 6K
variants analyzed. In all three cases, virus particles accumulated at
the plasma membrane. Although their morphology showed no anomaly, these
particles were unable to detach efficiently from cells. It is concluded
that the three mutations introduced in the 6K sequence confer defects
in virus budding, such that the pinching-off of the assembled virus
particles is inefficient. In turn, this defect leads to the formation
of virus plaques of smaller size (data not shown).
Enhanced Membrane Permeability Provoked by 6K and Its Variants in
E. coli Cells.--
The clearest activity that 6K shows when
individually expressed in cells is the capacity to increase membrane
permeability to a number of solutes (17). To assess the participation
of the interfacial region of 6K in this process, wt 6K and the three 6K
gene variants were cloned and expressed in E. coli cells
using an inducible system of gene expression (Fig.
4). Membrane permeability was analyzed by
the HB test (16, 17). As shown in Fig. 4A, expression of the
wt 6K gene not only permeabilized prokaryotic cells to the antibiotic
HB, but also induced rapid cell lysis, such that the synthesis of 6K
lasted only a few minutes upon 6K induction. By comparison, the three
6K variants showed a reduced permeabilization capacity and lower
toxicity after induction. In particular, the variant with both mutated
tripeptides (i.e. 9YLW11xAAA/18FWV20xAAA) maintained the
same expression level 60 min after induction, and membrane permeability
to HB did not appreciably change.
To assess further whether mutations introduced in the interfacial
sequence affected the capacity of 6K to interact with membranes, the
experiment shown in Fig. 4B was performed. After protein
labeling, the cells were broken, and the membrane-containing fraction
was obtained. The wt 6K protein seemed associated with the membrane fraction even after urea treatment. However, incubation of this membrane fraction with SDS detergent led to the release of 6K from
membranes. The data in Fig. 4B demonstrate that the
9YLW11xAAA/18FWV20xAAA variant behaves in a way akin to wt 6K.
Together, these results suggest that both wt 6K and its
9YLW11xAAA/18FWV20xAAA 6K variant are integral membrane proteins.
Therefore, the double modification of the interfacial sequence does not
hamper the ability of 6K to become an integral membrane protein,
although it does affect its ability to perturb the membrane
permeability barrier.
Membrane Permeability Induced by 6K Variants in BHK Cells--
The
potent permeabilizing capacity of 6K in prokaryotic cells was
recognized several years ago (17), but the action of this protein in
mammalian cells remains unknown. To address this matter, a system was
designed based on SV replicons that synthesize 6K product in large
amounts. This system consisted of cloning the 6K sequences adjacent to
the capsid, so that upon translation of the subgenomic mRNA, C
protein is liberated by its autocatalytic activity, and the rest of the
protein is made at normal levels. The two sites of proteolytic
cleavage, those between C-E3 and E2-6K, were maintained in this
construct to synthesize a genuine 6K protein with no extra amino acid
(see Fig. 5A). Using this approach, constructs were made with wt 6K or the three different variants placed just after C (Fig. 5A). BHK cells were
transfected with in vitro-transcribed RNAs, and after
16 h.p.e., protein synthesis was estimated in the absence or
presence of the aminoglycoside antibiotic HB (Fig. 5B). This
compound did not permeate into uninfected cells or into cells
transfected with RNA encoding only C protein (Fig. 5B).
Notably, HB readily entered into BHK cells transfected with full-length
RNA from wt SV, leading to a profound inhibition of viral translation.
The expression of wt 6K enhanced membrane permeability to HB, whereas
this effect was not as strong with the 6K variants. Thus, with respect
to synthesis-inhibition levels, it may be inferred that 9YLW11xAAA,
18FWV20xAAA, and 9YLW11xAAA/18FWV20xAAA substitutions provoked a
reduction in the capacity of wt 6K to induce HB entry by 25, 66, and
94%, respectively. These findings are consistent with the hypothesis
that the integrity of the interfacial sequence of 6K is crucial for the
correct activity of this protein with respect to enhancing membrane
permeability (Figs. 4 and 5) and virus budding (Fig. 3). Some
instability of the double 6K mutant was also observed, perhaps
indicating that the mutated region plays a part in governing protein
degradation in mammalian cells.
There are a number of virus-encoded proteins capable of enhancing
cell membrane permeability. These proteins can be classified into two
broad groups. One comprises viral glycoproteins with a particular
architecture in their transmembrane regions and the sequences proximal
to that domain (23-26). Oligomerization of these proteins may lead to
pore formation and membrane disturbance (27). The other is a group of
viral proteins that destabilize membranes and includes a number of
small and very hydrophobic proteins. These integral membrane
polypeptides are also able to oligomerize, promoting pore formation in
biological membranes. Consequently, they are known as viroporins (16,
17, 28-32). The alphavirus 6K protein is a typical viroporin that
contains a transmembrane region that anchors it to membranes during
protein synthesis. Despite its relative structural simplicity, 6K plays
several roles during the virus life cycle. Apart from its participation
in polyprotein processing, the major function of 6K stems from its
requirement for efficient virus budding from infected cells (6, 7, 4, 9-14). The exact molecular mechanisms of virus budding are not yet
fully understood. The fact that 6K enhances membrane permeability led
to the proposal that this activity facilitated the exit of viruses from
cells. Perhaps the destabilization of the plasma membrane by 6K
promotes the release of virus particles to the medium. This may rely
not only on the physical modification of the membrane but also on the
local dissipation of ionic gradients by the pore-formation capacity of 6K.
The 6K transmembrane region is thought to allow the early formation of
a hairpin structure embedded in the ER membrane. This structure
positions the polypeptide amino and carboxyl termini in the ER lumen
(8) (Fig. 6A). Little is known
about the stability of this predicted structure or the eventual
topology adopted by 6K embedded in the plasma membrane. Prediction of
membrane protein structure and stability has been recently implemented
with the introduction of the WW hydrophobicity-at-interface scale (20). The present work analyzed the interfacial hydrophobicity of alphavirus 6K. The predilection of a given sequence for membrane interfaces over
bulk apolar or polar phases seems to be dictated by the presence of
aromatic residues. WW analysis shows the presence of two stretches containing invariant Trp residues within the 6K protein ectodomain, with a high tendency to partition into membrane interfaces. The preservation of these interfacial sequences is crucial for enhanced membrane permeability by 6K protein. The 6K variants mutated in the
pretransmembrane region were unable to permeabilize membranes even
though they seemed to be membrane-integral products. Thus, the
integration of 6K into the membrane is insufficient for membrane destabilization, but this 6K amino-terminal region is also necessary to
permeabilize membranes and for efficient virus budding.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (IPTG). At 30 and 60 min after induction, proteins were
pulse-labeled for 15 min with [35S]Met-Cys in the
presence or absence of 0.5 mM HB. Cells were subsequently
harvested and the radiolabeled products analyzed by SDS-PAGE,
fluorography, and autoradiography. Membrane association of the 6K
proteins in these experiments was analyzed as follows. Radioactively
labeled cells were harvested by centrifugation, resuspended in buffer A
(50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mM EDTA, pH 8, 1 mM dithiothreitol) lysed by
sonication and centrifuged at 10,000 rpm for 15 min. The supernatant
fraction obtained was further subjected to ultracentrifugation at
100,000 rpm for 1 h. The pelleted fraction was resuspended in
buffer B (10 mM Tris-HCl, pH 9.3, 1 mM
-mercaptoethanol). Three aliquot samples of the pelleted membrane
fraction were processed as follows: 1) untreated as a control; 2)
treated with 8 M urea, and 3) treated with 0.5% SDS. After
further ultracentrifugation at 100,000 rpm for 1 h, equivalent
amounts of supernatant and pellet fractions were analyzed by SDS-PAGE
and fluorography.
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DISCUSSION
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Fig. 1.
A, multiple sequence alignment of divergent
alphavirus 6K proteins by ClustalW (36). Conserved Trp residues are
indicated in bold characters. Protein sequences: rrv, Ross River virus;
sfv, Semliki Forest virus; onnv, o'nyong-nyong virus; weev, western
equine encephalitis virus; sv, Sindbis virus; veev, Venezuelan equine
encephalitis virus; eeev, eastern equine encephalitis virus.
B, hydropathy plots (black bars) corresponding to
representative alphavirus 6K sequences. The plots (mean values of free
energies of transfer from membrane interfaces to water for a window of
five amino acids) were produced using the Wimley-White interfacial
hydrophobicity scale for individual residues (19). The white
bar diagram corresponds to the probability (scaled to the interval
0-9) of assigning a helical transmembrane region as predicted by the
predicted helical domains neural network (37). The segments
indicate TMD extensions based on the refined models.
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Fig. 2.
Hydropathy plots for the different 6K
variants studied. Hydropathy plots (mean values for a window of 5 amino acids) were elaborated using the Kyte-Doolittle hydropathy index
(KD) and Wimley-White interfacial hydrophobicity
(WW) scales for individual residues. The black
bar and white box above indicate the approximate
extensions of pretransmembrane and transmembrane sequences,
respectively.
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Fig. 3.
Electroporation of BHK cells by reconstituted
SV genomes with the 6K variants. A, protein synthesis in
electroporated BHK cells: cells were electroporated with in
vitro-transcribed RNA obtained from the different full-length
cDNAs clones as indicated in the figure. At 16 h.p.e., cells
were labeled for 30 min with [35S]Met-Cys, and processed
by SDS-PAGE, fluorography, and autoradiography. Arrows indicate the
migration positions of the different viral products. B,
electron microscopy of electroporated BHK cells: electron microscopy
analyses of cells electroporated with in vitro-transcribed
RNA from the different full-length cDNAs clones indicated in the
figure. WT, wild-type SV genome;
YLW/AAA and FWV/AAA, SV
genomes with the indicated mutations in the 6K gene. At 16 h.p.e.,
cells were processed for electron microscopy. Small arrows
show nucleocapsids and big arrowheads indicate budding
viruses still associated with the cell plasma membrane. Bars
correspond to 1 µm. The insets show at higher magnification
nucleocapsid-containing areas in the cytosol of BHK cells
electroporated with mutant YLW/AAA or FWV/AAA.
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Fig. 4.
Membrane permeability in E. coli cells that synthesize 6K proteins.
A, cultures of E. coli (BL21(DE3))
transformed with pET11-6K (wt, YLW/AAA, FWV/AAA, and YLW/FWV/AAA,AAA)
plasmids were induced to express the different 6K protein with 1 mM isopropyl-1-thio- -D-galactopyranoside. 30 or 60 min after induction, cells were labeled for 15 min with
[35S]Met-Cys in the presence (+) or absence (
) of 0.5 mM HB, and then processed for gel electrophoresis analyses
as indicated in Fig. 3A. B, interaction of 6K
proteins with membranes: the cultures indicated in the figure were
induced, labeled with [35S]Met-Cys, and processed to
analyze the membrane fraction, as indicated under "Experimental
Procedures." Lane 1, supernatant fraction after 100,000 rpm centrifugation; lane 2, pellet fraction after 100,000 rpm centrifugation; lane 3, supernatant fraction of
urea-treated sample 2; lane 4, pellet fraction of the
urea-treated sample 2; lane 5, supernatant fraction of the
SDS-treated sample 2; lane 6, pellet fraction of the
SDS-treated sample 2.
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Fig. 5.
Membrane permeabilization in BHK cells that
synthesize 6K proteins. A, schematic representation of the
full-length SVwt cDNA clone and the different SV-derived replicons
("rep") used in this experiment. B, protein
synthesis in BHK cells electroporated with the different RNAs. BHK
cells electroporated with in vitro-transcribed RNA from the
different constructs indicated in the figure were labeled at 16 h.p.e. with [35S]Met-Cys in the absence ( ) or presence
of 0.5 or 1 mM HB. Mock cells, as well as cells
electroporated with RNA from "rep"C, were used as
negative controls. Cells electroporated with RNA from pT7SVwt were used
as a positive control of permeabilization to HB. C,
percentage inhibition of protein synthesis exerted by HB in cells that
expressed the different 6K genes. Densitometric analyses of protein C
were used to calculate the inhibition of protein synthesis by HB.
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Fig. 6.
Schematic representation of 6K structure and
membrane topology. A, model for 6K protein inserted into the
ER membrane. According to the model proposed by Liljestrom and Garoff
(8), 6K is predicted to fold as a membrane-embedded hairpin constituted
by: 1) an outside-inside transmembrane domain (thick
cylinder); 2) an inside-outside transmembrane domain (narrow
cylinder); and 3) a Cys-rich loop that reverses chain direction.
One of the interfacial segments (gray ellipsoids) might
contribute to the outside-inside TMD in this state. The carboxyl- and
amino-terminal ends of the peptide chain would face the ER lumen and be
cleaved therein. B, the Del6K-revQ21L product consists
mainly of both interfacial segments that are not transferred into the
membrane core. C, model for 6K secreted to the plasma
membrane (PM). In this version, 6K contains an interfacial
helix (gray cylinder) followed by a single transmembrane
helix (white cylinder). The interfacial domain might disrupt
phospholipid cohesion at the external membrane monolayer (see also
models for poliovirus 3A protein interacting with membranes in Ref.
35).
We have previously reported (13) the isolation of an SV variant, Del6K-revQ21L, with a partially deleted 6K that displays an almost genuine interfacial domain (Figs. 2B and 6B). This variant shows correct proteolytic processing and transport but still exhibits defects in virus budding comparable with those observed when the 6K gene is completely oblated. Therefore, expression of the isolated interfacial region does not restore 6K function either. It would seem that a sequence immersed in the membrane interface followed by the transmembrane anchor represents the minimal structure required for functional 6K to induce membrane alterations (Fig. 6C).
Exposure to different lipid environments during protein trafficking might affect 6K structure and membrane topology. In particular, it has been proposed that cells rely on membrane thickness to sort proteins destined for the plasma membrane from the Golgi apparatus (33). Accordingly, the TMDs of plasma membrane proteins are longer than those of Golgi proteins (34). It is therefore possible that differences in membrane thickness, such as those that occur along the secretory pathway, might play a role in regulating 6K topology and structure. Computation of the free energy according to values determined by Wimley and White (19, 20) indicate that the inside-outside TMD (number 2 in model 6A) would not remain stably immersed in the membrane core (data not shown). It is therefore conceivable that a topology such as that shown in Fig. 6C, with the interfacial region (gray cylinder) followed by a single transmembrane helix (white cylinder) would be more favorably adopted by the secreted versions of 6K (see also the predictions in Fig. 1).
Several forms of peptide-chain embedding within the external membrane monolayer interface might influence 6K-induced membrane perturbation and permeabilization. In principle, the interfacial sequence might be required to disrupt interactions between lipid molecules and might be directly involved in the destabilization of membrane integrity (Fig. 6C). A similar mechanism has previously been proposed for poliovirus 3A protein (35). The differential surface increase of the external membrane monolayer might also contribute to membrane deformation and bending (positive curvature) at the points of virus budding (4, 7). The tendency to minimize energetically those perturbations might be exploited to assemble oligomeric transmembrane pores that actually induce the rupture of the permeability barrier. Moreover, the interfacial region might participate in the regulation of the opening of those transmembrane pores.
Finally, it should noted that the presence of complex helical
transmembrane regions might be of functional importance to viral proteins that induce membrane perturbations required for fusion or
permeabilization. Thus, an interfacial region similar to the 6K
sequence described here has been reported to occur in the region proximal to the transmembrane anchor of human immunodeficiency virus
gp41 fusion protein (24). A long interfacial sequence preceding the
transmembrane anchor seems to be a common structural motif in fusogenic
proteins belonging to several virus families. Our initial analyses
indicate that interfacial helices adjacent to membrane-spanning domains
might represent a motif conspicuously present also among members of the
viroporin protein family. A comparative study is under way in an effort
to establish the extent to which interfacial sequences contribute to
the pore-forming activity of this family of viral proteins.
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FOOTNOTES |
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* This work was supported by Comunidad Autónoma de Madrid (project number 08.2/0024.2/2000) and Dirección General de Investigación Científica y Tecnológica (project numbers PM99-0002 (to M. A. S., V. M. and L. C.) and BIO2000-0929 (to J. L. N.)). Centro de Biología Molecular was awarded an institutional grant by the Fundación Ramón Areces, Spain.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. Tel.: 34-913978451, Fax: 34-913974799, E-mail: masanz@cbm.uam.es.
¶ Supported by a CSIC-I3P fellowship financed by Fondo Social Europeo.
** Supported by the Basque Government (PI-1998-32) and the University of the Basque Country (UPV 042.310-G03/98).
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M206611200
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ABBREVIATIONS |
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The abbreviations used are: C, capsid; ER, endoplasmic reticulum; SV, Sindbis virus; wt, wild type; BHK, baby hamster kidney; h.p.e., hours post-electroporation; TMD, transmembrane domain; WW, Wimley-White; HB, hygromicin B.
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