From the Departments of Medical Microbiology and
¶ Biochemistry, Nijmegen Center for Molecular Life Sciences,
University Medical Center Nijmegen, P. O. Box 9100, 6500 HB Nijmegen,
The Netherlands and the § Department of Pathology,
University Medical Center Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands
Received for publication, July 31, 2002, and in revised form, September 17, 2002
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ABSTRACT |
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The 2B protein of enterovirus is
responsible for the alterations in the permeability of secretory
membranes and the plasma membrane in infected cells. The structural
requirements for the membrane association and the subcellular
localization of this essential virus protein, however, have not been
defined. Here, we provide evidence that the 2B protein is an integral
membrane protein in vivo that is predominantly localized at
the Golgi complex upon individual expression. Addition of
organelle-specific targeting signals to the 2B protein revealed that
the Golgi localization is an absolute prerequisite for the ability of
the protein to modify plasma membrane permeability. Expression of
deletion mutants and heterologous proteins containing specific domains
of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of
both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions
are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in
the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.
Enteroviruses (e.g. poliovirus, coxsackievirus,
ECHOvirus) belong to the family of picornaviridea, a group of
nonenveloped, cytolytic viruses that have a positive stranded RNA
genome of 7.5 kb. The enterovirus genome contains one large open
reading frame that is translated into a single, 220-kDa polyprotein.
Processing of the polyprotein by virus-encoded proteases yields the
structural P1 region proteins that encapsidate the viral RNA and the
non-structural P2 and P3 region proteins that are involved in
replication of the viral RNA. Processing of the P2 and P3 regions
yields the 2Apro, 2B, 2C, 3A, 3B, 3Cpro, and
3Dpol proteins and the more stable cleavage intermediates
2BC, 3AB, and 3CDpro, which have functions distinct from
their cleavage products. Although multiple functions have been
attributed to the mature viral proteins and the cleavage intermediates,
the exact function of most of the viral proteins in the replication
cycle is still largely unknown.
Host cell membranes are subject to a number of profound alterations
upon enterovirus infection. Enteroviruses gradually modify host cell
membrane permeability and rearrange intracellular membranes during
infection. The modification of the plasma membrane permeability is most
likely important for the lysis of the cell and the release of virus
progeny. Modifications of secretory pathway membranes are connected to
viral functions such as genome replication. The modification of host
cell membrane permeability is such that initially calcium is released
from intracellular stores (1). Ionic gradients maintained by the plasma
membrane are also disrupted (2). Also, later in infection, small
compounds such as hygromycin B, a small non-permeative translation
inhibitor, can efficiently enter the cell. The 2B protein has been
identified as the viral protein that is responsible for the alterations
in host cell membrane permeability that take place in
enterovirus-infected cells. Individual expression of the 2B protein was
shown to be sufficient for the release of calcium from intracellular
stores and the increase in plasma membrane permeability to both calcium
and hygromycin B (1, 3-6).
Another important membrane modification that can be observed in
enterovirus-infected cells is the massive proliferation and accumulation of membrane vesicles in the cytoplasm. These vesicles are
derived from the secretory pathway (7) and were shown to be the site at
which viral RNA replication takes place (8, 9). Individual expression
of the viral proteins has shown that the 2BC protein is responsible for
the induction of the vesicle formation (9-12), possibly together with
the 3A protein (13). The virus-induced inhibition of protein secretion
is another important modification, affecting trafficking of host cell
membranes. This inhibition may be advantageous for the virus by
preventing anti-viral host cell responses such as major
histocompatibility complex expression and secretion of interferon and
interleukines (14, 15). Individual expression of the enterovirus
non-structural proteins has shown that both the 2B protein and the 3A
protein are endowed with the ability to inhibit protein secretion (1,
3). The 3A protein was shown to inhibit ER-to-Golgi transport (16). The
step blocked by the 2B protein is still unknown.
The mechanism by which the enterovirus 2B protein exerts its effects on
membranes is as yet unknown. Moreover, it remains to be established
whether the different activities of the 2B protein (i.e.
membrane permeabilization, membrane rearrangement, and secretion inhibition) represent different functions or whether these activities are the result of a general membrane-disturbing activity of the 2B
protein. Our aim is to gain more insight into the structure and
function of the coxsackievirus 2B protein. The coxsackievirus 2B
protein is a small protein of 99 amino acids that, in the infected cell, is present at the virus-induced, secretory pathway-derived membrane vesicles at which the viral replication takes place (17). The
protein contains two hydrophobic regions (Fig.
1), of which one is predicted to form a
cationic amphipathic
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix with characteristics typical of the
group of the so-called membrane-lytic peptides (18). Mutations in the
amphipathic
-helix or the second hydrophobic region were shown to
have deleterious effects on viral RNA replication and virus growth,
indicating that the integrity of these regions is essential for an
early function in the viral life cycle (18, 19). Mutations in these
domains were also found to interfere with the ability of the 2B protein
to increase membrane permeability and to inhibit protein secretion (6).
This strongly suggests that the membrane-modifying activities of the 2B
protein are in some way required for the accumulation of the membrane
vesicles at which viral RNA replication takes place.
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Fig. 1.
The 2B protein contains
two hydrophobic regions, HR1 and HR2. A, hydropathy
plot of the 2B protein of CVB3, according to Kyte and Doolittle (45),
using a window size of 9 residues. B, amino acid sequence of
the two hydrophobic regions shown in the hydropathy plot. C,
helical wheel diagram of the putative amphipathic -helix formed by
HR1. The hydrophobic residues (boxed) are positioned at one
face of the
-helix, and the charged and polar residues are
positioned at the other face of the
-helix.
At present, the determinants for the membrane interaction and the
subcellular localization of the 2B protein have not been defined. In
this study, we have investigated the mode of membrane association
in vivo and the subcellular localization of the 2B protein.
Furthermore, by testing 2B deletion mutants, we have analyzed the
importance of specific domains for the mode of membrane interaction,
the subcellular localization, and the ability to increase membrane
permeability. Moreover, the correlation between the subcellular
localization and the membrane-active character of the 2B protein was determined.
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EXPERIMENTAL PROCEDURES |
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Cells and Media-- Buffalo green monkey (BGM)1 cells were grown in minimal essential medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 units of penicillin/ml, and 100 mg of streptomycin/ml. Cells were grown at 37 °C in a 5% CO2 incubator.
Antibodies and Conjugates-- Rabbit polyclonal anti-EGFP antiserum was a kind gift from Dr. J. Fransen, Department of Cell Biology and Histology, University Medical Center Nijmegen, Nijmegen, The Netherlands. Rabbit polyclonal anti-2B antiserum was obtained by immunizing specific pathogen-free rabbits with a recombinant MBP-2B fusion protein produced in Escherichia coli. Western blot analysis showed that the immune sera specifically recognized the MBP-2B and 2B-EGFP fusion proteins, whereas no reactivity of the preimmune sera was detected (data not shown). Mouse monoclonal anti-C-Myc (clone 9E10) and mouse monoclonal anti-Golgi 58K protein (clone 58K-9) antisera were obtained from Sigma. Rabbit polyclonal anti-calreticulin was obtained from Affinity Bioreagents, Inc. Fluorescein isothiocyanate-conjugated goat anti-rabbit polyclonal antibody, Texas Red-conjugated goat anti-mouse polyclonal antibody, and Texas Red-conjugated goat anti-rabbit polyclonal antibody were obtained from Jackson ImmunoResearch Laboratories. Peroxidase-conjugated goat anti-rabbit immunoglobulin was obtained form Dako Diagnostika.
Plasmids--
The coding sequences of the wild-type 2B protein
or 2B deletion mutants were amplified by PCR and cloned in the pEGFP
fusion vectors described below (Clontech). The
plasmid pCB3/T7 (20), which contains the full-length cDNA of CBV3,
was used as a template in the PCR reactions for the amplification of
the sequence encoding wild-type 2B, 2B(59C), 2B(
14C) and
2B(
20N) proteins lacking amino acids (aa) 41-99, aa 86-99,
and aa 1-20, respectively. The pCB3/T7-2B
HR1 and pCB3/T7-2B
HR2
deletion constructs (21) were used as templates in the PCR reactions
for the amplification of the sequences encoding the 2B(
HR1) and
2B(
HR2) proteins lacking the amphipathic
-helix (aa 34-56) or
the second hydrophobic region (aa 64-80), respectively.
2B-EGFP and EGFP-2B Fusion Proteins-- For the construction of the p2B-EGFP plasmid, the 2B coding sequence was amplified using a forward primer that introduced a SaII restriction site (restriction sites are in italics) and a start codon (underlined) preceded by a Kozak sequence (p115-16, 5'-ctcctgtggctggctagcgtcgacgccaccatgggagtgaaggactatgtggaa-3') and a reverse primer that introduced a BamHI restriction site (p115-20, 5'-ccagctggatccttggcgttcagccatagg-3'). The PCR product was cloned into pEGFP-N3 digested with SalI and BamHI to yield p2B-EGFP. For the construction of the pEGFP-2B plasmid, the 2B coding sequence was amplified by PCR using primer p115-16 and a reverse primer that introduced a stop codon (underlined) and a SmaI restriction site (p115-7, 5'-aagccacccgggctattggcgttcagccatagg-3'). The PCR product was cloned into pEGFP-C1 digested with SmaI and SalI to yield pEGFP-2B.
2B-EGFP Deletion Mutants--
For the construction of the
p2B(59C)-EGFP plasmid, the coding sequence of the N-terminal 40 aa
of the 2B protein was amplified using p115-16 and a reverse primer that
introduced an EcoCRI restriction site (p169-3,
5'-taaggcttttagagagagctctaagatggagtc-3'). The PCR product
was cloned into pEGFP-N2 digested with NheI and
SmaI to yield p2B(
59C)-EGFP.
For the construction of the 2B(14C)-EGFP plasmid, the coding
sequence of aa 1-85 of the 2B protein was amplified using forward primer p115-16 and a reverse primer that introduced a BamHI
restriction site (p115-29,
5'-ggggggggggaccggtgccccggggaccggcggatcctgtttgagccaccgccacgg-3'). The PCR product was cloned into pEGFP-N3 digested with SalI
and BamHI to yield p2B(
14C)-EGFP.
For the construction of the p2B(20N)-EGFP plasmid, the coding
sequence of aa 21-99 of the 2B protein was amplified using a forward
primer that introduced a SalI restriction site and a start
codon (underlined) preceded by a Kozak sequence (p115-30, 5'-ggggggggggtcgacgccatcatggtatgtgagcaagtcaacctc-3')
and a reverse primer p115-20. The PCR product was cloned into pEGFP-N3
digested with SalI and BamHI to yield
p2B(
20N)-EGFP.
For the construction of the p2B(HR1)-EGFP and p2B(
HR2)-EGFP
plasmids, the coding sequences of proteins 2B(
HR1) and 2B(
HR2) were amplified using primers p115-16 and p115-20. The PCR products were
cloned into p2B-EGFP digested with SalI and BamHI
to yield p2B(
HR1)-EGFP and p2B(
HR2)-EGFP, respectively.
For the construction of the plasmids pHR1-EGFP and pHR2-EGFP, the coding sequences of the regions encompassing these regions (aa 30-62 and aa 56-85, respectively) were amplified using primers that introduced a SalI restriction site and a start codon (underlined) preceded by a Kozak sequence at the 5'-end and a BamHI restriction site at the 3'-end. The HR1 coding sequence was amplified using p115-47 (5'-aacctcgtcgacgccaccatgtcactagtgggtcaagactcc-3') and p115-49 (5'-agtcacggattcgtcatcgtggttcctcaccac-3'), and the HR2 coding region was amplified using p115-48 (5'-tcagccgtcgacgccaccatggtggtgaggaaccacgat-3') and p115-50 (5'-atattgggatccctgtttgagccaccgccacgg-3'). The region encompassing both HR1 and HR2 (aa 30-85) was amplified using p115-47 and p115-50. The pHR1-EGFP, pHR2-EGFP, and p(HR1+HR2)-EGFP plasmids were made by cloning the PCR products digested with SalI and BamHI into the pEGFP-N3 vector, digested with the same enzymes. All sequences that were amplified by PCR were checked by sequence analysis.
2B-Myc Constructs--
Plasmids p2B-Myc, p2B(20N)-Myc,
p2B(
14C)-Myc, p2B(
HR1)-Myc, and p2B(
HR2)-Myc were constructed
by deleting the EGFP sequence (using BamHI and
NotI) from the corresponding 2B-EGFP plasmids and replacing
it with a PCR product encoding the C-Myc tag (aa EQKLISEEDL) followed
by a stop codon.
ER-targeted 2B Construct-- For the construction of the plasmids p2B-EGFP-KKAA and p2B-EGFP-AAAA, the EGFP coding region was amplified using a forward primer containing a BamHI restriction site (p318-17, 5'-ggggggggattcatcgccaccgtcagcaagggcgaggag-3') and a reverse primer containing a stop codon (underlined), a NotI restriction site, and a sequence encoding either the AAAAAAKKAA or the AAAAAAAAAA tag (p382-3, 5'-ggggggggcggccgcttacgccgctttcttagctgcggctgccgcgcccttgtacagctcgtccatgcc-3', and p382-4, 5'-ggggggggcggccgcttacgccgcagcagcagctgcggctgccgcgcccttgtacagctcgtccatgcc-3', respectively). The PCR product was cloned into p2B-EGFP digested with BamHI and NotI to yield p2B-EGFP-KKAA and 2B-EGFP-AAAA, respectively.
Golgi-targeted 2B Construct--
For the construction of
Golgi-targeted 2B-EGFP, a construct was generated that contained an
additional SmaI restriction site between the 2B and the EGFP
coding regions. The coding sequence of the 2B protein was amplified
using p115-16 and a reverse primer that introduced a SmaI
restriction site to the 5'-end of the BamHI restriction site
(p318-25, 5'-gggggggggggatcccccgggttggcgttcagccatagg-3'). The PCR product was cloned into p2B-EGFP digested with SalI
and BamHI to yield p2B-EGFP(+SmaI). The coding
sequence of the N-terminal 103 amino acids of
-1,2-N-acetylglucosaminyl-transferase I (NGAT-I) was
amplified from pNGAT-I-GFP (a kind gift from Dr. Graham Warren, Cell
Biology Laboratory, Imperial Cancer Research Fund, London, UK) using a
forward primer that introduced an EcoRV restriction site
(p318-14, 5'-gggggggatatcctgaagaagcagtctgcagg-3') and a
reverse primer that introduced a BamHI restriction site
(p381-13, 5'-ggggggggatcccgccggcgcgggggtcacagg-3'). The PCR product was
cloned into p2B-EGFP(+SmaI) that was digested with
SmaI and BamHI to yield p2B-Golgi-EGFP.
EGFP-Golgi--
The plasmid pEGFP-Golgi was constructed by
replacing the ECFP coding region of pECFP-Golgi
(Clontech) with the EGFP coding region of pEGFP-N1
using the BamHI and NotI restriction sites. In
this construct, EGFP is fused to the N-terminal 81 amino acids of human
-1,4-galactosyltransferase, which targets the EGFP protein to the
trans-medial region of the Golgi apparatus.
Transfections-- BGM cell monolayers were grown to 70% confluency in 6-well plates or on coverslips in 24-well plates and transfected with 5 µg of plasmid DNA/well of a 6-well plate or with 1 µg of plasmid DNA/well of a 24-well plate. Transfections were carried out using FuGENE 6 reagent (Roche Molecular Biochemicals) according to the instructions of the manufacturer. For each transfection reaction, the Fugene reagent was mixed with serum-free medium and incubated for 5 min at room temperature. This mixture was added dropwise to the plasmid DNA preparation and incubated for 15 min at room temperature. The DNA/FuGENE mixture was added dropwise to the cells. Cells were grown at 37 °C until further analysis.
Membrane Association--
BGM cells grown in 6-well plates were
transfected with plasmids encoding the proteins EGFP, wild-type
2B-EGFP, or the 2B-EGFP deletion mutants. At 40 h after
transfection, cells were washed twice with ice-cold TES (20 mM Tris (pH 7.4), 1 mM EDTA, 100 mM NaCl). Samples were kept on ice during the entire procedure. Cells were
scraped in 0.5 ml of ice-cold 1:10 TES, collected by centrifugation for
10 min at 4,500 × g, and resuspended in 250 µl of
1:10 TES. Cells were incubated for 15 min and broken by 30 strokes in a Dounce homogenizer. Nuclei and cell debris were removed by
centrifugation for 10 min at 4,500 × g. Membrane and
cytoplasmic fractions were separated by centrifugation for 1 h at
150,000 × g at 4 °C. Supernatants were removed and
stored at 80 °C until further analysis. Pellet fractions were
either resuspended in one supernatant volume of buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM
EDTA, 0.1 M phenylmethylsulfonyl fluoride, 1% Nonidet
P-40, 0.05% SDS) and stored at
80 °C until further analysis or
resuspended in 200 µl of PBS, 0.1 M
Na2CO3 (pH 11.5), 0.5 M EDTA, 1 M NaCl, 4 M urea, or 1% Triton X-100. Resuspended pellet fractions were incubated on ice for 1 h and centrifuged for 1 h at 150,000 × g and 4 °C.
The supernatant fractions and the pellet fractions, which were
resuspended in 200 µl of lysis buffer, were analyzed by Western blot.
Western Blot Analysis-- Supernatant and pellet fractions of each sample were run on a 12.5% SDS-polyacrylamide gel and transferred to a nitro-cellulose membrane (Bio-Rad). EGFP fusion proteins were stained with the anti-EGFP polyclonal antiserum (dilution 1:10,000) and peroxidase-conjugated goat anti-rabbit immunoglobulins (dilution 1:1,000) and visualized using Lumi-Lightplus Western blotting substrate (Roche Molecular Biochemicals) according to the instructions of the manufacturer.
Immunofluorescence and Confocal Laser Scanning Microscopy-- At 24 h after transfection, BGM cells grown on coverslips were fixed with 4% paraformaldehyde in PBS (pH 7.4). Cells were permeabilized using PBS, 0.1% Triton X-100. Antibodies and conjugates were diluted with PBS, 0.1% Triton X-100, 2% normal goat serum. Primary antibodies were diluted 1:200 (anti-2B), 1:200 (anti-C-Myc), 1:75 (anti-Golgi 58K), or 1:150 (anti-calreticulin). Conjugates were diluted 1:75. Incubations with the primary antibody were carried out overnight at 4 °C, and incubations with the secondary antibody were carried out for 1 h at 4 °C. Cells were washed with PBS, 0.1% Triton X-100 between incubation steps and mounted in Mowiol (Sigma). Cells were analyzed by confocal laser scanning microscopy (CLSM) (Leica TCS NT, Leica Lasertechnik GmbH, Heidelberg, Germany).
Hygromycin B Assay--
BGM cells grown in 6-well plates were
transfected in duplicate with plasmids encoding the proteins EGFP,
wild-type 2B-EGFP, or the 2B-EGFP deletion mutants. At 40 h after
transfection, one of the duplicate wells was starved of methionine for
15 min in the presence of 500 µg/ml hygromycin B, whereas the other
well was starved of methionine for 15 min in the absence of hygromycin B. The cells were then pulse-labeled for 1 h with
[35S]methionine (50 µCi/well) in the presence or
absence of hygromycin B, washed with ice-cold PBS, and lysed in 1 ml of
lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl,
1 mM EDTA, 0.1 M phenylmethylsulfonyl fluoride,
1% Nonidet P-40, 0.05% SDS). Anti-EGFP rabbit polyclonal antiserum
(1:1,000) was added to the cell lysate, and the lysate/antiserum solution was incubated at 4 °C for 18 h. To collect the
antibody-protein complexes, the samples were incubated for 2 h
with protein A-Sepharose (Amersham Biosciences), washed two times with
dilution buffer (0.01 M Tris (pH 8.0), 0.14 M
NaCl, 0.1% bovine serum albumin, 0.1% Triton X-100), washed once with
TSA (0.01 M Tris (pH 8.0), 0.14 M NaCl), washed
once with 0.05 M Tris (pH 6.8), and precipitated. Samples
were resuspended in 35 µl of SDS-sample buffer, boiled for 5 min, and
analyzed by SDS-PAGE. The amount of radiolabeled anti-EGFP-precipitated protein was quantified using a phosphorimaging device (Bio-Rad Multi-Analyst version 1.0.1), and the ratios of the
amount of protein synthesized in the presence and absence of hygromycin
B were determined.
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RESULTS |
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Construction and Characterization of N- and C-terminal Fusions of EGFP to the 2B Protein-- Tagged 2B proteins were used because (i) an anti-2B antiserum might not recognize all 2B deletion mutants due to possible loss of epitopes and (ii) the deletion mutants may become too small (<10 kDa) to be efficiently detected. Fusion proteins of wild-type and mutant 2B to EGFP were used because they may be used in subcellular localization studies in living cells, and the resulting 36-kDa fusion protein is efficiently recognized and detectable by an anti-EGFP serum.
The fusion of a fluorescent protein at the N terminus or C terminus of
a protein may affect its biological properties. Therefore, the
possible effects of the EGFP fusion on the membrane-active character
and the subcellular localization of the 2B protein were studied. The
ability of 2B-EGFP and EGFP-2B to increase plasma membrane permeability
of transfected cells was studied by analyzing the entry of hygromycin
B, a small inhibitor of translation that under physiological conditions
poorly passes the plasma membrane. Cells were transfected in duplicate
wells with the indicated constructs and pulse-labeled in the absence or
presence of hygromycin B. The radiolabeled EGFP fusion proteins were
immunoprecipitated and analyzed by SDS-PAGE to measure the entry of
hygromycin B (Fig. 2A). In
EGFP-expressing cells, the amount of protein synthesis was similar in
the absence or presence of hygromycin B, indicating that the EGFP
protein does not increase plasma membrane permeability to hygromycin B. Expression of the 2B-EGFP protein resulted in the increased entry of
hygromycin B, as was reflected by the almost complete inhibition of
protein synthesis in the presence of hygromycin B. The increase in
plasma membrane permeability induced by the 2B-EGFP protein was similar
to that induced by the untagged 2B protein. The EGFP-2B protein showed
a reduced ability to increase plasma membrane permeability as compared
with the untagged 2B protein.
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The subcellular localization of the 2B-EGFP and EGFP-2B fusion proteins was compared with that of the untagged 2B protein, which was stained with the anti-2B antiserum (Fig. 2B). The 2B-EGFP fusion protein was predominantly present in the juxtanuclear Golgi region, similar to the untagged 2B protein. In contrast, a small portion of the 2B-EGFP was observed in a reticular, ER-like pattern. The subcellular localization of the EGFP-2B protein differed substantially from that of the untagged 2B protein and showed a resemblance to the ER. Based on these results, it was decided to use the 2B-EGFP protein in the subsequent experiments.
In Vivo Membrane Association of 2B-EGFP--
The membrane
association properties of the 2B protein were studied using an in
vivo system in which cytosolic and membrane fractions of EGFP- or
2B-EGFP-expressing cells were collected and analyzed on Western blot.
As expected, the cytosolic EGFP protein was found exclusively in the
supernatant fraction (Fig. 3A). The 2B-EGFP fusion
protein was found exclusively in the pellet fraction, indicating that
the 2B protein is associated with membranes. The mode of membrane
association was further investigated by extracting the membrane
fractions with buffers that discriminate between peripheral membrane
proteins (which are attached to membranes by ionic interactions with
membrane-integral proteins or phospholipid head groups) and integral
membrane proteins (which are embedded in the phospholipid bilayer).
Fig. 3B shows the supernatant and pellet fractions of the
membrane fractions following extraction with the indicated buffers. The
2B-EGFP protein remained attached to membranes upon extraction with PBS
(control) and buffers that extract peripheral membrane proteins by
increasing the ionic strength of the buffer (1 M NaCl),
creating mild chaotropic salt conditions (4 M urea), or
chelating divalent cations (0.5 M EDTA). Moreover, the
protein was still detected in the pellet fraction when membranes were
extracted using alkaline conditions (0.1 M
Na2CO3, pH 11.5), one of the most potent
methods to discriminate between peripheral and membrane-integral
proteins (22). In addition, alkaline conditions convert membranes to
sheets (but do not solubilize membranes) and release soluble proteins
that are trapped inside membranous vesicles. The resistance of 2B-EGFP
to alkaline extraction therefore argues against a peripheral
association of the 2B-EGFP protein at the lumenal side of
endo-membranes. The 2B-EGFP protein was found in the supernatant
fraction only upon solubilization of membranes using 1% Triton X-100,
a non-ionic detergent that releases integral membrane proteins. Taken
together, these findings provide evidence that the 2B protein is
embedded in the phospholipid bilayer, rather than evidence that it is
peripherally associated with membranes.
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In Vivo Membrane Association of 2B-EGFP Deletion
Mutants--
Deletion mutants of the 2B protein were designed to
identify the regions that are important for its mode of membrane
association. Wild-type 2B (99 aa) and deletion mutants of 2B are
depicted in Fig. 3C. Protein 2B(59C) contains only the
N-terminal 40 amino acids of 2B, protein 2B(
14C) lacks the
C-terminal 14 amino acid residues, and protein 2B(
20N) has a
deletion of the N-terminal 20 amino acids. In addition, deletion
mutants were generated that lacked either the amphipathic
-helix (aa
34-56), which will further be referred to as the first hydrophobic
region HR1 or the second hydrophobic region (aa 64-80), which will
further be referred to as HR2.
Cytosolic and membrane fractions of cells transfected with
2B-EGFP deletion mutants were prepared and analyzed as described above
for the wild-type 2B-EGFP protein. Fig. 3C shows that
protein 2B(59C)-EGFP is predominantly present in the cytosolic
fraction, indicating that the N-terminal 40 amino acids of the 2B
protein are not sufficient to mediate membrane association. The
2B(
14C)-EGFP and 2B(
20N)-EGFP proteins were found predominantly
in the pellet fraction, demonstrating that the N and the C terminus of
the protein do not contribute to the membrane association of the
2B protein. Also, remarkably, the 2B(
HR1)-EGFP and 2B(
HR2)-EGFP
proteins were found predominantly in the membrane fraction, indicating that the presence of either one of the hydrophobic regions of the 2B
protein was sufficient to mediate membrane binding.
The membrane-associated 2B-EGFP deletion mutants were further
investigated to determine their mode of membrane association. Membrane
fractions were extracted using PBS, 0.1 M
Na2CO3, pH 11.5, and 1% Triton X-100 (Fig.
3D). The 2B(14C)-EGFP and 2B(
20N)-EGFP proteins
behaved like the wild-type 2B protein and were found predominantly in
the membrane fraction after the extraction with 0.1 M
Na2CO3, suggesting that the N and C terminus of
the 2B protein have little influence on its mode of membrane
association. Both protein 2B(
HR1)-EGFP and protein 2B(
HR2)-EGFP
behaved differently upon Na2CO3 extraction.
Substantial amounts of these proteins were found in the supernatant
fraction upon Na2CO3 extraction, whereas both
proteins were almost exclusively detected in the pellet fraction upon
PBS extraction. These results indicate that both hydrophobic regions
are required for the correct membrane association of the 2B protein.
When the membranes were solubilized using 1% Triton X-100, the
wild-type 2B-EGFP protein as well as 2B(
14C)-EGFP, 2B(
20N)-EGFP,
and 2B(
HR2)-EGFP were predominantly present in the supernatant
fraction. In contrast, the majority of the 2B(
HR1)-EGFP protein
remained present in the pellet fraction upon Triton X-100 extraction,
indicating that 2B(
HR1)-EGFP was pelleted in a membrane-independent
manner upon solubilization of membranes. This result suggests that the
2B(
HR1)-EGFP protein is prone to form aggregates in the absence of
membranes (due to the Triton X-100 extraction). It cannot be excluded
that the 2B(
HR1)-EGFP protein also (partially) formed aggregates in
the Na2CO3 extraction experiment. It is
therefore difficult to draw conclusions regarding the membrane
association properties of 2B(
HR1), other than that it behaves
differently from the wild-type 2B protein, suggesting that this region
is an important determinant for the membrane association properties of
the 2B protein. In the subcellular localization experiments documented
below, however, evidence will be provided that the 2B(
HR1)-EGFP
protein is present as a membrane-associated protein in
vivo. The results of the in vivo membrane association experiments are summarized in Table
I.
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Subcellular Localization of Protein 2B Deletion Mutants--
The
importance of specific domains of the 2B protein for its subcellular
localization was investigated in living cells expressing 2B-EGFP
deletion mutants (Fig. 4). The wild-type
2B-EGFP protein was predominantly present in the juxtanuclear Golgi
region and was additionally found in an ER-like reticular pattern,
similar to what was observed in fixed cells (Fig. 2B). The
2B(59C)-EGFP was present throughout the cell, staining both the
cytoplasm and the nucleus, consistent with the observation that the
protein is not membrane-associated (Fig. 3C). Localization
of the 2B(
14C)-EGFP and 2B(
20N)-EGFP was essentially the same as
that of the wild-type 2B-EGFP protein (data not shown). The
2B(
HR1)-EGFP and 2B(
HR2)-EGFP proteins showed an altered
localization as compared with the wild-type 2B-EGFP protein. The
2B(
HR1)-EGFP protein was present in an ER-like network, and staining
of the juxtanuclear Golgi region was virtually absent. The
2B(
HR2)-EGFP protein was observed in the juxtanuclear Golgi region;
however, staining of the ER-like network was more apparent than for
wild-type 2B-EGFP.
|
A more detailed examination of the subcellular localization was
undertaken in fixed cells by investigating colocalization of the 2B
protein with markers for the Golgi and the ER. In these experiments,
the 2B deletion mutants were analyzed as Myc-tagged proteins. This
procedure was followed because the subcellular localization of the
Myc-tagged 2B protein better resembled that of the untagged 2B
protein (compare Figs. 2B and
5A). Colocalization experiments with the Golgi complex were performed using the EGFP-Golgi fusion protein in which EGFP is targeted to the trans-medial region of
the Golgi complex by fusion to the N-terminal 81 amino acids of human
-1,4-galactosyltransferase. Colocalization experiments with the ER
were performed using the anti-calreticulin antiserum. The 2B-Myc,
2B(
14C)-Myc, and 2B(
20N)-Myc proteins colocalized with the
EGFP-Golgi marker and were virtually absent outside of the Golgi region
(Fig. 5, A-C, respectively), indicating that the N and the
C terminus of the 2B protein have little or no influence on its
subcellular localization. Interestingly, 2B(
HR1)-Myc staining was
completely different from the wild-type 2B-Myc protein. The 2B(
HR1)-Myc protein did not colocalize with EGFP-Golgi (Fig. 5D) but was exclusively present in a network-like pattern
that was identical to the anti-calreticulin-stained ER (Fig.
5F). Also, the 2B(
HR2)-Myc staining differed from that of
the wild-type 2B-Myc protein. The 2B(
HR2)-Myc protein was present in
the Golgi complex (Fig. 5E), but also a substantial part was
observed in a network-like pattern that was identified as the ER (Fig.
5G).
|
To further investigate the role of HR1 and HR2 in the Golgi
localization, the ability of these domains to provide Golgi
localization in the absence of other determinants of the 2B protein was
analyzed. Initially, Myc-tagged HR1 and HR2 constructs were generated.
However, it was found that HR1-Myc and HR2-Myc proteins were too small to be preserved during fixation (data not shown). Therefore,
heterologous proteins were constructed in which regions of the 2B
protein encompassing either HR1 or HR2, or both HR1 and HR2, were fused
to the N terminus of the EGFP protein. The Golgi complex was stained
using the anti-Golgi 58K marker. Colocalization studies of these
proteins with this endogenously expressed Golgi marker showed that the
presence of HR1 could indeed partially target the EGFP protein to the
juxtanuclear Golgi region (Fig.
6A), indicating that HR1 acts
as a Golgi targeting signal in the absence of other domains of the 2B
protein. The HR2-EGFP protein was present in a reticular network that
showed a close resemblance to the ER (Fig. 6B). Its absence
from the Golgi complex suggests that HR2 does not contain any Golgi
targeting signals. In contrast to the HR1-EGFP protein, which was
partially localized in the ER, the (HR1+HR2)-EGFP fusion protein was
predominantly present in the Golgi complex (Fig. 6C), giving
support for an additional role of HR2 in Golgi localization. Taken
together, these findings indicate that, although the amphipathic
-helix is the Golgi targeting signal, the second hydrophobic region
is an important additional determinant for the Golgi localization of
both the 2B protein and the heterologous EGFP protein. The results of
the subcellular localization experiments are summarized in Table I.
|
Membrane-permeabilizing Activity of 2B-EGFP Deletion Mutants-- In the experiments described above, we have shown that the 2B protein is an integral membrane protein that is predominantly localized at the Golgi complex and that these properties are largely dependent on the presence of both hydrophobic regions of the protein. To investigate the importance of different regions of the 2B protein for its membrane-active character, we also tested the 2B-EGFP deletion mutants for their ability to increase membrane permeability to hygromycin B.
First, a time course experiment was performed to define the time point
at which plasma membrane permeability induced by the 2B-EGFP protein
was maximal (Fig. 7A). From
24 h after transfection to 40 h after transfection, an
increased influx of hygromycin B was observed, resulting in almost
complete inhibition of protein synthesis in its presence at 40 h
after transfection. When cells were analyzed at 48 h after
transfection, no further increase in plasma membrane permeability to
hygromycin B was observed. Therefore, all subsequent experiments were
performed at 40 h after transfection.
|
Fig. 7B shows the results of the hygromycin B assay for the
2B-EGFP deletion mutants. All mutants were tested in three independent assays, the amount of protein synthesized was determined by
phosphorimaging, and the ratio of protein synthesis in the presence and
absence of hygromycin B was calculated. Cells expressing wild-type
protein 2B-EGFP showed an increased entry of hygromycin B, reflected by the reduction in protein synthesis to 10% of protein synthesis in
cells expressing the wild-type 2B-EGFP protein in the absence of
hygromycin B. Cells expressing protein 2B(59C)-EGFP showed no influx
of hygromycin B, indicating that the N-terminal 40 amino acids of the
2B protein are not sufficient to increase plasma membrane permeability.
Hygromycin B entry in cells expressing the 2B(
14C)-EGFP protein was
approximately equal to that of cells expressing the wild-type 2B
protein. In cells expressing 2B(
20N)-EGFP, protein synthesis in the
presence of hygromycin B was ~45% of that in the absence of
hygromycin B, reflecting that plasma membrane permeability was reduced
as compared with that of cells expressing the wild-type 2B-EGFP
protein. Cells expressing either the 2B(
HR1)-EGFP or
2B(
HR2)-EGFP fusion protein showed no entry of
hygromycin B, indicating that the ability of these proteins to increase
plasma membrane permeability was completely disrupted. Thus, the
combined presence of both hydrophobic regions is not only required for the membrane-integral character of the 2B protein but also for its
membrane-active function, suggesting that the membrane-integral topology is essential for its ability to modify membrane permeability. The results of the membrane permeabilization experiments are summarized in Table I.
Functional Characterization of Organelle-targeted 2B
Proteins--
The 2B protein is predominantly present in the Golgi
complex of transfected cells. We investigated the
importance of the Golgi localization for the ability to increase plasma
membrane permeability. For this purpose, the 2B-EGFP protein was
targeted exclusively to the ER or to the Golgi complex by fusion of
specific targeting signals. It was shown previously that membrane
proteins acquired ER localization by the addition of a tag sequence
containing the KKAA dilysine motif (AAAAAAKKAA), which acts as an ER
retention signal in mammalian cells (23). The fusion of the KKAA motif to the 2B-EGFP protein indeed abolished the Golgi localization and
resulted in a typical ER localization pattern (Fig.
8A). The presence of a control
tag of 10 alanine residues, containing no additional localization
signals, did not affect the subcellular localization of the 2B-EGFP
protein (Fig. 8A). To confer complete Golgi localization to
the 2B-EGFP protein, a construct was generated in which the 2B protein
was fused at the N terminus of the NGAT-I-GFP construct (which
contains the targeting information of the
-1,2-N-acetylglucosaminyl-transferase I, a type I
med-trans Golgi membrane protein) (24). The resulting 2B-Golgi-EGFP protein was exclusively present in the Golgi complex (Fig. 8A).
|
To analyze the importance of the subcellular localization of the 2B
protein for its membrane-active function, the entry of hygromycin B was
analyzed in cells expressing the targeted 2B proteins. Fig.
8B shows that the ER-targeted 2B-EGFP-KKAA protein was
severely impaired in its ability to increase plasma membrane permeability to hygromycin B. The 2B-EGFP-AAAA protein behaved like the
untagged protein, indicating that the decrease in hygromycin B entry
was not the result of the fusion of a tag to the C terminus of 2B-EGFP.
Rather, the altered subcellular localization of the protein was
responsible for this result, suggesting that the Golgi localization of
the 2B protein is important for its effect on plasma membrane
permeability. This finding was further substantiated by the observation
that the 2B-Golgi-EGFP protein increases plasma membrane permeability
to hygromycin B to the same extent as the untagged 2B-EGFP protein.
These data indicate that the Golgi localization is a critical
determinant for the ability of the 2B protein to modify permeability of
the plasma membrane.
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DISCUSSION |
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The enterovirus 2B protein is responsible for the alterations in the permeability of secretory membranes and the plasma membrane that are observed in infected cells (1, 3, 5, 6). The molecular mechanism by which the 2B protein disturbs host cell membranes is largely unknown. In this study, we have investigated the structural and functional requirements for the in vivo membrane association and the membrane-active function of this important virus protein.
The membrane association of the 2B protein was resistant against extraction with buffers that release peripherally associated membrane proteins, indicating that one or more domains of the protein are embedded in the phospholipid bilayer rather than that the protein associates with membranes through electrostatic interactions with membrane proteins or polar lipid head groups. Expression of deletion mutants and heterologous proteins showed that each of the hydrophobic regions HR1 and HR2 alone could mediate membrane binding. However, in contrast to the wild-type 2B protein, a substantial amount of the 2B proteins lacking either HR1 or HR2 was released upon Na2CO3 extraction, indicating that both hydrophobic regions are required for the correct membrane association. Moreover, the presence of both HR1 and HR2 was absolutely required for the membrane-active function of the 2B protein, consistent with previous reports (6, 11). Together, these findings suggest that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex and that the formation of this complex is required for the membrane association and membrane-active function of the 2B protein.
These findings provide important new insights into the molecular
architecture of the 2B protein in the membrane. The first hydrophobic
region of the 2B protein has been predicted to form a cationic
amphipathic -helix that shows similarities with the group of lytic
polypeptides (18). Amphipathic
-helical lytic polypeptides may form
multimers that build membrane-integral pores by exposing their
hydrophobic sides to the lipid bilayer, whereas their hydrophilic faces
form an aqueous interior (25-27). The 2B protein has been shown to
form homo-multimers by yeast two-hybrid analysis (28), mammalian
two-hybrid analysis (21), and in living mammalian cells using
fluorescence resonance energy transfer microscopy (29). The behavior of
the 2B protein as an integral membrane protein and the observation that
each of the hydrophobic regions HR1 and HR2 can independently mediate
membrane binding lend support to the idea that multimers of protein 2B
build membrane-integral pores and that each of these hydrophobic
regions is involved in the formation of these pore complexes. Fig.
9 shows a model of a multimeric pore
complex formed by 2B proteins. According to this model, HR1 and HR2
interact with each other to form a "helix-loop-helix" hairpin motif
that traverses the lipid bilayer. The hydrophobic regions are separated
by a hydrophilic loop sequence (RNHDD in the CVB3 2B protein).
Presumably both the N and C terminus of the 2B protein reside at the
cytosolic side of the membrane. This suggestion is supported by the
observation that a cytosolic ER retention signal (KKAA) (23, 30) at the
C terminus of the 2B protein is functional in conferring ER retention
to the 2B protein. Furthermore, the lack of glycosylation at
NX(S/T) glycosylation signals introduced at different
positions in the region upstream HR1 argues that the N terminus is
localized at the cytosolic side of the
membrane.2 Finally,
localization of the N and C terminus at the cytosolic side of the
membrane is consistent with the need of proteolytic liberation of the
2B protein from the viral polyprotein by the viral protein
3Cpro, a cytosolic protease. Both the membrane association
and the membrane-active function were independent of the C-terminal
15 amino acids. Although dispensable for the membrane association, the N-terminal 20 amino acids were found to be of importance for the
membrane-active function of the 2B protein, indicating that the
molecular structure and function of the 2B protein depend on more than
just the presence of HR1 and HR2 and therefore require further
investigation.
|
Upon expression in the absence of other viral proteins, the CVB3 2B
protein was localizing in the Golgi complex, consistent with the Golgi
localization described for the 2B protein of the closely related
poliovirus (31). Retention of the 2B protein in the ER by fusion of an
ER retention signal severely inhibited the ability to increase plasma
membrane permeability. This activity, however, was unaffected when the
2B protein was exclusively targeted to the Golgi complex by fusion of a
Golgi targeting signal. This strongly suggests that the Golgi complex
is the target organelle of the 2B protein to exert its effect on the
plasma membrane. It is remarkable that a virus protein that is
localized at the Golgi complex can modify plasma membrane permeability.
The mechanism underlying this activity is as yet unknown. We predict
that this activity is somehow linked to the ability of the 2B protein
to increase Golgi membrane permeability. Previously, we have shown that
expression of the 2B protein resulted in a decrease in the amount of
calcium that could be released from thapsigargin-sensitive stores (1).
Because at that time only the ER was recognized as a
thapsigargin-sensitive store (32), we concluded that the 2B protein
modified ER membrane permeability. Recently, however, the Golgi complex
was also identified as a thapsigargin-sensitive intracellular calcium
store (33). This finding, together with the identification of the Golgi
complex as the main target organelle of the 2B protein, suggests that
the 2B protein causes an increased Golgi membrane permeability and a
general disruption of the ionic gradients maintained by the
Golgi membrane. Alterations in ionic gradients maintained by membranes
of the Golgi complex have been demonstrated to result in functional
disturbances of the Golgi complex. Disruption of cationic gradients by
monensin, an H+/Na+ ionophore that mediates
cation diffusion over Golgi membranes, results in the inhibition of
complex glycosylation in the Golgi complex (reflected by the
inability of secretory proteins to acquire resistance to
endo--N-acetylglucosaminidase H treatment) and inhibition
of protein secretion (34). Analogous to the effects of monensin,
expression of the 2B protein also results in the inhibition of complex
glycosylation (as demonstrated by the
endo-
-N-acetylglucosaminidase H sensitivity of
glycoproteins in 2B-expressing cells) and inhibition of protein
secretion (3). We propose that the disruption of the ionic content of
the Golgi complex by 2B is responsible for this effect. How the
disturbance of the Golgi milieu leads to an increase in plasma membrane
permeability remains to be established. Possibly, the 2B-induced defect
in transport of membrane proteins or lipids beyond the Golgi complex
results in destabilization and permeabilization of the plasma membrane.
However, it cannot be excluded that the effect on the plasma membrane
is caused by the disturbance of another Golgi function(s).
Having identified the Golgi complex as the main target organelle of the
2B protein, the question of what determines the Golgi localization of
the 2B protein arises. In this study, the amphipathic -helix was
identified as a Golgi targeting signal that could confer partial Golgi
localization to the heterologous EGFP protein in the absence of other
domains of the 2B protein. However, the presence of both the
amphipathic
-helix and the second hydrophobic domain was found to be
required for efficient Golgi targeting. The Golgi localization of the
2B protein might depend on the length of the transmembrane domain(s),
as suggested by the "bilayer-thickness model" (35-40). HR1 and HR2
are predicted to consist of 20 and 18 aa respectively, which is
similar to the transmembrane domain of some Golgi resident proteins but
shorter than the transmembrane domain of plasma membrane proteins (35,
36, 41). Alternatively, the "kin-recognition" model suggests that
Golgi residents form large, oligomeric complexes that become too large
to be transported as they reach the correct Golgi compartment (42, 43).
It is unlikely that homo-multimerization reactions of the 2B protein (21, 28, 29) are involved in determining its Golgi localization as 2B
deletion mutants that lacked either HR1 or HR2 were not transported
beyond the Golgi complex, although they were unable to form
homo-multimers (28). Another possible explanation is that the 2B
protein is not transported further along the secretory pathway because
of the lack of specific signals, as was suggested for some trans-Golgi
network markers (35). The possibility that trafficking of the 2B
protein out of the Golgi complex is blocked as a consequence of the
inhibition of protein secretion that is induced by the 2B protein
itself (3, 6) is unlikely as 2B deletion mutants lacking either HR1 or
HR2 are not transported beyond the Golgi complex, although their
function was severely impaired.
How do our findings relate to the situation in virus-infected cells, where the 2B protein is present at the virus-induced membrane vesicles that build the viral RNA replication complex? Initially, these membrane vesicles are derived from the ER (9). Later in infection, however, the Golgi complex has disappeared (12, 31, 44), and Golgi membranes contribute to the vesicle population (7, 44), suggesting that the Golgi complex is gradually used up to produce the membrane vesicles that build the viral replication complex. Therefore, our findings that identified Golgi membranes as the main target of the 2B protein are in agreement with the localization of the 2B and 2BC proteins at the ER- and Golgi-derived membrane vesicles in infected cells.
The exact function of the 2B protein in the early steps in viral RNA
replication remains to be established. The viability of viruses that
carry mutations in the 2B protein closely correlates with the ability
of the 2B protein to increase membrane permeability and inhibit protein
secretion (1, 6), suggesting that the destabilization and/or
permeabilization of secretory pathway membranes by the 2B protein is
somehow required for the accumulation of membrane vesicles or the
formation of the membranous replication complex. Additional studies are
needed to gain better insight into the membrane-active functions of the
2B and 2BC proteins and how these functions might aid in the processes
that are required to facilitate the formation of membrane-associated
replication complexes.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. J. Fransen (Department of Cell Biology and Histology, University Medical Center Nijmegen, Nijmegen, The Netherlands) for the kind gift of the anti-EGFP antiserum.
![]() |
FOOTNOTES |
---|
* This work was partly supported by grants from the European Communities (INTAS 2012).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.:
31-24-3617574; Fax: 31-24-3614666; E-mail:
f.vankuppeveld@ncmls.kun.nl.
Published, JBC Papers in Press, September 18, 2002, DOI 10.1074/jbc.M207745200
2 A. S. de Jong and F. J. M. van Kuppeveld, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
BGM, Buffalo
green monkey;
GFP, green fluorescent protein;
EGFP, enhanced GFP;
ECFP, enhanced cyan fluorescent protein;
aa, amino acids;
NGAT, -1,2-N-acetylglucosaminyl-transferase;
TES, 20
mM Tris (pH 7.4), 150 mM EDTA, 100 mM NaCl;
PBS, phosphate-buffered saline;
CLSM, confocal
laser scanning microscopy;
ER, endoplasmic reticulum.
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