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Originally published In Press as doi:10.1074/jbc.M511766200 on March 15, 2006

J. Biol. Chem., Vol. 281, Issue 20, 14144-14150, May 19, 2006
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The Coxsackievirus 2B Protein Increases Efflux of Ions from the Endoplasmic Reticulum and Golgi, thereby Inhibiting Protein Trafficking through the Golgi*

Arjan S. de Jong{ddagger}, Henk-Jan Visch§, Fabrizio de Mattia{ddagger}, Michiel M. van Dommelen{ddagger}, Herman G. Swarts§, Tomas Luyten, Geert Callewaert, Willem J. Melchers{ddagger}, Peter H. Willems§, and Frank J. van Kuppeveld{ddagger}1

From the Departments of {ddagger}Medical Microbiology and §Biochemistry, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, 6500 HB Nijmegen, The Netherlands and Department of Physiology, Catholic University Leuven, B-3000 Leuven, Belgium

Received for publication, November 1, 2005 , and in revised form, March 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Coxsackievirus infection leads to a rapid reduction of the filling state of the endoplasmic reticulum (ER) and Golgi Ca2+ stores. The coxsackievirus 2B protein, a small membrane protein that localizes to the Golgi and to a lesser extent to the ER, has been proposed to play an important role in this effect by forming membrane-integral pores, thereby increasing the efflux of Ca2+ from the stores. Here, evidence is presented that supports this idea and that excludes the possibility that 2B reduces the uptake of Ca2+ into the stores. Measurement of intra-organelle-free Ca2+ in permeabilized cells revealed that the ability of 2B to reduce the Ca2+ filling state of the stores was preserved at steady ATP. Biochemical analysis in a cell-free system further showed that 2B had no adverse effect on the activity of the sarco/endoplasmic reticulum calcium ATPase, the Ca2+-ATPase that transports Ca2+ from the cytosol into the stores. To investigate whether 2B specifically affects Ca2+ homeostasis or other ion gradients, we measured the lumenal Golgi pH. Expression of 2B resulted in an increased Golgi pH, indicative for the efflux of H+ from the Golgi lumen. Together, these data support a model that 2B increases the efflux of ions from the ER and Golgi by forming membrane-integral pores. We have demonstrated that a major consequence of this activity is the inhibition of protein trafficking through the Golgi complex.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Enteroviruses (e.g. poliovirus, coxsackievirus, ECHOvirus) belong to the family of Picornaviridea, a large family of nonenveloped, cytolytic viruses that contain a single-stranded RNA genome of positive polarity. Upon infection, enteroviruses induce a number of dramatic alterations in their host cell, which serve to create the appropriate conditions for viral RNA replication and/or prevent antiviral host cell responses. One of these alterations is the modification of intracellular Ca2+ homeostasis. We have previously shown that infection of HeLa cells with coxsackievirus results in a reduction of the amount of Ca2+ that can be released from the intracellular stores using thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA),2 the Ca2+-ATPase that transports Ca2+ from the cytosol into the stores. In addition, a gradual increase in the cytosolic Ca2+ concentration ([Ca2+]cyt) was observed due to the influx of extracellular Ca2+ (1).

The enterovirus 2B protein, one of the nonstructural proteins involved in viral RNA replication, plays a major role in the alterations in intracellular Ca2+ homeostasis that take place in enterovirus-infected cells (1, 2). The mechanism by which 2B, or its precursor 2BC, exerts its effects is largely unknown. Ca2+ homeostasis in the intracellular stores (i.e. endoplasmic reticulum (ER) and Golgi) is the net result of the activity of the SERCA on the one hand and the continuous passive Ca2+ leak from these organelles that exists under normal conditions on the other hand (3). Thus, the reductions in the Ca2+ filling state of the stores in 2B-expressing cells may result from a decrease in Ca2+ uptake (through reduced SERCA action) or from an increase in Ca2+ efflux.

The enterovirus 2B protein is a small (97–99 amino acids) membrane-integral protein (4) that upon individual expression localizes to the Golgi and, to a lesser extent, to the ER (4, 5). The enterovirus 2B protein contains two hydrophobic regions (HR1 and HR2), the first of which is predicted to form a cationic amphipathic {alpha}-helix (6). The {alpha}-helix displays characteristics typical for the group of the socalled lytic polypeptides, which may build membrane-integral pores by forming transmembrane multimers (7, 8). Accumulating evidence indicates that 2B forms homo-multimers, in which both hydrophobic regions span the phospholipid bilayer, suggesting that 2B increases the efflux of Ca2+ from the stores by forming transmembrane pores (9–11). However, the possibility that 2B reduces the uptake of Ca2+ has not yet been excluded.

In this study, we investigated ER and Golgi ion homeostasis in 2B-expressing cells. Using organelle-targeted aequorins, we have provided evidence that 2B decreases the Ca2+ content of both the ER and Golgi complex without affecting the Ca2+ uptake. Moreover, measurement of the Golgi pH with a Golgi-targeted pH-sensitive GFP variant revealed that expression of 2B also results in an increased Golgi pH, indicative of the efflux of H+ from the lumen of the Golgi. Together these data support the idea that 2B increases the efflux of ions from the ER and Golgi. Finally, we have shown that this activity of 2B results in the inhibition of protein trafficking through the Golgi complex.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Media—Buffalo green monkey (BGM) kidney cells were grown in minimal essential medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum, 100 units of penicillin/ml, and 25 µgof streptomycin/ml. Cells were grown at 37 °C in a 5% CO2 incubator.

Antibodies, Conjugates, and Reagents—Mouse monoclonals anti-c-Myc (clone 9E10), anti-Golgi 58K, and rabbit polyclonal anti-calreticulin were obtained from Sigma-Aldrich. Texas Red-conjugated goat anti-mouse polyclonal antibody and Texas Red-conjugated goat anti-rabbit polyclonal antibody were from Jackson ImmunoResearch Laboratories. Coelenterazine-W and coelenterazine-N were from Molecular Probes, thapsigargin from LC Services (Woburn, MA), and ionomycin and monensin from Sigma-Aldrich.

Plasmids—Expression plasmids p2B-EGFP and p2B-Myc (4), pER-AEQ (12), and pGolgi-AEQ (13) have been described. The pHluorin and pHluorin-TGN constructs (14) were a kind gift from Drs. G. Miesenbock and J. Rothman, Memorial Sloan-Kettering Cancer Center, New York. pHluorin-Golgi was constructed by cloning the coding sequence of the N-terminal 81 amino acids of human beta 1,4-galactosyltransferase, which contains the targeting information for the med-trans region of the Golgi complex, immediately upstream the pHluorin coding region. The coding sequence of the N-terminal 81 amino acids of human beta 1,4-galactosyltransferase was amplified by PCR using the EGFP-Golgi plasmid (Clontech) as template. The forward primer contained an NheI restriction site (underlined) and a start codon preceded by a Kozak sequence (p318–22; 5'-gggggggctagcgccaccatgaggct tcgggagccgc-3'), the reverse primer contained an EcoRI restriction site (underlined) (p318–23; 5'-gggggggaattcggtggcgaccggtggatcctt-3'). pVSV-G(ts045)-GFP (15) was a kind gift from Drs. P. Keller and K. Simons, Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

Plasmid DNA Transfections—BGM cell monolayers were grown to 70% confluency and transfected using FuGENE 6 reagent (Roche Applied Science) as described previously (4). For aequorin measurements, cells were seeded onto 13-mm coverslips in 12-well plates and transfected with 0.8 µg of aequorin construct with 2 µg of p2B-Myc or 2 µg of non-relevant plasmid DNA (control) per well. For SERCA activity measurements, cells were grown in two 6-well plates and transfected with 5 µg of either p2B-EGFP or pEGFP-N1 DNA (control) per well. For the pHluorin measurements, cells were seeded onto 22-mm coverslips in 6-well plates and transfected with 1.5 µg of pHluorin construct with or without 3 µg of p2B-Myc DNA/well. For the immunofluorescence assays, cells were seeded onto 12-mm coverslips in 24-well plates and transfected using 1 µg of pVSV-G-GFP with or without 2 µg of wild-type or mutant p2B-Myc DNA/well. Cells were grown at 37 °C until further analysis at the indicated times.

Luminescence Monitoring of Ca2+ in ER and Golgi Complex—The cDNA of ER- and Golgi-targeted aequorins with reduced Ca2+ affinity (12, 13) were digested from pcDNA1 with KpnI/NotI and MluI/NotI, respectively, and ligated into pcDNA3.0 (Invitrogen). BGM cells seeded on 13-mm glass coverslips were transfected and used for luminescence measurements as described previously (16). Cells were reconstituted with coelenterazine-N (5 µM) in Ca2+-free Hepes-Tris medium (132 mM NaCl, 4.2 mM KCl, 1 mM MgCl2, 5.5 mM D-glucose, 0.5 mM EGTA, and 10 mM Hepes, pH 7.4) containing ionomycin (1 µM) to deplete the intracellular stores for 1.5 h at 4 °C. Next, coverslips were washed thoroughly with Ca2+-free medium containing 2% (w/v) bovine serum albumin to remove ionomycin and placed in a luminometer. To selectively permeabilize the plasma membrane, cells were perfused with Ca2+-free intracellular medium (10 mM Hepes, 120 mM KCl, 5 mM NaCl, 2.5 mM MgCl2,2mM EGTA, 2.5 mM ATP, pH 7.05) containing 20 µg/ml of saponin for 2 min at 37 °C. ATP-dependent Ca2+ uptake into the stores was started by adding 0.55 mM CaCl2, resulting in a free Ca2+ concentration of 0.1 µM. At the end of the measurement, cells were lysed with 100 µM digitonin in the presence of 10 mM CaCl2 and aequorin photon emission was converted off-line into Ca2+ concentration values according to Montero et al. (12). Values from multiple experiments were expressed as average ± S.E.

SERCA Activity Measurements—SERCA activity was determined using a radiochemical method (17). Transfected cells were harvested in PBS supplemented with 10 mM EDTA at 40 h posttransfection. After washing with PBS, cells were sorted by fluorescence-activated cell sorter analysis using an Altra Hypersort flow cytometer (Beckman Coulter) equipped with an Argon laser running at 20 milliwatts. Forward scatter, side scatter, and cell fluorescence were analyzed and viable GFP-positive cells were high speed sorted (15,000 cells/s) using a band-pass filter of 525/530 nm and a dichroic mirror of 550 nm. GFP-positive cells were washed with homogenization buffer (250 mM sucrose, 2 mM EDTA, 50 mM Tris-Ac, pH 7.0) and lysed by resuspension in 0.1 ml of H20 and freezing and thawing in liquid nitrogen three times. The homogenate was diluted with 0.2 ml of homogenization buffer. Aliquots of 20 µl were incubated at 37 °C for 40 min in a reaction medium containing 50 mM Tris-Ac (pH 7.0), 5 mM MgCl2,1mM Tris-azide, 0.5 mM EGTA, 0.5 mM EDTA, 5 µM ionomycin, 150 mM KCl, 3 mM ({gamma}-32P)ATP (specific activity 100–500 mCi/mmol), and 0.5 mM CaCl2 (free [Ca2+]5 µM). The reactions were stopped by adding 500 µl of 10% (w/v) charcoal in 6% (w/v) trichloroacetic acid, and the mixture was centrifuged for 30 s at 10,000 x g. To 200 µl of clear supernatant containing the liberated inorganic phosphate (32Pi), 4 ml of OptiFluor (Canberra Packard, Tilburg, The Netherlands) was added, and the mixture was analyzed by liquid scintillation analysis. Blanks were prepared by incubation of the reaction medium in the absence of homogenate. ATPase activity was determined in the presence and absence of 10 µM thapsigargin, and SERCA activity is presented as the difference between the mock-treated and the thapsigargin-treated homogenates.

pHluorin Measurements—Targeted pHluorin constructs were used to measure pH in cytosol, med-trans region of the Golgi complex, and the TGN at the single cell level. Coverslips containing pHluorin-expressing BGM cells were mounted in a Leiden chamber (18) and placed on the stage of an inverted microscope (Axiovert 200 M; Carl Zeiss, Jena, Germany) equipped with a x63, 1.25 NA plan-Neofluar objective. During measurement, cells were perfused with Krebs-Ringer bicarbonate medium. pHluorin was excited at 395 and 470 nm, respectively, using a monochromator (Polychrome IV; TILL Photonics, Gräfelfing, Germany), and fluorescence was monitored at 508 nm. Fluorescence light was directed by a 505DRLPXR dichroic mirror (Omega Optical Inc.) through a 515ALP emission filter (Omega Optical Inc.) onto a Cool-SNAP HQ monochrome CCD-camera (Roper Scientific, Vianen, The Netherlands). Hardware was controlled with Metafluor 6.0 software (Universal Imaging Corp.) running on a PC equipped with 1 Gb RAM and Windows XP Professional.

Immunofluorescence and Confocal Laser Scanning Microscopy—BGM cells grown on coverslips were fixed with 4% (w/v) paraformaldehyde in PBS (pH 7.4) at the indicated time points posttransfection. Cells were permeabilized using PBS/0.1% (v/v) Triton X-100. Primary antibodies and conjugates were diluted 1:200 with PBS/0.1% (v/v) Triton X-100/2% (w/v) normal goat serum (NGS). 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% (v/v) Triton X-100 between incubation steps and mounted in mowiol (Sigma-Aldrich). Cells were analyzed using confocal laser scanning microscopy (Leica TCS NT; Leica Lasertechnik GmbH, Heidelberg, Germany).

Calculations—Data are presented as mean values ± S.D. Differences were tested for significance by means of the Student's t-test.


Figure 1
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  		FIGURE 1.
The 2B protein modifies ER and Golgi Ca2+ homeostasis at steady-state ATP levels. Ca2+ levels were determined in the ER (A) and the Golgi complex (B) of permeabilized 2B-expressing cells and control cells. BGM cells were transfected with the indicated aequorin and p2B-Myc or a control plasmid and processed for luminometric analysis of populations of cells. At the indicated times, the organelles were depleted of Ca2+, and the aequorins were reconstituted with coelenterazine. Cells were subsequently transferred to the luminometer chamber. For the ER and Golgi measurements, cells were perfused with 20 µg/ml of saponine for 2 min at 37 °C to permeabilize the plasma membrane and subsequently perfused with 0.1 µM free Ca2+ and 2.5 mM ATP. Cytosolic measurements were performed with non-permeabilized cells that were perfused with 1 mM Ca2+. Aequorin luminescence was collected and calibrated into [Ca2+] values as described under "Experimental Procedures." Representative traces are shown of control cells (black) and 2B-expressing cells (gray). The bar indicates when Ca2+ was added. The mean [Ca2+] ± S.D. of four individual experiments is shown. *, p < 0.005.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The 2B Protein Disturbs ER and Golgi Ca2+ Homeostasis without Affecting Ca2+ Uptake—Previously, we demonstrated that 2B expression resulted in a reduced filling state of the intracellular Ca2+ stores in intact HeLa cells (2). Here, we investigated the effect of 2B on ER and Golgi Ca2+ levels in BGM kidney cells. Measurements of organelle [Ca2+] were performed in cells that were permeabilized with saponin and then incubated in an intracellular medium containing a high ATP concentration (saponin specifically permeabilizes the plasma membrane without affecting endomembranes). By following this approach, it can be excluded that possible effects on organelle [Ca2+] are caused by reduced SERCA activity due to ATP depletion. The [Ca2+]ER and [Ca2+]Golgi were determined by bioluminometric analysis of cell populations using the Ca2+-sensitive photoprotein aequorin that was targeted to these organelles by fusion to specific targeting signals (12, 13). BGM cells were cotransfected with expression constructs of either ER- or Golgi-targeted aequorin together with either p2B-Myc or a control plasmid. Upon perfusion of the permeabilized cells with intracellular medium containing 0.1 µM free Ca2+ and 2.5 mM ATP, the organelle [Ca2+] increased rapidly by the action of the SERCA (Fig. 1, A and B, left panels). No increase in organelle [Ca2+] was observed under these conditions in the absence of saponin, indicating that the cells were efficiently permeabilized (data not shown). Moreover, the increase in luminescence observed upon addition of ATP and Ca2+ was blocked by the SERCA inhibitor cyclopiazonic acid, indicating that Ca2+ is actively stored in the ER and Golgi compartment (data not shown). After ~40 s, a steady state [Ca2+] was reached at which the amount of Ca2+ pumped into the organelle by the SERCA was equal to the Ca2+ leak. In control cells, the mean steady-state [Ca2+]ER was 650 µM (Fig. 1A, right panel). In 2B-expressing cells, however, this value was significantly (p < 0.005) decreased to 430 and 395 µM at 18 and 40 h posttransfection, respectively. Expression of the 2B protein had a similar effect on [Ca2+]Golgi (Fig. 1B, right panel). In control cells, the mean steady-state [Ca2+]Golgi was 520 µM, whereas in 2B-expressing cells this value was significantly (p < 0.005) decreased to 340 and 325 µM at 18 and 40 h posttransfection, respectively. These results demonstrate that 2B expression reduces the Ca2+ content of both the ER and Golgi complex in BGM kidney cells and that this effect is not caused by ATP depletion.


Figure 2
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  		FIGURE 2.
The 2B protein does not inhibit SERCA activity. BGM cells were transfected with pEGFP-N1 (control) or p2B-EGFP expression plasmids. At 40 h posttransfection, SERCA activity was determined using an in vitro radiochemical ATPase assay. Fluorescent cells were sorted using fluorescence-activated cell sorter analysis and homogenized. The ATPase activity of the homogenates (2.5 x 106 cells) was determined by analyzing the hydrolysis of radiolabeled ATP in the absence and presence of 10 µM SERCA-inhibitor thapsigargin. The ATPase activity in the absence of thapsigargin was calculated relative to the ATPase activity in the presence of thapsigargin, which was set at 100%. The difference between these two measurements represents SERCA ATPase activity and is indicated by an arrow. The mean ATPase activity of three independently performed experiments is shown. No difference is observed in the thapsigargin-sensitive ATPase activity in control cells and 2B-expressing cells.

 
To address the possibility that the 2B protein reduced [Ca2+]ER and [Ca2+]Golgi by inhibiting the SERCA pump, we performed an in vitro radiochemical analysis of SERCA activity (17). BGM cells were transfected with pEGFP-N1 (control) or p2B-EGFP. At 40 h posttransfection, fluorescent cells were sorted by fluorescence-activated cell sorter analysis and subsequently homogenized. ATPase activity of the homogenate was determined by analyzing ({gamma}-32P)ATP hydrolysis, counting the amount of liberated radiolabeled inorganic phosphate (32Pi). Homogenates were assayed in the absence and presence of 10 µM thapsigargin and at an ambient-free [Ca2+]of5 µM. The total amount of liberated 32Pi was equal in EGFP- and 2B-EGFP-expressing cells (45 ± 3 and 46 ± 10 nmol/h/106 cells, respectively), demonstrating that 2B does not affect overall ATPase activity. In each experiment, the total ATPase activity was set at 100%, to which the activity measured in the presence of thapsigargin was related. Fig. 2 shows that the thapsigargin-sensitive ATPase activity was not different between control cells and 2B-expressing cells and amounted to ~45% of the total activity. This indicates that the 2B protein does not inhibit the SERCA pump. Together with the observation that reduced Ca2+ filling of the ER and Golgi complex occurs also when ATP is not rate-limiting, this argues against an effect of 2B on Ca2+ uptake.


Figure 3
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  		FIGURE 3.
The 2B protein increases the pH of the Golgi complex. A and C, BGM cells were transfected with the indicated constructs, fixed at 18 h posttransfection, stained with the anti-c-Myc antiserum, and processed for CLSM analysis. B and D, BGM cells were transfected with the indicated constructs and processed for digital imaging microscopy of individual cells. At the indicated times posttransfection, the 395/470-nm excitation ratio of individual cells, which is indicative for the pH value, was determined as described under "Experimental Procedures." The mean value ± S.D. of two experiments is shown. R395/470, 395/470-nm excitation ratio. *, p < 0.05; **, p < 0.005. E and F, BGM cells were co-transfected with pHluorin-Golgi and either an empty control plasmid or the 2B-Myc expression plasmid. Cells were fixed at 40 h posttransfection, stained with antisera against ER-localized calreticulin (E) or Golgi-localized 58K (F), and processed for CLSM analysis. Bar,10 µm.

 
The 2B Protein Disturbs the pH of the Golgi Complex—We next investigated whether the 2B-induced increase in ER and Golgi membrane permeability is Ca2+-specific or also applies to other ions. Therefore, we analyzed the effect of 2B expression on the pH of the Golgi complex, reasoning that non-selective alterations in membrane permeability would result in a disturbance of the proton gradient that exists over the Golgi membranes. This proton gradient is the consequence of the gradual acidification of secretory pathway organelles by the activity of the vacuolar H+-ATPase (19). Intra-organelle pH measurements were performed by digital imaging microscopy of individual cells expressing targeted ratiometric pHluorin constructs. pHluorin is a variant of wild-type (non-enhanced) GFP that displays a pH-dependent fluorescence excitation spectrum due to specific substitution mutations (14). The excitation spectrum of pHluorin displays two excitation peaks, one at 395 and one at 470 nm. At low pH values, emission intensity is low at 395 nm and high at 470 nm, resulting in a low 395/470-nm fluorescence emission ratio at 510 nm (R395/470). At high pH values, emission intensity is high at 395 nm and low at 470 nm, resulting in a higher R395/470 value. Thus, the R395/470 increases as a function of the pH and can be used to determine pH differences (14).

First, we investigated whether the ratiometric pHluorins allowed us to sensitively detect in vivo pH differences in BGM cells. To this end, we determined the R395/470 emission ratio of the cytosol (pHcyt ~ 7.2), the med-trans region of the Golgi complex (pHGolgi ~ 6.4), and the trans-Golgi network (pHTGN ~ 5.9) (pH values described in Ref. 19). Cells were transfected with pHluorin expression constructs targeted either to the cytosol (pHluorin-cyt), the med-trans region of the Golgi complex (pHluorin-Golgi), or the trans-Golgi network (pHluorin-TGN). Fig. 3A shows the localization of the targeted pHluorins. At 40 h posttransfection, the R395/470 fluorescence emission ratio of the different subcellular compartments was determined. The mean R395/470 of pHluorin-cyt was 1.04 ± 0.02 (Fig. 3B), indicative of the neutral pH that exists in the cytosol. The mean R395/470 values obtained with pHluorin-Golgi (0.59 ± 0.10) and pHluorin-TGN (0.44 ± 0.07) were significantly lower (p < 0.005), reflecting the lower pH values in these compartments.

Next, we investigated the effect of 2B expression on the pH of the Golgi complex. Previous work showed that the 2B protein predominantly localizes to the med-trans region of the Golgi complex (4). Therefore, we used the med-trans Golgi-targeted pHluorin (pHluorin-Golgi) to investigate the effect of 2B expression on the pH in this compartment. In this part of the study we used the 2B-Myc protein because fluorescence derived from 2B-EGFP would contaminate the pHluorin signals. Colocalization of 2B-Myc and pHluorin-Golgi was confirmed by confocal laser scanning microscopy analysis (Fig. 3C). Fig. 3D demonstrates that in 2B-expressing cells the R395/470 was increased to 0.72 ± 0.09 at 18 h posttransfection (p < 0.05) and to 0.81 ± 0.12 at 40 h posttransfection (p < 0.005), suggesting that the 2B protein increases the pH of the Golgi. Expression of a control protein (i.e. Golgi-targeted aequorin) had no effect on Golgi pH (Fig. 3D), indicating that the increased Golgi pH in 2B-expressing cells is not a mere consequence of the overexpression of a Golgi-localized membrane protein. To exclude the possibility that the observed higher R395/470 ratio is due to an altered localization of the pHluorin probe (e.g. in earlier, higher pH compartments), we analyzed the subcellular localization of pHluorin-Golgi in the absence and presence of 2B-Myc at 40 h posttransfection. Fig. 3, E and F, shows that the subcellular localization of pHluorin-Golgi was not altered by 2B expression.


Figure 4
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  		FIGURE 4.
The 2B protein inhibits VSV-G trafficking through the Golgi complex. BGM cells were transfected with pVSV-G-GFP or cotransfected with pVSV-G-GFP and p2B-Myc. Cells were incubated at 40 °C for 18 h to accumulate VSV-G-GFP in the ER. Cells were additionally incubated for 2 h at 40 °C (A) or at 32 °C to allow VSV-G-GFP trafficking along the secretory pathway (B–D). Cells were fixed, stained with the anti-c-Myc antiserum, and processed for CLSM analysis. A, VSV-G-GFP accumulation in the ER upon incubation at 40 °C. B, VSV-G-GFP exposure on the plasma membrane upon subsequent incubation at 32 °C. C, VSV-G-GFP accumulation in the Golgi complex upon treatment with 5 µM monensin for 20 h. D–F, VSV-G-GFP accumulation in the Golgi complex upon co-expression with the 2B-Myc protein. Bar,10 µm.

 
Together, these data demonstrate that the 2B protein not only causes the efflux of Ca2+ from the intracellular stores but also significantly alters the pH of the Golgi complex. Importantly, the increase in Golgi pH was also observed in coxsackievirus-infected BGM cells at 5 h postinfection (data not shown).

The 2B Protein Inhibits Protein Transport through the Golgi Complex—Alterations in lumenal ion concentrations of secretory pathway organelles can have deleterious effects on protein transport. For instance, the monensin-induced increase in pH of the Golgi complex has been shown to result in the inhibition of protein trafficking through the Golgi complex (20). Recent evidence suggests that there may also be a role for Ca2+ in vesicular traffic. The release of Ca2+ from the lumen of transport vesicles is involved in many soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent fusion events (21). Here, we investigated whether the 2B-induced disturbances of ion homeostasis in the lumen of the ER and Golgi complex affect protein trafficking. As a marker for protein trafficking, we used the temperature-sensitive mutant of the envelope glycoprotein of vesicular stomatitis virus (VSV-G-ts045, hereafter referred to as VSV-G) fused to the N terminus of GFP. At 40 °C, the temperature-sensitive fusion protein (VSV-G-GFP) is misfolded and accumulates in the ER (Fig. 4A). Upon shifting the temperature to 32 °C, the protein is correctly refolded and transported via the secretory pathway to the plasma membrane (Fig. 4B) (22). Fig. 4C shows that treatment of BGM cells with monensin resulted in accumulation of VSV-G-GFP in the Golgi region, consistent with earlier observations reported in the literature (20).

To test the effect of 2B on vesicular trafficking, BGM cells were transfected with expression plasmids encoding VSV-G-GFP and 2B-Myc. Cells were incubated at 40 °C for 18 h to accumulate VSV-G-GFP in the ER and subsequently at 32 °C for 2 h to allow its transport out of the ER. Cells were fixed, stained with anti-c-Myc, and processed for CLSM. In 2B-expressing cells, VSV-G-GFP colocalized with the 2B-Myc protein in the med-trans region of the Golgi complex (Fig. 4, D–F), demonstrating that 2B expression resulted in the inhibition of protein trafficking through the Golgi complex. Inhibition of protein trafficking by 2B was evident as early as 18 h posttransfection (data not shown).


Figure 5
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  		FIGURE 5.
Mutations in the putative pore-forming regions of 2B interfere with its ability to inhibit VSV-G trafficking. BGM cells were cotransfected with pVSV-G-GFP and the indicated mutant p2B-Myc plasmids, incubated at 40 °C for 18 h, and additionally incubated for 2 h at 32 °C. Cells were fixed, stained with the anti-c-Myc antiserum, and processed for CLSM analysis. The K41L/K44L/K48L mutant (A) and I64S/V66S mutant (B) were impaired in the ability to inhibit VSV-G-GFP trafficking. The ins (5)linker mutant (C), the ins (34)linker (D), and the ins (94)linker mutant (E) inhibited VSV-G-GFP trafficking through the Golgi complex. Bar,10 µm.

 
To investigate whether the ability of 2B to inhibit protein transport is connected to its ability to increase ion permeability of ER and Golgi membranes, we analyzed VSV-G-GFP trafficking in BGM cells expressing 2B mutants that were previously shown to be defective in increasing ion permeability of these membranes (2). The mutants in question, K41L/K44L/K48L and I64S/V66S, have mutations in the hydrophobic regions HR1 and HR2 that are implicated in pore formation (23). BGM cells were transfected with expression plasmids of the fluorescent VSV-G-GFP fusion protein and mutant 2B-Myc constructs and processed as described above. The K41L/K44L/K48L mutant, which localized to the ER, did not inhibit VSV-G-GFP trafficking through the Golgi complex, resulting in VSV-G-GFP exposure on the plasma membrane (Fig. 5A). The same lack of inhibition was obtained with the I64S/V66S mutant, which localized to the Golgi complex (Fig. 5B). These findings are in agreement with the idea that the ability of 2B to increase ER and Golgi membrane permeability is required for its inhibitory effect on protein transport. Consistent with this idea, VSV-G-GFP trafficking through the Golgi complex was also inhibited in HeLa and Chinese hamster ovary cells (data not shown), two other cell types in which 2B expression results in an increase in ER and Golgi membrane permeability (2).

We also analyzed VSV-G-GFP trafficking in cells expressing 2B mutants that carried linker insertions in regions outside the hydrophobic regions HR1 and HR2. The mutants in question, ins (5)linker, ins (34)linker, and ins (94)linker mutants, contain a 9-amino acid linker inserted at the indicated amino acid position (23). All three mutants localized to the Golgi complex and inhibited VSV-G-GFP trafficking through this complex (Fig. 5, C, D, and E, respectively). These data indicate that regions outside the putative pore-forming hydrophobic regions are not essential for the transport-inhibiting function of 2B.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we investigated the mechanism by which the coxsackievirus alters ER and Golgi membrane permeability. Using organelle-targeted forms of the Ca2+-sensitive photoprotein aequorin and the pH-sensitive GFP variant pHluorin, we demonstrated that expression of the viral 2B protein results in a reduction of the free Ca2+ concentration in both the ER and Golgi complex and an increase of the pH in the Golgi complex. The 2B-induced decrease in ER and Golgi Ca2+ concentration occurred under conditions that ATP is not rate-limiting. Therefore, the decrease in Ca2+ content of the intracellular stores is not the result of a decrease in cellular ATP content. In addition, measurements of maximal SERCA activity in a cell-free system showed no difference between control and 2B-expressing cells. This indicates that SERCA action is not altered by expression of the 2B protein. Based on these findings, we conclude that the 2B-induced reduction of the steady-state Ca2+ level in intracellular stores is due to increased Ca2+ leakage rather than decreased Ca2+ uptake. Together with the observation that 2B expression results in a reduced H+ concentration in the Golgi complex, these findings lend support to the idea that the 2B protein increases ion permeability of ER and Golgi membranes by forming membrane-integral pores. Previous work with MBP-2B fusion proteins showed an increase in permeability of large unilamellar vesicles to a number of different compounds (11). The molecular mass cut off was estimated to be between 660 and 1000 kDa, suggesting that, in addition to ions, low molecular mass compounds may pass through the putative pores formed by 2B.

The finding that 2B-Myc decreases ER and Golgi Ca2+ levels in BGM cells is in line with previous results in HeLa cells expressing 2B-GFP (2). This latter protein localizes at both ER and Golgi membranes (4, 10). The 2B-Myc protein is mainly observed in the Golgi complex, similar to untagged 2B (4), yet it affects ER and Golgi Ca2+ levels to a similar extent. It is unknown how 2B-Myc affects the ER Ca2+ content. It is possible that a small portion of 2B-Myc is localized at ER membranes, yet not detected by immunofluorescence, and forms pores in these membranes, suggesting a direct effect on ER Ca2+ level. Another, not mutually exclusive, explanation is that Golgi-localized 2B-Myc exerts an indirect effect on the ER Ca2+ content. Indeed, there is evidence that ER and Golgi Ca2+ levels are functionally linked. Bcl-2, a major antiapoptotic protein that exerts part of its function from its localization at the ER, reduces the Ca2+ content of both ER and Golgi, although it does not localize to this latter organelle (24). Further studies are required to investigate whether 2B reduces the ER Ca2+ level in a direct or indirect manner.

What consequences do the 2B-induced disturbances in ion homeostasis have for the host cell? Ca2+ is a highly versatile second messenger that is involved in many different functions of the cell. Ca2+ signaling events are tightly regulated and mostly function through the generation of repetitive brief Ca2+ pulses (25). The reduction in organelle Ca2+ levels, and the subsequent increase in cytosolic Ca2+ in 2B-expressing cells (1), may affect Ca2+ signaling events. Ca2+ plays an important role in many physiological processes, including cell proliferation cell death (26). In fact, we recently demonstrated that the 2B-induced reduction in the Ca2+ content of ER and Golgi stores resulted in the down-regulation of Ca2+ signaling between the ER and mitochondria and that this down-regulation of Ca2+ signaling plays an important role in the protection of HeLa cells against certain apoptotic stimuli (2). This apoptosis-suppressing function of 2B may be of great importance for the virus to prevent premature curtailment of the viral life cycle. In addition to the alterations in Ca2+ signaling, the disturbance of the pH in the Golgi complex may be of importance for the viral regulation of the balance between cell death and survival. For instance, the interaction of the papilloma virus E5 oncoprotein with the 16-kDa subunit of the vacuolar H+-ATPase and the subsequent increase in the Golgi pH of E5-expressing cells was shown to be essential for the oncogenic transformation of the host cell (27).

Here, we identified another important consequence of the 2B-induced disturbance in ER and Golgi ion homeostasis. We found that the 2B-induced alterations in membrane permeability of the ER and Golgi complex are paralleled by inhibition of protein trafficking through the Golgi complex. Mutations in the putative pore-forming hydrophobic regions that disrupted the ability of 2B to increase ER and Golgi membrane permeability also impaired its ability to inhibit protein transport. According to our model, this means that 2B forms pores to alter the permeability of ER and Golgi membranes, which in turn leads to inhibition of protein trafficking. Accordingly, mutations outside the putative pore-forming regions did not interfere with the ability of 2B to accumulate secretory proteins in the Golgi complex. The inhibition of protein trafficking through the Golgi may be the result of alterations in the lumenal ionic conditions, which may inhibit the sugar-modifying enzymes of the Golgi complex (i.e. glycosyltransferases and glycosidases). Consistent with this, expression of poliovirus 2B was reported to accumulate secretory glycoproteins in an endoglycosidase H-sensitive glycosylation state (28). Accumulation of improperly glycosylated proteins in the Golgi complex is in agreement with the role of Golgi complex in the quality control of protein folding (29, 30). The inhibition of transport by 2B may be due to its effect on Golgi pH, similar as has been described for the Na+/H+-ionophore monensin (20). The transport inhibition may also be due to the effects of 2B on the Ca2+ concentration in the lumen of the ER and Golgi complex. Ca2+ plays an important role in protein folding (31) and has been implicated in membrane fusion events, which require the local release of lumenal Ca2+ (21, 32). By reducing the lumenal ER and Golgi Ca2+ concentrations, the 2B protein may interfere with such membrane fusion events.

The 2B-induced alterations in Golgi ion homeostasis and the resulting inhibition of protein trafficking may eventually lead to changes in Golgi morphology. At later time points posttransfection, we found that in cells expressing high levels of 2B the Golgi appeared more diffuse (data not shown). A similar observation was reported in cells expressing high levels of poliovirus 2B (5).

To our knowledge, the enterovirus 2B protein is the first viral protein that interferes with protein trafficking through the Golgi complex by forming non-selective pores that allow the diffusion of ions from the Golgi lumen. Another viral protein that interferes with protein transport is the influenza virus M2 protein, yet this protein disturbs the pH of the Golgi complex and the TGN by forming a specific proton channel (33). Perturbation of the pH of the Golgi and TGN by influenza M2 was shown to slow trafficking of various glycoproteins (influenza hemagglutinin, F glycoprotein of paramyxovirus SV5, and {gamma}-glutamyltranspeptidase) along the secretory pathway (34, 35).

The relevance of the 2B-induced inhibition of protein transport for the viral life cycle remains to be established. Enteroviruses most likely inhibit secretory pathway transport to down-regulate innate immune responses (secretion of cytokines) as well as adaptive immune responses (exposure of peptide-loaded MHC-I molecules) and to interfere with the recycling of (labile) death receptors (e.g. tumor necrosis factor receptor) to the cell surface (36–38). Both the enterovirus 2B and 3A proteins have been shown to interfere with vesicular protein trafficking when expressed individually (1, 39, 40). It is difficult to dissect the relative importance of the inhibitory effects of 2B and 3A for the evasion of anti-viral immune responses in infected cells. For this, enteroviruses must be obtained that carry mutations in 2B or 3A that interfere with the transport-inhibiting activity of these proteins but that do not affect virus growth. Until now, only 3A mutants have been obtained that fulfill these requirements (39, 41). Kirkegaard and co-workers (39) demonstrated that such a 3A mutant displayed a reduced ability to inhibit cytokine secretion and MHC-I exposure, confirming the importance of the transport inhibition induced by the 3A protein for the evasion of anti-viral immune responses in infected cells. Unfortunately, no enterovirus 2B mutants have yet been described with a reduced ability to inhibit protein transport but that show a wild-type growth, most likely because the effect of 2B on ER and Golgi membrane permeability is connected to its function in viral RNA replication (see below). It is therefore as yet impossible to define the importance of the transport-inhibiting function of the 2B protein for the suppression of immune responses by the host cell.

Is the ability of 2B to increase ion permeability of ER and Golgi membranes required for its function in viral RNA replication? Mutations in the 2B protein cause early defects in viral RNA replication (6, 42, 43). Moreover, our previous reports suggest that there is a close correlation between the ability of 2B mutants to alter membrane permeability and to support viral RNA replication (23, 40). Infected cells contain both the 2B protein and its relatively stable precursor 2BC. We found that expression of the 2BC protein, but not of the 2C protein, also results in the accumulation of VSV-G-GFP in the juxtanuclear Golgi region.3 The 2BC protein has been identified as the viral protein that is responsible for the accumulation of the membrane vesicles at which viral RNA replication takes place (44–46). These replication vesicles have been proposed to be anterograde transport vesicles that accumulate in infected cells (47). The 2B(C)-induced increase in membrane permeability of these anterograde transport vesicles might be involved in their cytoplasmic accumulation, possibly because the increase in the lumenal pH, which was also observed in coxsackievirus-infected cells (data not shown), inhibits their trafficking or because the reduced lumenal Ca2+ concentrations abolish Ca2+-dependent membrane fusion events that require the local release of lumenal Ca2+ (21, 32). Alternatively, it has been suggested that the membranes that support viral replication are of autophagic origin (46, 48). It is also possible that the 2B(C)-induced changes in organelle Ca2+ and H+ concentrations lead to cellular stress, culminating in the accumulation of autophagic vacuoles. Further research is required to characterize the functional relevance of the poreforming capacity of the 2B protein for the viral life cycle.


   FOOTNOTES
 
* This work was partly supported by grants from the Netherlands Organization for Scientific Research (NWO-VIDI-917.46.305), the M. W. Beijerink Virology Fund from the Royal Netherlands Academy of Sciences, and the European Communities (INTAS 2012). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: 31-24-3617574; Fax: 31-24-3614666; E-mail: f.vankuppeveld{at}ncmls.ru.nl.

2 The abbreviations used are: SERCA, sarco/endoplasmic reticulum calcium ATPase; ER, endoplasmic reticulum; GFP, green fluorescent protein; EGFP, enhanced GFP; BGM, Buffalo green monkey; PBS, phosphate-buffered saline; NGS, normal goat serum; TGN, trans-Golgi network; VSV, vesicular stomatitis virus. Back

3 A. S. de Jong and F. J. van Kuppeveld, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Henri Dijkman for advice and support with the CLSM studies, Drs. P. Keller and K. Simons for the kind gift of the pVSV-G(ts045)-GFP construct, and Drs. G. Miesenbock and J. Rothman for the kind gift of the pHluorin and pHluorin-TGN constructs.



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