This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moffat, K.
Right arrow Articles by Wileman, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moffat, K.
Right arrow Articles by Wileman, T.

 Previous Article  |  Next Article 

Journal of Virology, February 2007, p. 1129-1139, Vol. 81, No. 3
0022-538X/07/$08.00+0     doi:10.1128/JVI.00393-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Inhibition of the Secretory Pathway by Foot-and-Mouth Disease Virus 2BC Protein Is Reproduced by Coexpression of 2B with 2C, and the Site of Inhibition Is Determined by the Subcellular Location of 2C{triangledown}

Katy Moffat,1 Caroline Knox,2,{dagger} Gareth Howell,1,{ddagger} Sarah J. Clark,1 H. Yang,1 Graham J. Belsham,1,§ Martin Ryan,2 and Thomas Wileman1*

Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Surrey GU24 0NF, United Kingdom,1 University of St Andrews, School of Biology, Centre for Biomolecular Sciences, North Haugh, St Andrews KY16 9ST, United Kingdom2

Received 24 February 2006/ Accepted 7 November 2006


arrow
ABSTRACT
 
Infection of cells with picornaviruses can lead to a block in protein secretion. For poliovirus this is achieved by the 3A protein, and the consequent reduction in secretion of proinflammatory cytokines and surface expression of major histocompatibility complex class I proteins may inhibit host immune responses in vivo. Foot-and-mouth disease virus (FMDV), another picornavirus, can cause persistent infection of ruminants, suggesting it too may inhibit immune responses. Endoplasmic reticulum (ER)-to-Golgi apparatus transport of proteins is blocked by the FMDV 2BC protein. The observation that 2BC is processed to 2B and 2C during infection and that individual 2B and 2C proteins are unable to block secretion stimulated us to study the effects of 2BC processing on the secretory pathway. Even though 2BC was processed rapidly to 2B and 2C, protein transport to the plasma membrane was still blocked in FMDV-infected cells. The block could be reconstituted by coexpression of 2B and 2C, showing that processing of 2BC did not compromise the ability of FMDV to slow secretion. Under these conditions, 2C was located to the Golgi apparatus, and the block in transport also occurred in the Golgi apparatus. Interestingly, the block in transport could be redirected to the ER when 2B was coexpressed with a 2C protein fused to an ER retention element. Thus, for FMDV a block in secretion is dependent on both 2B and 2C, with the latter determining the site of the block.


arrow
INTRODUCTION
 
The genomes of small RNA viruses have a relatively low coding capacity. In the case of the Picornaviridae, e.g., poliovirus (PV), genomes of approximately 7.5 kb encode a single polyprotein (P1-P2-P3) that is predominantly processed by the viral 3C protease to about 11 mature proteins (VP1 to VP4, 2Apro, 2B, 2C, 3A, 3B, 3Cpro, and 3Dpol). The proteins derived from the polyprotein initiate genome replication and then package the RNA genome to enable infection of new cells. It is likely that the same viral proteins counteract innate and acquired immune responses that would otherwise limit the spread of the infection in vivo. These are highly complex processes, and it is currently a challenge to understand how they are controlled by so few proteins. The functional capacity of the viral polyprotein can be increased when precursor proteins have properties that are different from those of the fully processed products. The precursor forms of the P2 proteins (P2 and 2BC-P3), rather than fully processed forms (2A, 2B, 2C, and 2BC) are, for example, required to initiate negative-strand RNA synthesis (13), and PV 3CD, rather than the 3C protease, shows efficient processing of the P1 capsid protein. Thus, cleavage of P3 at the 3B/3C junction generates 3CD to process P1, while cleavage of P3 at the 3C/3D junction generates the 3D polymerase. At the same time, 3CD functions with 3D to stimulate uridylylation of VPg (18, 22).

The functional capacity of the polyprotein is further increased when cellular changes that occur during viral replication impact negatively on cellular pathways that are critical for innate and acquired immune responses to the virus. Picornavirus infection often shuts down host translation to release ribosomes for the translation of viral RNA. Inhibition of translation induced by viral proteases, e.g., 2A, can suppress synthesis of antiapoptotic proteins, for example, NF-{kappa}B, and induce apoptosis to increase virus release (2, 11). Proteolytic cleavage of the p65-relA subunit of NF-{kappa}B by the 3C protease may further reduce the proinflammatory activity of NF-{kappa}B during infection (20). Infection of cells with picornaviruses also leads to a block in secretion. The block in the secretory pathway by PV appears to be mediated by PV 3A, which, when expressed alone in cells, reduces secretion of ß-interferon, interleukin 6, and interleukin 8 and lowers surface expression of major histocompatibility complex (MHC) class I (3, 6, 8). PV 3A also increases the survival of cells in the presence of tumor necrosis factor alpha by reducing surface expression of the tumor necrosis factor receptor (21). In this way the block in secretion has the capacity to defend the virus from elements of the innate and acquired immune responses in vivo.

Foot-and-mouth disease virus (FMDV) is a picornavirus which causes an economically important disease of pigs and ruminants. The spread of the virus can be limited by vaccination, but vaccination does not prevent the establishment of persistent infections in which live virus can be isolated from the upper respiratory tracts of animals. This failure to produce sterile immunity is the major limitation for the use of vaccination to control outbreaks of FMDV (1). The establishment of a persistent infection suggests that FMDV can inhibit immune defenses, and this is supported by the observation that infection reduces cell surface expression of MHC class I (23). In common with many picornaviruses, FMDV causes rearrangement of cellular membrane compartments. These membranes, which contain nonstructural proteins associated with replication and newly synthesized viral RNA, accumulate next to the nucleus within a fragmented Golgi apparatus (14, 17). Since these structures could disrupt the functioning of the Golgi apparatus, we have investigated the effect of the nonstructural proteins of FMDV on the secretory pathway (16). In contrast to the action of the PV 3A protein described above, the FMDV 3A protein did not block the secretory pathway, but endoplasmic reticulum (ER)-to-Golgi apparatus transport was blocked by FMDV 2BC (16). This was an intriguing observation, because during infection 2BC protein is processed to 2B and 2C by the 3C protease, raising the possibility that processing by 3C could circumvent the block in secretion imposed by 2BC. This is supported by our observation that ER-to-Golgi apparatus transport was not blocked by 2B or 2C expressed individually. For this reason, we have looked at the relationship between 2BC processing and subsequent effects on the secretory pathway in more detail. The results show that even though 2BC is processed rapidly during infection, protein transport to the plasma membrane is still blocked in cells infected with FMDV. The block in transport can be reconstituted by coexpression of 2C and 2B. Under these conditions, the 2C protein located to the Golgi apparatus, and the block in transport occurred in the Golgi apparatus. The block by 2B and 2C could be redirected to the ER if 2C was directed to the ER by fusion to an ER retention element. The results show that for FMDV a block in secretion is dependent on expression of both 2B and 2C, and the site of the block is determined by the subcellular location of 2C.


arrow
MATERIALS AND METHODS
 
Plasmids. The FMDV nonstructural proteins 2Bv5 (which contains a V5 C-terminal tag) and 2C were expressed using the pcDNA 3.1 expression vector (Invitrogen, Paisley, United Kingdom). Vesicular stomatitis virus (VSV) TsO45 glycoprotein (G-protein) fused to yellow fluorescent protein (GYFP) was generated from VSV TsO45 glycoprotein fused to cyan fluorescent protein from Jennifer Lippincott-Schwartz (National Institutes of Health, Bethesda, MD) and has been described previously (16).

Transfection of cells. Vero cells (ECACC 84113001; ECACC, Wiltshire, United Kingdom) were grown in 5% CO2 at 37°C in HEPES-buffered Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 20 mM L-glutamine, 100 SI units/ml penicillin, and 100 µg/ml streptomycin. Cells were grown to 70% confluence and transfected with plasmid DNA (0.5 µg/well in a 24-well plate) using Transfast (Promega, Southampton, United Kingdom) in DMEM for 1 h at 37°C.

Virus infection of cells. FMDV 01BFS was provided by Terry Jackson at the Institute for Animal Health. Virus (multiplicity of infection = 5 PFU per cell) was adsorbed to near-confluent BHK-21 (ECACC 85011433; ECACC, Wiltshire, United Kingdom) cells at 4°C for 0 to 0.5 h in DMEM. The cells were washed with phosphate-buffered saline (PBS) to remove unattached virus. To initiate virus infection, fresh culture medium containing 10% fetal calf serum was then added, and incubation continued at 37°C. For VSV TsO45 G-protein temperature shift experiments, virus was added to cells which were then immediately grown at 40°C without adsorption at 4°C.

Antibodies. Rabbit antibody ß-COP and rabbit anti-ERp57 have been described previously (16). Mouse anti-FMDV 2C (1C8, isotype immunoglobulin G1 [IgG1], and 3F7, isotype IgG2a) were a kind gift of E. Brocchi (IZSLE, Brescia, Italy). Rabbit antibody DM12, which recognizes FMDV 2C, was provided by D Mackay, Institute for Animal Health, Pirbright, United Kingdom. Mouse anti-V5 (isotype IgG2a) was purchased from Invitrogen (Invitrogen, Paisley, United Kingdom). Mouse antiactin was purchased from Sigma (Sigma, Poole, United Kingdom). Mouse anti-GM130 was a kind gift of M. Lowe (University of Manchester, Manchester, United Kingdom) and P. Monaghan (Institute for Animal Health).

Metabolic labeling and immunoprecipitation. BHK-21 cells grown to approximately 90% confluence in 175-cm2 flasks were infected with FMDV for 2 h, trypsinized, starved in methionine- and cysteine-free Eagle's medium for 20 min, and then labeled with 10 MBq of 35S-Promix (Amersham Life Sciences, Chalfont St Giles, United Kingdom) per ml in the same medium for 5 min. Cells were chased by replacing the labeling medium with normal culture medium. At appropriate times after incubation at 37°C, cells were washed and lysed on ice in immunoprecipitation buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 10 mM iodoacetamide, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml [each] leupeptin, pepstatin, chymostatin, and antipain) containing 1% Nonidet P-40. Antigens were immunoprecipitated by overnight incubation with antibodies immobilized with protein A or G coupled to Sepharose 4B. Proteins were resolved in sodium dodecyl sulfate (SDS)-polyacrylamide gels and detected by autoradiography.

Western blotting. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto a Protran BA85 cellulose nitrate membrane (Schleicher & Schuell, Dassel, Germany). The membrane was blocked for 1 h in 5% Marvel, 10% normal goat serum, and PBS-Tween 20, incubated with the primary antibody, washed in PBS-Tween 20, and incubated with goat antimouse antibody conjugated to horseradish peroxidase (Southern Biotechnology Associates, Inc., Birmingham, AL); antibodies were diluted in 10% normal goat serum-5% Marvel PBS-Tween 20. After washes, the Western blot was revealed by using enhanced chemiluminescence detection reagents (Amersham Life Science, Buckinghamshire, England).

Indirect immunofluorescence and microscopy. Cells were seeded onto 13-mm glass coverslips (Agar Scientific, Stanstead, United Kingdom) and transfected in situ. Samples were fixed in 4% (wt/vol) paraformaldehyde in PBS for 45 min at room temperature, washed in PBS, and stored at 4°C. Cells were permeabilized and incubated in blocking buffer 1 (50 mM Tris [pH 7.4], 150 mM NaCl, 1% [wt/vol] gelatin, 1% [vol/vol] Nonidet P-40, 30% [vol/vol] normal goat serum) with shaking for 15 min at room temperature. Primary antibodies were diluted in blocking buffer 1 and incubated with coverslips for 30 min at room temperature. Samples were then washed three times in 0.5% Tween 20 (Sigma, Poole, United Kingdom) in PBS. Primary antibodies were detected with Alexa 405-, Alexa 488-, Alexa 568-, or Alexa 633-conjugated species-specific immunoglobulins (Molecular Probes, through Invitrogen, Paisley, United Kingdom) diluted 1:200 in blocking buffer 1. DNA was stained with 50 ng/ml of 4',6'-diamidino-2-phenylindole (Sigma). Coverslips were mounted in Vectashield (Vector Laboratories, Peterborough, United Kingdom) and imaged with a Leica TCS SP2 confocal microscope.

Subcellular membrane fractionation. BHK-21 cells were infected with FMDV for 2.5 h, trypsinized, resuspended in 3 ml of homogenization buffer (HB) (containing 10 mM HEPES [pH 7.4], 1 mM EDTA, 0.25 M sucrose, 1 µg/ml [each] of leupeptin, pepstatin, chymostatin, and antipain [Roche, Lewes, United Kingdom]), and homogenized by 20 passages through a 25-gauge syringe needle. Whole cells and nuclei were removed by centrifugation at 6,000 rpm for 2 min at 4°C in a Sorval Fresco centrifuge. Postnuclear membranes were collected at 33,000 rpm for 1 h at 4°C in a Beckman L8-M ultracentrifuge and resuspended in 1 ml of HB buffer. Discontinuous iodixanol (OptiPrep, 60% wt/vol; Gibco) gradients were prepared in a 12-ml SW40 centrifugation tube. Gradients were generated by underlaying 2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 20.0, and 30.0% iodixanol. The postnuclear membranes were loaded on the top of the gradient and centrifuged at 39,000 rpm for 2.5 h at 4°C in a Beckman L8-M ultracentrifuge. One-milliliter fractions were solubilized and analyzed by immunoblotting to determine the distribution of 2BC, 2C, ERp57, and GM130.


arrow
RESULTS
 
FMDV infection inhibits ER-to-Golgi apparatus traffic. Our previous work (16) showed that FMDV 2BC blocks ER-to-Golgi apparatus transport and cell surface expression of a model membrane protein, the TsO45 G-protein of vesicular stomatitis virus. The same temperature shift experiment was used to see if a block in transport also occurred in cells infected with FMDV (Fig. 1). Cells expressing TsO45 GYFP were incubated at the nonpermissive temperature (40°C) and infected with FMDV for 2 h (Fig. 1 A to C). Cells were then shifted to the permissive temperature (32°C) for 3 h and analyzed by fluorescence microscopy, where infected cells were indicated by staining for 2C (Fig. 1E and H). At 40°C (A and C), the G-protein was located to the ER and nuclear envelope. After 3 h at 32°C (D to I), examination of cells lacking the 2C signal (see arrows in D and G) showed the G-protein at perinuclear sites indicative of the Golgi apparatus. In contrast, in cells infected with FMDV, the G-protein remained in reticular structures in the cytoplasm and the nuclear envelope but was absent from the cell surface. The location of the G-protein in infected cells was studied in more detail using antibodies recognizing the luminal ER protein ERp57 (Fig. 2). In control cells at 40°C (panels A to C), the G-protein colocalized with ERp57, and when cells were incubated at 32°C for 30 min (panels D to F), the G-protein moved to perinuclear vesicular structures lacking ERp57, indicating movement to the Golgi apparatus. In infected cells incubated at 32°C (panels G to I), the G-protein remained colocalized with ERp57, indicating retention in the ER. At higher magnification (panels J to M), colocalization between the ER and G-protein was denoted by arrows a and c; no 2C staining is visible in this location. Where 2C staining was detected, denoted by arrow b, little ERp57, or G-protein, was present (see also Fig. 1, panel F).


Figure 1
View larger version (38K):
[in this window]
[in a new window]

  		FIG. 1. FMDV infection prevents surface expression of the TsO45 G-protein. Vero cells expressing the TsO45 GYFP were incubated for 48 h at 40°C and infected with FMDV for 2 h. Cells were either fixed immediately (A to C) or cooled to 32°C for 3 h (D to I) prior to processing for immunofluorescence. G-protein was visualized through the natural fluorescence of YFP, pseudocolored green (A, D, G), and FMDV infection was indicated by positive immunostaining for 2C, mouse monoclonal 3F7 (B, E, H), visualized by goat secondary antibody coupled to Alex 568 (red). Panels D to F show high-magnification images, while panels G to I show lower magnification. Surface and perinuclear G-protein are indicated by arrows in cells negative for 2C. Merged images are shown in panels C, F, and I.


Figure 2
View larger version (47K):
[in this window]
[in a new window]

  		FIG. 2. FMDV infection prevents movement of TsO45 G-protein from ER membranes. Vero cells expressing TsO45 GYFP were infected with FMDV and subjected to the temperature shift experiment described in the legend to Fig. 1. The ER was visualized using an antibody specific for ERp57 and secondary antibody coupled to Alexa 633 (cyan), and FMDV infection was visualized using antibody against 2C, mouse monoclonal 3F7, and secondary antibody coupled to Alexa 568 (red). Panels A to C show cells incubated at 40°C. Panels D to F show movement of G-protein to a perinuclear location in cells negative for 2C following a temperature shift to 32°C for 30 min. Panels G to H show an infected cell 30 min after the temperature shift experiment. Here the G-protein colocalizes with ERp57-positive membranes. Panels J to M show magnified details taken from the region of interest indicated in image G.

The results show that the G-protein remains localized with the ER marker in infected cells. This implies that the virus prevents movement to the Golgi apparatus. The Golgi apparatus is disassembled in cells infected by picornaviruses, however, and can move away from its characteristic perinuclear location. We cannon rule out the possibility that infection causes Golgi and ER markers to become closely aligned during infection, such as would occur if Golgi and ER membranes were drawn into the replication complex. Even so, the G-protein stain remained reticular and characteristic of the ER, and little G-protein colocalized with 2C, a marker for the replication complex.

The effect of FMDV on ER and Golgi membranes was tested by subcellular membrane fractionation (Fig. 3A). Infection had little effect on the density of ER membranes containing ERp57, which migrated predominantly in fractions 1 to 4 of the gradient. Interestingly, in control cells the Golgi marker, GM130, located to the center of the gradient in fraction 5, but in infected cells GM130 was found in two peaks, one in denser fractions 2 and 3 and the other in light membranes in fractions 8 and 9. The results showed that FMDV infection changed the density of membranes containing GM130, consistent with dispersal of the Golgi apparatus. The heavier GM130 signal comigrated with the ER marker and fractions containing 2C. The 2BC signal was very weak but could be seen following overexposure of the Western blot, where it largely cofractionated with 2C. The reason for the increase in density seen for GM130 is unknown, but we cannot rule out fusion of the Golgi apparatus with the ER, which would be worthy of further study. Alternatively, Golgi membranes may increase in density because they accumulate 2C and/or other viral proteins involved in replication. The latter seems unlikely, since we have been unable to see any colocalization between FMDV 2C and Golgi markers in infected cells (14).


Figure 3
View larger version (40K):
[in this window]
[in a new window]

  		FIG. 3. Membrane association and processing of FMDV 2BC protein during infection. Panel A. Subcellular membrane fractionation of cells infected with FMDV. Uninfected control BHK cells or BHK cells infected with the FMDV O1BFS strain for 2.5 h were homogenized, and postnuclear membrane fractions were separated on a discontinuous iodixanol density gradient. Fractions taken from the bottom of the gradient upwards were examined by immunoblotting for the Golgi marker GM130, ER marker ERp57, and FMDV 2C (mouse monoclonal 1C8). 2BC levels were low and were visualized by overexposure of immunoblots. Panel B. Processing of FMDV 2BC. BHK cells were infected with the FMDV O1BFS strain for 2 h, metabolically labeled with [35S]methionine-cysteine, and then chased in complete growth medium for the indicated times (minutes). Cells were then lysed and proteins immunoprecipitated using DM12 antibody specific for 2C. Samples were resolved by SDS-PAGE and detected by autoradiography. 2BC and 2C are indicated by arrows. Panel C. Steady-state levels of 2BC and 2C. BHK cells were infected with the FMDV O1BFS strain for 2 h. Cells were lysed at 15-min intervals (lanes 1 to 5). Proteins were separated by SDS-PAGE and analyzed for 2BC, 2C, and actin by Western blotting. NI indicates cells taken before infection.

2BC is processed rapidly in cells infected with FMDV. During infection with FMDV, the 2BC protein is processed by the 3C protease to 2B and 2C, which, when expressed individually, are unable to block secretion (16). These observations suggested either that 2BC is processed relatively slowly during infection and/or that 2B and 2C can act together to block transport following processing of 2BC by the 3C protease. The rate of movement of different proteins from the ER to the Golgi apparatus varies considerably; however, maximal rates of transport suggest a half-time of 10 to 15 min for delivery of a membrane protein from the ER to the Golgi apparatus (10, 12). To determine if 2BC would be converted to 2B and 2C during this time period, the proteolytic processing of 2BC in cells infected with the O1BFS strain of FMDV was analyzed by pulse-chase immunoprecipitation. Cells were pulse-labeled with 35S-Promix for 5 min and lysed at increasing times. The 2BC precursor and 2C present in the lysate were immunoprecipitated using an antibody specific for FMDV 2C. Figure 3B shows that even during the 5-min pulse-labeling, the 55-kDa 2BC protein was processed to 2C, as indicated by the appearance of the 36-kDa 2C protein. Significantly, the majority of the 2BC precursor was cleaved during the first 5-min chase, and levels of 2BC continued to decrease during the following 20 min; by 60 min (not shown), FMDV 2BC was barely detectable. The results showed that a substantial proportion of 2BC was processed to 2B and 2C during the time frame required to transport the fastest proteins from the ER to the Golgi apparatus. The steady-state levels of 2BC and 2C were examined over the course of an FMDV infection. Infected cells were lysed at 15-min intervals starting from 2 h postinfection and analyzed by Western blotting (Fig. 3C). 2C was easily detectable at 2 h 30 min, whereas 2BC was barely visible and remained a minor band on the blot until 3 h. After 3 h, levels of 2BC increased but were still much lower than those of 2C. The Western blots support the results of the pulse-chase immunoprecipitation experiments and suggest that processing of 2BC to 2C occurs rapidly, particularly early during infection. Studies have shown that MHC class I proteins move from the ER (28) to the Golgi apparatus with a half-time between 20 and 55 min depending on the allele. Cells infected with FMDV nevertheless show defects in surface expression of MHC class I (23). This suggests that a transport block is maintained by the low relative levels of 2BC that exist during infection and/or that 2B and 2C may act together to block ER-to-Golgi apparatus transport.

FMDV 2B and 2C act together to block the secretory pathway. The effects of FMDV 2B and 2C, expressed separately or together, on the movement of the TsO45 G-protein to the cell surface are shown in Fig. 4. When cells expressing 2B or 2C separately were cooled to 32°C for 3 h, the G-protein (panels C and F) moved to perinuclear structures suggestive of the Golgi apparatus and was also detected at the plasma membrane (arrows). Two examples of cells coexpressing 2B and 2C are shown for comparison in panels G to I and J to L. G-protein was not detected on the surface of these cells, while neighboring cells expressing 2B alone (arrows) show surface staining of the G-protein. Interestingly, there was a pronounced perinuclear accumulation of G-protein in cells coexpressing 2B and 2C (Fig. 4, panels I and L), which differed from the accumulation of the G-protein in vesicular structures associated with the ER we observed previously in cells expressing 2BC (16). The distribution of 2B and 2C in these cells was therefore compared with markers for the ER and Golgi apparatus (Fig. 5). Most areas highly enriched for 2B (red) contained the ER marker ERp57 (B) but were separate from areas that stained strongly for 2C (panel D and arrows in magnified inset). The Golgi marker, ß-COP (G), was moderately dispersed compared with results for control cells and largely separate from 2B (I). Most of the ß-COP signal colocalized with 2C (F; also arrow a, magnified insert); even so, there was not complete colocalization, and 2C (blue) signal separate from ß-COP (green) could be seen (arrow b in magnified insert). Interestingly, the structure indicated by arrow b was positive for 2B and 2C, showing that proportions of the two proteins colocalize in cells in an area enriched for ß-COP. Taken together, the results suggested that when 2B and 2C were coexpressed, the transport of the G-protein was arrested in perinuclear structures near the Golgi apparatus, rather than in the ER.


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		FIG. 4. FMDV 2B and 2C act together to block the secretory pathway. Vero cells expressing TsO45 GYFP were transfected with vectors expressing 2Bv5 (A to C), 2C (D to F), or 2Bv5 and 2C (G to L) and maintained at 40°C for 48 h. They were then cooled to 32°C for 3 h and processed for immunofluorescence microscopy. 2Bv5 was detected by an antibody to the epitope tag and secondary antibody coupled to Alexa 568 (red), and 2C was detected using antibody 3F7 and secondary antibody coupled to Alexa 633 (cyan). G-protein was detected through the natural fluorescence of YFP, pseudocolored green. Arrows show surface staining for the G-protein in cells expressing 2Bv5 or 2C alone. Panels G to I and J to L show two examples where surface staining for the G-protein is seen in cells expressing 2Bv5 alone (arrows) but not in cells expressing 2Bv5 and 2C.


Figure 5
View larger version (42K):
[in this window]
[in a new window]

  		FIG. 5. FMDV 2C locates to structures associated with the Golgi apparatus when expressed with 2B. Vero cells expressing 2Bv5 and 2C were analyzed using ERp57 antibody specific for the ER (A to E) or ß-COP to identify the Golgi apparatus (F to J). The merged images in C and E show that 2Bv5 (anti-v5) (red) and ERp57 (green) overlap (C) but are separate from 2C (1C8) (E). The region of interest indicated in panel E was split into separate colors, and an arrow indicates a 2C-positive structure that is negative for 2Bv5 (red) or ERp57 (green). The merged images in panels H and J show that 2C and ß-COP overlap (H) but are largely separate from 2Bv5 (J). The region of interest in J was split into separate colors. Arrow a indicates a structure positive for 2C and ß-COP and negative for 2Bv5; arrow b indicates a structure positive for 2C and 2Bv5 but negative for ß-COP.

FMDV 2C redirected to the ER is unable to block protein traffic unless coexpressed with 2B. The N terminus of 2B contains two hydrophobic stretches thought to tether 2BC to the cytoplasmic face of the ER. To see if ER tethering alone would allow 2C to block transport of the TsO45 G-protein at the ER, the 2C protein was fused to an ER retention signal taken from the transmembrane domain of the CD3 {varepsilon} chain of the T-cell antigen receptor to generate 2C{varepsilon}. Panel A of Fig. 6 shows again that the unmodified 2C protein largely codistributed with ß-COP; the stain was a little scattered, suggesting that 2C may disrupt the Golgi apparatus, as seen in infected cells (14). 2C was nevertheless separate from G-protein (panel B) retained in the ER at 40°C; moreover, 2C failed to block transport of the G-protein to the surfaces of cells when cooled to 32°C (panel C). The 2C{varepsilon} protein showed reticular staining that colocalized with the ER marker ERp57 (panel D) and G-protein at 40°C (panel E), indicating that the ER targeting sequence redirected FMDV 2C to the ER. When cells expressing 2C{varepsilon} and TsO45 GYFP were then incubated at 32°C for a further 3 h, there was strong immunostaining of the G-protein at the plasma membrane, showing that, as seen for 2C, the 2C{varepsilon} protein alone was also unable to block surface expression of the G-protein (panel F). The effect of coexpressing FMDV 2B with 2C{varepsilon} was therefore examined (Fig. 7). In cells cultured at 40°C, the G-protein (image C) located to reticular structures with some punctate staining and colocalized with the 2C{varepsilon} protein (image B). We have noticed during these experiments that the staining pattern produced by 2B can vary from a reticular to a more punctate membranous stain (image A). However, when the punctate 2B staining was observed at high magnification (panels E to H), the majority of 2B was found to overlap with the 2C{varepsilon} and ERp57 stain, suggesting the punctate 2B still located to a region of the ER. When cells expressing 2B and 2C{varepsilon} were incubated at 32°C for a further 3 h (panels I to L), 2B and 2C{varepsilon} colocalized at the nuclear envelope and to reticular structures in the cytoplasm. The G-protein was absent from the surface of the cell and was retained in structures that contained both 2B and 2C{varepsilon}. Taken together, the results showed that a block in transport by 2C requires 2B and that the site of the block is dependent on the location of 2C.


Figure 6
View larger version (62K):
[in this window]
[in a new window]

  		FIG. 6. FMDV 2C redirected to the ER is unable to block protein traffic. TsO45 GYFP was coexpressed in Vero cells with 2C (A to C) or 2C fused to the ER retention sequence of the CD3{varepsilon} chain (D to F). 2C and 2C{varepsilon} were detected by mouse monoclonal 3F7 and secondary antibody coupled to Alexa 488 or 568. Panels A and B show cells grown at 40°C for 48 h and stained for 2C (green) and ß-COP (red) in A or 2C (red) and G-protein (green) in B. Panel C shows movement of G-protein to perinuclear sites in cells positive for 2C, following cooling to 32°C for 3 h. Panels D and E show cells grown at 40°C for 48 h and stained for 2C{varepsilon} (green) and ERp57 (red) in D or 2C{varepsilon} (red) and TsO45 YFP (green) in E. Panel F shows movement of G-protein to perinuclear sites in cells positive for 2C following cooling to 32°C for 3 h.


Figure 7
View larger version (72K):
[in this window]
[in a new window]

  		FIG. 7. FMDV 2C redirected to the ER blocks protein traffic when coexpressed with 2B. Vero cells transfected with vectors expressing TsO45 GYFP, 2C{varepsilon}, or 2Bv5 were grown at 40°C for 48 h (panels A to D). They were then moved to 32°C for a further 3 h (panels I to L). 2Bv5 was detected by an antibody to the epitope tag and secondary antibody coupled to Alexa 405 (red), and 2C was detected using mouse monoclonal 1C8 and secondary antibody coupled to Alexa 568 (cyan). G-protein was detected through the natural fluorescence of YFP pseudocolored green. The region of interest, image D, was magnified and split into separate colors. Arrows indicate that the vesicular nature of 2B overlaps with the reticular staining of 2C{varepsilon} and G-protein.


arrow
DISCUSSION
 
This work was stimulated by our observation that the FMDV 2BC precursor protein, but not 2B or 2C, blocks ER-to-Golgi apparatus transport when expressed individually in cells (16). Given that 2BC is processed to inactive 2B and 2C during infection, it was important to determine whether the secretory pathway was compromised in cells infected with FMDV. Experiments using the TsO45 G membrane protein of VSV showed that the secretory pathway was indeed blocked in cells infected by FMDV, and the G-protein failed to reach the plasma membrane. Subsequent experiments sought to determine how a block in secretion could be maintained under conditions in which 2BC was processed to 2C and 2B. Proteins move at different rates between the ER and the Golgi apparatus, with half-times ranging from 15 min to several hours (10), and it was possible that 2BC processing could be slow compared with the rate of ER-to-Golgi apparatus transport. A mean rate constant of 2.8% per minute at 32°C has been calculated for ER-to-Golgi apparatus transport of the G-protein in living cells (12), meaning that half the protein synthesized at any one time would take approximately 18 min to reach the Golgi apparatus. Steady-state levels of 2BC were very low early during infection, and cleavage of the majority of 2BC to 2B and 2C proteins in infected cells occurred within 5 min of synthesis. Since this was some threefold faster than the transport of the G-protein from the ER to the Golgi apparatus, it raised the possibility that 2B and 2C may act together to block the secretory pathway. This was verified by showing that coexpression of 2B and 2C prevented surface expression of the G-protein.

While this demonstrated that 2C required 2B to block the secretory pathway, the coexpressed 2B and 2C proteins arrested transport in a compartment containing ß-COP, suggesting inhibition within the Golgi apparatus rather than the ER, as seen in cells expressing 2BC (16). An explanation for this difference came from analysis of the cellular location of 2C. When coexpressed with 2B, the 2C protein located to a perinuclear compartment containing ß-COP, suggesting the Golgi apparatus. In contrast, the 2BC precursor of 2C locates to vesicular structures associated with the ER (16). The site of block therefore seemed to be determined by the location of 2C. The 2B protein has hydrophobic stretches that target 2B (and 2BC) to the ER. Interestingly, when 2C was tethered to the cytoplasmic face of the ER by an alternative ER targeting sequence derived from the CD3 {varepsilon} chain, it failed to prevent surface expression of the G-protein unless coexpressed with 2B.

Even though the function of 2C was dependent on 2B, it was difficult to find evidence for large-scale colocalization of 2B and 2C in cells; even so, a few structures containing both 2B and 2C were seen in the vicinity of the Golgi apparatus. We were unable to detect interactions between 2B and 2C by coimmunoprecipitation experiments in infected cells or in cells coexpressing 2B and 2C (not shown). Protein linkage mapping of PV nonstructural proteins carried out using yeast two-hybrid analysis has shown a relatively strong interaction between 2B and 2C, but the interaction was much weaker in glutathione S-transferase pull-down experiments using in vitro-translated products (5, 24). A mammalian two hybrid study using the P2 proteins of coxsackievirus B3 has demonstrated multimerization of 2B and 2BC and heteromeric complexes containing 2BC and 2B and 2C (7). The same assay, however, failed to detect an interaction between 2B and 2C. It is possible that when 2BC is cleaved to 2B and 2C in infected cells, they may interact, and the ER tethering function of 2B may prevent binding of 2C to the Golgi apparatus. However, if interactions between 2B and 2C do occur, they are likely to be weak, and this may explain our failure to coprecipitate FMDV 2B and 2C.

Much work has focused on the ability of the 3A proteins of PV and coxsackievirus to block ER-to-Golgi apparatus transport (3, 4, 9, 25-27). It is interesting to note that the 2B and 2BC proteins of PV also slow the movement of proteins through the Golgi apparatus (9), and in the context of our results, the ability of PV 2BC to block traffic through the Golgi apparatus also required the N-terminal region of 2B, because 2C alone was unable to affect secretion (9). More recently the 2BC precursor of CVB3 has also been shown to have an inhibitory effect on protein movement through the Golgi apparatus (4). This inhibitory effect of 2BC was greater than that seen for the CVB3 2B subunit, suggesting again that both 2B and 2C work together to prevent secretion.

Picornaviruses may affect the secretory pathway in different ways, because their nonstructural proteins have different amino acid sequences and possibly different functions. The FMDV 3A protein has 50 extra C-terminal amino acids compared with its PV counterpart and lacks the proline and lysine residues in the N-terminal domain that are critical in PV 3A for blocking secretion (3, 26). The 2B proteins of PV and FMDV also differ. The FMDV protein is one-third longer at the C terminus, and hydrophobicity plots suggest this may produce a third hydrophobic domain which is absent from PV 2B. A failure to block secretion has also been reported recently for 3A proteins of human rhinovirus 14, enterovirus 71, hepatitis A virus, and Theiler's virus (3). These viruses either do not need to block secretion, because inflammatory and immune responses are advantageous to their life cycle, or as we have demonstrated for FMDV, they may use 2BC and its products to block ER-to-Golgi apparatus transport in infected cells. For FMDV the block has the potential to contribute to the development of persistent infections seen in ruminants that recover from acute viral infection, and it will be interesting to see if 2BC and coexpressed 2B and 2C have direct effects on MHC class I trafficking, antigen presentation, and/or cytokine secretion. This strategy is not limited to picornaviruses. Recent work shows that the secretory pathway is compromised in cells infected with African swine fever virus (19) and in cells expressing the nonstructural proteins of hepatitis C virus (15), and in common with FMDV, both viruses cause persistent infections and have proved difficult to control through vaccination.


arrow
ACKNOWLEDGMENTS
 
This work was supported by BBSRC grants 49/CO7867, 49/C14570, and 201/S14654 to Graham Belsham, Thomas Wileman, and Martin Ryan and the BBSRC Bioimaging Initiative 201/S11230.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: School of Medicine, Health Policy and Practice, University of East Anglia, Norwich NR4 7TJ, United Kingdom. Phone: 01603 591546. Fax: 01603 593752. E-mail: t.wileman{at}uea.ac.uk. Back

{triangledown} Published ahead of print on 22 November 2006. Back

{dagger} Present address: Rhodes University, Department of Biochemistry, Microbiology and Biotechnology, Grahamstown 6140, South Africa. Back

{ddagger} Present address: University of Leeds, Institute of Molecular and Cellular Biology, Leeds LS2 9JT, United Kingdom. Back

§ Present address: Danish Institute for Food and Veterinary Research, Lindholm, 4771 Kalvehave, Denmark. Back


arrow
REFERENCES
 
    1
  1. Alexandersen, S., Z. Zhang, and A. I. Donaldson. 2002. Aspects of the persistence of foot-and-mouth disease virus in animals—the carrier problem. Microbes Infect. 4:1099-1110.[CrossRef][Medline]
    2. 2
  2. Barco, A., E. Feduchi, and L. Carrasco. 2000. Poliovirus protease 3C(pro) kills cells by apoptosis. Virology 266:352-360.[CrossRef][Medline]
    3. 3
  3. Choe, S. S., D. A. Dodd, and K. Kirkegaard. 2005. Inhibition of cellular protein secretion by picornaviral 3A proteins. Virology 337:18-29.[CrossRef][Medline]
    4. 4
  4. Cornell, C. T., W. B. Kiosses, S. Harkins, and J. L. Whitton. 2006. Inhibition of protein trafficking by coxsackievirus b3: multiple viral proteins target a single organelle. J. Virol. 80:6637-6647.[Abstract/Free Full Text]
    5. 5
  5. Cuconati, A., W. Xiang, F. Lahser, T. Pfister, and E. Wimmer. 1998. A protein linkage map of the P2 nonstructural proteins of poliovirus. J. Virol. 72:1297-1307.[Abstract/Free Full Text]
    6. 6
  6. Deitz, S. B., D. A. Dodd, S. Cooper, P. Parham, and K. Kirkegaard. 2000. MHC I-dependent antigen presentation is inhibited by poliovirus protein 3A. Proc. Natl. Acad. Sci. USA 97:13790-13795.[Abstract/Free Full Text]
    7. 7
  7. de Jong, A. S., I. W. Schrama, P. H. Willems, J. M. Galama, W. J. Melchers, and F. J. van Kuppeveld. 2002. Multimerization reactions of coxsackievirus proteins 2B, 2C and 2BC: a mammalian two-hybrid analysis. J. Gen. Virol. 83:783-793.[Abstract/Free Full Text]
    8. 8
  8. Dodd, D. A., T. H. Giddings, Jr., and K. Kirkegaard. 2001. Poliovirus 3A protein limits interleukin-6 (IL-6), IL-8, and beta interferon secretion during viral infection. J. Virol. 75:8158-8165.[Abstract/Free Full Text]
    9. 9
  9. Doedens, J. R., and K. Kirkegaard. 1995. Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A. EMBO J. 14:894-907.[Medline]
    10. 10
  10. Fries, E., L. Gustafsson, and P. A. Peterson. 1984. Four secretory proteins synthesized by hepatocytes are transported from endoplasmic reticulum to Golgi complex at different rates. EMBO J. 3:147-152.[Medline]
    11. 11
  11. Goldstaub, D., A. Gradi, Z. Bercovitch, Z. Grosmann, Y. Nophar, S. Luria, N. Sonenberg, and C. Kahana. 2000. Poliovirus 2A protease induces apoptotic cell death. Mol. Cell. Biol. 20:1271-1277.[Abstract/Free Full Text]
    12. 12
  12. Hirschberg, K., C. M. Miller, J. Ellenberg, J. F. Presley, E. D. Siggia, R. D. Phair, and J. Lippincott-Schwartz. 1998. Kinetic analysis of secretory protein traffic and characterization of Golgi to plasma membrane transport intermediates in living cells. J. Cell Biol. 143:1485-1503.[Abstract/Free Full Text]
    13. 13
  13. Jurgens, C., and J. B. Flanegan. 2003. Initiation of poliovirus negative-strand RNA synthesis requires precursor forms of p2 proteins. J. Virol. 77:1075-1083.[CrossRef][Medline]
    14. 14
  14. Knox, C., K. Moffat, S. Ali, M. Ryan, and T. Wileman. 2005. Foot-and-mouth disease virus replication sites form next to the nucleus and close to the Golgi apparatus, but exclude marker proteins associated with host membrane compartments. J. Gen. Virol. 86:687-696.[Abstract/Free Full Text]
    15. 15
  15. Konan, K. V., T. H. Giddings, Jr., M. Ikeda, K. Li, S. M. Lemon, and K. Kirkegaard. 2003. Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus. J. Virol. 77:7843-7855.[Abstract/Free Full Text]
    16. 16
  16. Moffat, K., G. Howell, C. Knox, G. J. Belsham, P. Monaghan, M. D. Ryan, and T. Wileman. 2005. Effects of foot-and-mouth disease virus nonstructural proteins on the structure and function of the early secretory pathway: 2BC but not 3A blocks endoplasmic reticulum-to-Golgi transport. J. Virol. 79:4382-4395.[Abstract/Free Full Text]
    17. 17
  17. Monaghan, P., H. Cook, T. Jackson, M. Ryan, and T. Wileman. 2004. The ultrastructure of the developing replication site in foot-and-mouth disease virus-infected BHK-38 cells. J. Gen. Virol. 85:933-946.[Abstract/Free Full Text]
    18. 18
  18. Nayak, A., I. G. Goodfellow, and G. J. Belsham. 2005. Factors required for the uridylylation of the foot-and-mouth disease virus 3B1, 3B2, and 3B3 peptides by the RNA-dependent RNA polymerase (3Dpol) in vitro. J. Virol. 79:7698-7706.[Abstract/Free Full Text]
    19. 19
  19. Netherton, C. L., M. C. McCrossan, M. Denyer, S. Ponnambalam, J. Armstrong, H. H. Takamatsu, and T. E. Wileman. 2006. African swine fever virus causes microtubule-dependent dispersal of the trans-Golgi network and slows delivery of membrane protein to the plasma membrane. J. Virol. 80:11385-11392.[Abstract/Free Full Text]
    20. 20
  20. Neznanov, N., K. M. Chumakov, L. Neznanova, A. Almasan, A. K. Banerjee, and A. V. Gudkov. 2005. Proteolytic cleavage of the p65-RelA subunit of NF-kappaB during poliovirus infection. J. Biol. Chem. 280:24153-24158.[Abstract/Free Full Text]
    21. 21
  21. Neznanov, N., A. Kondratova, K. M. Chumakov, B. Angres, B. Zhumabayeva, V. I. Agol, and A. V. Gudkov. 2001. Poliovirus protein 3A inhibits tumor necrosis factor (TNF)-induced apoptosis by eliminating the TNF receptor from the cell surface. J. Virol. 75:10409-10420.[Abstract/Free Full Text]
    22. 22
  22. Paul, A. V., E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer. 2000. Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg. J. Virol. 74:10359-10370.[Abstract/Free Full Text]
    23. 23
  23. Sanz-Parra, A., F. Sobrino, and V. Ley. 1998. Infection with foot-and-mouth disease virus results in a rapid reduction of MHC class I surface expression. J Gen. Virol. 79:433-436.[Abstract]
    24. 24
  24. Teterina, N. L., E. Levenson, M. S. Rinaudo, D. Egger, K. Bienz, A. E. Gorbalenya, and E. Ehrenfeld. 2006. Evidence for functional protein interactions required for poliovirus RNA replication. J. Virol. 80:5327-5337.[Abstract/Free Full Text]
    25. 25
  25. Wessels, E., D. Duijsings, K. H. Lanke, S. H. van Dooren, C. L. Jackson, W. J. Melchers, and F. J. van Kuppeveld. 2006. Effects of picornavirus 3A proteins on protein transport and GBF1-dependent COP-I recruitment. J. Virol. 80:11852-11860.[Abstract/Free Full Text]
    26. 26
  26. Wessels, E., D. Duijsings, R. A. Notebaart, W. J. Melchers, and F. J. van Kuppeveld. 2005. A proline-rich region in the coxsackievirus 3A protein is required for the protein to inhibit endoplasmic reticulum-to-Golgi transport. J. Virol. 79:5163-5173.[Abstract/Free Full Text]
    27. 27
  27. Wessels, E., R. A. Notebaart, D. Duijsings, K. Lanke, B. Vergeer, W. J. Melchers, and F. J. van Kuppeveld. 2006. Structure-function analysis of the coxsackievirus protein 3A: identification of residues important for dimerization, viral RNA replication, and transport inhibition. J. Biol. Chem. 281:28232-28243.[Abstract/Free Full Text]
    28. 28
  28. Williams, D. B., S. J. Swiedler, and G. W. Hart. 1985. Intracellular transport of membrane glycoproteins: two closely related histocompatibility antigens differ in their rates of transit to the cell surface. J. Cell Biol. 101:725-734.[Abstract/Free Full Text]

Journal of Virology, February 2007, p. 1129-1139, Vol. 81, No. 3
0022-538X/07/$08.00+0     doi:10.1128/JVI.00393-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Burgon, T. B., Jenkins, J. A., Deitz, S. B., Spagnolo, J. F., Kirkegaard, K. (2009). Bypass Suppression of Small-Plaque Phenotypes by a Mutation in Poliovirus 2A That Enhances Apoptosis. J. Virol. 83: 10129-10139 [Abstract] [Full Text]  
  • Paton, D. J., Sumption, K. J., Charleston, B. (2009). Options for control of foot-and-mouth disease: knowledge, capability and policy. Phil Trans R Soc B 364: 2657-2667 [Abstract] [Full Text]  
  • Armer, H., Moffat, K., Wileman, T., Belsham, G. J., Jackson, T., Duprex, W. P., Ryan, M., Monaghan, P. (2008). Foot-and-Mouth Disease Virus, but Not Bovine Enterovirus, Targets the Host Cell Cytoskeleton via the Nonstructural Protein 3Cpro. J. Virol. 82: 10556-10566 [Abstract] [Full Text]  
  • de Jong, A. S., de Mattia, F., Van Dommelen, M. M., Lanke, K., Melchers, W. J. G., Willems, P. H. G. M., van Kuppeveld, F. J. M. (2008). Functional Analysis of Picornavirus 2B Proteins: Effects on Calcium Homeostasis and Intracellular Protein Trafficking. J. Virol. 82: 3782-3790 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moffat, K.
Right arrow Articles by Wileman, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Moffat, K.
Right arrow Articles by Wileman, T.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»