1 MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK
2 Division of Virology, University of Glasgow, Institute of Virology, Church Street, Glasgow G11 5JR, UK
Correspondence
Richard J. Sugrue
r.sugrue{at}vir.gla.ac.uk
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ABSTRACT |
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INTRODUCTION |
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The formation of the RSV envelope occurs within lipid-raft platforms on the surface of virus-infected cells (Brown et al., 2002b; McCurdy & Graham, 2003
; Jeffree et al., 2003
) and the mature virus can be visualized as long filamentous structures on the cell surface (Parry et al., 1979
; Roberts et al., 1995
; Brown et al., 2002a
). The virus encodes three membrane-bound glycoproteins, namely the fusion (F), attachment (G) and small hydrophobic (SH) proteins. The F protein mediates fusion of the virus and cell membranes, and the G protein is involved in virus attachment. The biological properties of the F and G glycoproteins and the role that they play during virus replication are relatively well understood, but the functional significance of the SH protein during virus replication remains unclear.
The amino acid sequence of the SH protein is highly conserved among all RSV A subtypes (Chen et al., 2000) and the SH protein of the RSV A2 strain is expressed as several different forms in virus-infected cells (Olmsted & Collins, 1989
; Collins et al., 1990
; Anderson et al., 1992
). In the human RSV A2 subtype, the full-length translated SH protein contains 65 amino acids and appears in a variety of forms depending upon its glycosylation status. These are a 7·5 kDa non-glycosylated form (SH0), a 1315 kDa N-linked glycosylated form (SHg) and a polylactosaminoglycan-modified form of the protein (SHp) that ranges in size from 2130 kDa. Evidence suggests that SH0 is first modified by N-linked glycosylation to SHg, which is subsequently transformed into SHp by the addition of polylactosaminoglycan. In addition, a fourth form of the SH protein can be detected in which initiation of translation occurs at an alternative methionine, giving rise to a 4·6 kDa truncated form of the non-glycosylated protein (SHt).
The role played by the SH protein during RSV replication is at present unclear. Studies which have employed reverse genetics to produce viruses in which the SH gene has been deleted, appear to suggest that it is dispensable for virus growth but may play a role in evading the host's immune system (Bukreyev et al., 1997). In contrast, several publications have indicated that this protein may play an ancillary role in virus-mediated cell fusion (Heminway et al., 1994
; Perez et al., 1997
; Techaarpornkul et al., 2001
) and in this regard, the ability of the SH protein to interact with the F protein in virus-infected cells has been demonstrated (Feldman et al., 2001
). A greater understanding of its biological properties should aid our understanding of the role played by the SH protein during virus replication. This report examines the cellular distribution of the SH protein in virus-infected cells. The results provide evidence for an association of the SH protein with lipid-raft membrane structures, although only low levels of the protein are associated with the envelope of mature virus filaments. Furthermore, the Golgi complex appears to be a major site of SH protein accumulation within the cell.
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METHODS |
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Antibodies.
The SH protein monoclonal antibody (MAbSH) was prepared as follows. A branched peptide (SH5264) corresponding to the C-terminal 13 amino acids (NKTFELPRARVNT) of the RSV A2 strain (Collins & Wertz, 1985) was synthesized on a tetravalent (Lys)2-Lys-
-Ala-Wang resin (Novabiochem) using standard 9-fluorenylmethoxycarbonyl solid-phase chemistry and an Advanced ChemTech 348 (automated peptide synthesizer). The resulting peptide, SH5264, was coupled to a mouse T-cell epitope from sperm whale myoglobin (NKALELFRKDIAAKYKE) using standard procedures. This peptide was used to immunize BALB/c mice and monoclonal antibodies were prepared using standard protocols. The tissue culture medium was harvested from MAbSH-expressing hybridoma cells, concentrated and filtered through a 0·2 µm filter prior to use.
The F protein monoclonal antibody (MAb169) was prepared from recombinant F protein expressed in E. coli (H. Rixon, S. Graham and R. Sugrue, unpublished data). MAb19 and anti-M were gifts from Geraldine Taylor (IAH, Compton, UK) and Paul Yeo (MRC Virology Unit, Glasgow, UK), respectively. The Golgi-specific marker, GM130, was provided by Martin Lowe (School of Biological Sciences, University of Manchester, UK), the Calnexin antibody was purchased from Stressgen and cholera toxin B subunit conjugated to FITC (CTX-BFITC) was purchased from Sigma.
Radioimmunoprecipitation (RIP).
The radiolabelling and RIP procedures were performed as previously described (Rixon et al., 2002). Briefly, mock- and RSV-infected Vero cells were incubated at 33 °C for 18 h, after which the DMEM plus 2 % FCS was replaced with DMEM minus either methionine or glucose and the cells were incubated for a further 18 h in the presence of either 100 µCi (3·70 MBq) [35S]methionine ml1 or 150 µCi (5·55 MBq) [3H]glucosamine ml1, respectively. Immunoprecipitated proteins were separated by using 15 % SDS-PAGE and the [35S]methionine-labelled proteins were detected by using a Bio-Rad personal Fx phosphorimager while the [3H]glucosamine-labelled proteins were detected by fluorography.
Immunofluorescence.
Cells were seeded on 13 mm glass coverslips and incubated overnight at 37 °C prior to infection with RSV. Mock- or RSV-infected cells were fixed with 3 % paraformaldehyde for 30 min at 4 °C. The fixative was removed and the cells were washed once with PBS+1 mM glycine and then four times with PBS. The cells were incubated at 25 °C for 1 h with primary antibody after which they were washed and incubated for a further 1 h either with anti-mouse or anti-rabbit IgG (whole molecule) conjugated either to FITC, TRITC or cy5 (1/100 dilution). The stained cells were mounted on slides using Citifluor and visualized in a Zeiss Axioplan 2 confocal microscope. The images were processed using LSM 510 v2.01 software.
Flotation gradient analysis of detergent-insoluble lipid-raft membranes.
Cell sheets were washed twice with PBSA (20 mM sodium phosphate, 150 mM NaCl pH 7·2) and then twice with TM buffer (10 mM Tris/HCl pH 7·4; 1 mM MgCl2). The cells (1x108) were scraped into TM buffer supplemented with an EDTA-free protease-inhibitor mixture (Roche Molecular Biochemicals) and Dounce homogenized (80 strokes). Unbroken cells and nuclei were removed by centrifugation at 1000 g for 5 min. The 1000 g supernatant was centrifuged at 45 000 g for 15 min to pellet the cell membranes, which were then washed in TM buffer. The membranes were resuspended at 4 °C in PBSA containing 1 % Triton X-100 by using a Dounce homogenizer, after which they were incubated for a further 1·5 h at 4 °C. The homogenate was added to an equal volume of 80 % (w/v) sucrose in 10 mM Tris/HCl, pH 7·4; 150 mM NaCl; 1 mM EDTA (TNE). The solubilized membranes (in 40 % sucrose) were placed at the bottom of a centrifuge tube and overlaid successively with 7 ml 35 % sucrose and 2 ml 5 % sucrose (in TNE). After centrifugation at 210 000 g for 18 h, the gradient was fractionated and the individual fractions were analysed by Western blotting for the presence of specific proteins using appropriate antibodies.
Western blotting.
Protein samples were separated using SDS-PAGE and were transferred by Western blotting onto a PVDF membrane. After transfer, membranes were washed with PBSA and blocked for 18 h at 4 °C in PBSA containing 1 % Marvel and 0·05 % Tween 20. They were then washed twice in PBSA, prior to incubation with the appropriate primary antibody for 1 h, washed four times in PBSA containing 0·05 % Tween 20 and probed either with goat anti-mouse or anti-rabbit IgG (whole molecule) peroxidase conjugate (Sigma). The protein bands were visualized by using the ECL protein detection system (Amersham). Apparent molecular masses were estimated using Rainbow protein markers (Amersham) in the molecular mass range 14·3220 kDa.
Immuno-transmission electron microscopy (I-TEM).
Cell monolayers were pelleted in BEEM capsules and fixed for 5 h at 4 °C with 0·5 % glutaraldehyde in PBS after which the fixed pellet was extensively washed with PBS and dehydrated by transfer through a gradient of 30, 50, 70, 90 and 100 % ethanol. The cell pellet was subsequently infiltrated with Unicryl (TAAB Laboratories) and the resin polymerized by UV irradiation at 15 °C. Ultrathin sections were placed on nickel-coated grids and incubated either with MAbSH (1/50 dilution), or anti-M (1/100 dilution) for 5 h at 25 °C. The grids were washed with PBS and incubated for 2 h either with anti-rabbit or anti-mouse IgG (whole molecule) 5 or 10 nm colloidal gold conjugate (Sigma) or anti-rabbit IgG (whole molecule) 20 nm colloidal gold conjugate (British BioCell) as appropriate. They were then washed in PBS and fixed in osmium tetroxide vapour for 2 h prior to staining with uranyl acetate (saturated in 50 : 50 ethanol/water), washing in distilled water and counter-staining with lead citrate.
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RESULTS AND DISCUSSION |
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The specificity of MAbSH for the SH protein was confirmed by Western blotting analysis (Fig. 1A). Probing RSV-infected cell lysates with MAbSH revealed the presence of a major protein band of approximately 10 kDa and a minor band of approximately 5 kDa. The protein profiles and sizes of the products detected are similar to the SH protein species that have been previously described (Olmsted & Collins, 1989
; Collins et al., 1990
; Anderson et al., 1992
). The size of the 10 kDa species is consistent with it being the non-glycosylated form of the SH protein (SH0) and the 5 kDa species is similar to the expected size of the truncated form of the SH protein (SHt). No protein bands corresponding to SHg or SHp were detected by Western blotting, confirming previous findings that these glycosylated forms represent minor amounts of the total expressed SH protein (Olmsted & Collins, 1989
; Anderson et al., 1992
). As expected, no protein bands were detected in mock-infected cells by Western blotting, thus demonstrating the specificity of MAbSH.
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In this study, two monoclonal antibodies were used to detect the RSV F protein, namely MAb19, whose use has been previously described (Taylor et al., 1992; Brown et al., 2002a
, b
), and MAb169 (see Methods). Analysis of virus-infected cell lysates by Western blotting with MAb169 showed a single protein band whose size is consistent with that of the F1 subunit of the F protein (Fig. 1D
). No protein bands were detected in lysates prepared from mock-infected cells, thus demonstrating the specificity of this reagent.
The SH protein interacts with lipid-raft membrane structures
Although several publications have shown that the SH protein is an integral membrane protein (Olmsted & Collins, 1989; Collins & Mottet, 1993
), there is little information about how the protein is distributed within the virus-infected cell. Mock- and virus-infected cells were labelled with MAbSH and analysed by fluorescence microscopy (Fig. 2
A). No significant staining was observed in mock-infected cells whereas in virus-infected cells, although MAbSH exhibited a diffuse staining pattern across the cell, the staining appeared to concentrate in the perinuclear region. This observation, together with the known association of the SH protein with cellular membranes (Olmsted & Collins, 1989
), suggested that the SH protein may accumulate at specific locations in the secretory pathway. Therefore, infected cells were examined by fluorescence microscopy after double-labelling with MAbSH and cellular markers specific for the endoplasmic reticulum (ER) and Golgi complex. Cells stained with GM130 exhibited a perinuclear pattern of labelling that was characteristic for Golgi staining (Fig. 2B
). Although the SH protein partly co-localized with ER markers such as Calnexin (Fig. 2C
), the co-localization between the SH protein and the Golgi marker GM130, was much stronger. The strong level of co-localization between GM130 and the SH protein (Fig. 2B
, yellow staining pattern) suggested that despite being detected in the cytoplasm, a large proportion of the total expressed SH protein was located in the early compartments of the secretory pathway and in particular the Golgi compartment.
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Recent evidence has suggested that RSV assembly occurs within lipid-raft structures on the cell surface (Brown et al., 2002a, b
; McCurdy & Graham, 2003
; Jeffree et al., 2003
) and several reports have demonstrated that specific proteins are sorted into lipid-raft domains in the Golgi network (reviewed by van Meer & Simons, 1988
; Brown & London, 1998
; Ikonen, 2001
). It was therefore interesting to determine if the SH protein was able to interact with lipid rafts, since very little of the protein was detected in structures which are associated with mature virus (see below). In this experiment, the cholera toxin B subunit (CTX-B) was used to detect the presence of the raft lipid, GM1, within the cell. CTX-B binds to GM1 at the cell surface and is then transported via endocytosis to the Golgi apparatus (Lencer et al., 1992
, 1995
, 1999
; Le & Nabi, 2003
). Exposure of cells to CTX-B at 4 °C allows the detection of cell surface GM1 since at this temperature CTX-B is not internalised. At higher temperatures (e.g. between 25 °C and 37 °C), the GM1-bound CTX-B is internalized and transported to the Golgi complex. In this way, CTX-B-conjugated to FITC (CTX-BFITC) can be used to locate GM1-enriched microdomains at both the cell surface and within the Golgi compartment depending upon the temperature at which the cells are exposed to CTX-B. Virus-infected cells were exposed to CTX-BFITC under normal culturing conditions (i.e. 33 °C), which allowed internalization and uptake of CTX-BFITC, after which the cells were fixed and labelled with MAbSH and GM130 and visualized using a three-colour analysis that has been previously described (Henderson et al., 2002
). In this way, the distribution of GM1, the Golgi compartment and the SH protein was compared in the same cell. This revealed that GM1 (green), GM130 (blue) and the SH protein (red) co-localized strongly in the perinuclear region of the cell (Fig. 3
A, indicated by white staining pattern in the merged image), providing evidence that the SH protein is located within regions of the Golgi compartment that contain the raft lipid GM1. The presence of the SH protein within GM1-rich regions of the cell can be seen clearly by comparing the SH protein and GM1 distribution within the cell, revealing a significant level of co-localization between CTX-BFITC and MAbSH (Fig. 3B
, indicated by yellow staining in the merged image). However, no significant co-localization of the SH protein with GM1 was detected at the surface of virus-infected cells following exposure of the cells to CTX-BFITC at 4 °C (data not shown).
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Low levels of the SH protein are incorporated into the virus envelope
It is currently unclear to what extent the SH protein interacts with mature RSV filaments. The cellular distribution of the SH protein in infected cells was therefore examined using two complementary techniques, fluorescence microscopy and immuno-transmission electron microscopy (I-TEM).
Previously, fluorescent staining of RSV filaments had been observed following labelling of infected cells with antibodies against the F and G glycoproteins (Roberts et al., 1995; Brown et al., 2002a
; Jeffree et al., 2003
) and the M protein (Henderson et al., 2002
). However, in the fluorescence microscopic analysis described above (Fig. 2
), no obvious staining of the virus filaments with MAbSH was observed. The absence of detectable SH protein in virus filaments studied by confocal microscopy was confirmed by comparing the surface-labelling pattern of the SH and F proteins (Fig. 5
A, plates A and B). Non-permeabilized virus-infected cells were labelled either with MAbSH or MAb19 and the labelling pattern visualized by confocal microscopy. Low-level surface-labelling for the SH protein was observed, which appeared to be punctate in appearance rather than filamentous. This contrasted with the surface-labelling of cells stained with MAb19 which revealed the presence of clearly defined virus filaments.
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The fluorescence microscopy data suggested that the SH protein was not present within mature virus filaments. However, a major limitation of light microscopy is its lower resolution compared with that of electron microscopy and thus it seemed possible that fluorescence microscopy may not detect low levels of virus-associated SH protein. Previous studies which employed an indirect biochemical analysis suggested that low levels of the SH0 and SHp forms may be present within purified RSV particles (Collins et al., 1990; Anderson et al., 1992
). However, a major problem in interpreting the results with this type of approach is that RSV-infectivity remains largely cell-associated (Roberts et al., 1995
) and purification of infectious RSV is a difficult process. It is possible that low levels of contaminating virus proteins may also co-purify with the RSV particles, which is particularly relevant since the failure to detect the SH protein in virus filaments by fluorescence microscopy suggested that if it was present in mature virus filaments, it was only present at low levels. To overcome these two potential problems, immuno-transmission electron microscopy (I-TEM) was employed to analyse RSV-infected cells in situ. In this study, infected cells were processed for I-TEM as previously described (Brown et al., 2002a
) and the SH protein was detected using MAbSH. In the I-TEM images, the mature virus filaments were visible as were the virus inclusion bodies (Fig. 6
A and B). The thin sections prepared from virus-infected cells were labelled with MAbSH and the presence of bound antigen was detected using 5 nm colloidal gold-labelled second antibody. Although it was possible to visualize the SH protein in virus filaments, only between one and four gold particles per filament were detected, despite extensive examination of the samples. The significance of this low level of gold signal within the virus filaments is unclear. However, it is unlikely to be due to non-specific binding of the gold conjugate since gold label was not observed in sections in which either MAbSH was omitted or MAbSH was replaced with a non-specific mouse IgG (Fig. 6C and D
).
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I-TEM was used to examine and compare the relative distribution of the SH and M proteins. Although such a comparison may not provide an accurate quantitative measure of SH and M protein levels, it was reasoned that the levels of each protein in the virus-induced structures, i.e. virus filaments and IBs, could be compared qualitatively. The relative distribution of the M and SH proteins within virus filaments was examined in sections labelled with both anti-M and MAbSH, and the presence of the bound antibody detected using 5 and 10 nm colloidal gold-conjugated second antibodies, respectively (Fig. 7A and B). An abundance of labelling with anti-M was identified by the relatively large amount of 5 nm gold probe (highlighted by white arrow) associated with mature virus filaments and newly budded virus. In contrast, very little 10 nm gold probe (highlighted by black arrow) appeared to be associated either with the mature virus filaments or with newly budding virus.
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Although direct evidence has been provided that a low level of the SH protein is present within mature RSV filaments, our results show that raft membranes within the Golgi complex are a major site of SH protein accumulation during virus infection. Although the function of the SH protein has not been defined, it is possible that it may modify some property of the Golgi complex to aid virus protein transport through the secretory pathway. In this respect, it is interesting to note that several small membrane proteins encoded by other viruses are able to alter the protein transport machinery of the host-cell, a list that includes the M2 and BM2 proteins of influenza virus. The M2 protein of influenza A virus modifies the acidity of the Golgi compartment (Sugrue et al., 1990; Ciampor et al., 1992
), a process that has been shown, in polarized cells, to decrease the rate of transport of glycoproteins to the apical cell surface (Sakaguchi et al., 1996
; Henkel & Weisz, 1998
; Henkel et al., 1998
, 2000
). More recent work has demonstrated that the BM2 protein of influenza B virus is able to elicit similar effects to those observed for the M2 protein (Mould et al., 2003
). However, at present, the effect exerted by the SH protein on the physiology of the host cell, and in particular the secretory pathway (e.g. the Golgi complex), remains to be established as does its functional significance within the virus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Brown, D. A. & London, E. (1998). Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 14, 111136.[CrossRef][Medline]
Brown, G., Aitken, J., Rixon, H. W. McL. & Sugrue, R. J. (2002a). Caveolin-1 is incorporated into mature respiratory syncytial virus particles during virus assembly on the surface of virus-infected cells. J Gen Virol 83, 611621.
Brown, G., Rixon, H. W. McL. & Sugrue, R. J. (2002b). Respiratory syncytial virus assembly occurs in GM1-rich regions of the host-cell membrane and alters the cellular distribution of tyrosine phosphorylated caveolin-1. J Gen Virol 83, 18411850.
Bukreyev, A., Whitehead, S. S., Murphy, B. R. & Collins, P. L. (1997). Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse. J Virol 71, 89738982.[Abstract]
Chen, M. D., Vazquez, M., Buonocore, L. & Kahn, J. S. (2000). Conservation of the respiratory syncytial virus SH gene. J Infect Dis 182, 12281233.[CrossRef][Medline]
Ciampor, F., Bayley, P. M., Nermut, M. V., Hirst, E. M. A., Sugrue, R. J. & Hay, A. J. (1992). Evidence that the amantadine-induced M2-mediated conversion of influenza A virus hemagglutinin to the low pH conformation occurs in an acidic trans Golgi compartment. Virology 188, 1424.[Medline]
Collins, P. L. & Wertz, G. W. (1985). The 1A protein gene of human respiratory syncytial virus: nucleotide sequence of the mRNA and a related polycistronic transcript. Virology 141, 283291.[Medline]
Collins, P. L. & Mottet, G. (1991). Post-translational processing and oligomerization of the fusion glycoprotein of human respiratory syncytial virus. J Gen Virol 72, 30953101.[Abstract]
Collins, P. L. & Mottet, G. (1993). Membrane orientation and oligomerization of the small hydrophobic protein of human respiratory syncytial virus. J Gen Virol 74, 14451450.[Abstract]
Collins, P. L., Olmsted, R. A. & Johnson, P. R. (1990). The small hydrophobic protein of human respiratory syncytial virus: comparison between antigenic subgroups A and B. J Gen Virol 71, 15711576.[Abstract]
Feldman, S. A., Crim, R. L., Audet, S. A. & Beeler, J. A. (2001). Human respiratory syncytial virus surface glycoproteins F, G and SH form an oligomeric complex. Arch Virol 146, 23692383.[CrossRef][Medline]
Garcia, J., Garcia-Barreno, B., Vivo, A. & Melero, J. A. (1993). Cytoplasmic inclusions of respiratory syncytial virus-infected cells: formation of inclusion bodies in transfected cells that coexpress the nucleoprotein, the phosphoprotein, and the 22K protein. Virology 195, 243247.[CrossRef][Medline]
Garcia-Barreno, B., Delgado, T. & Melero, J. A. (1996). Identification of protein regions involved in the interaction of human respiratory syncytial virus phosphoprotein and nucleoprotein: significance for nucleocapsid assembly and formation of cytoplasmic inclusions. J Virol 70, 801808.[Abstract]
Ghildyal, R., Mills, J., Murray, M., Vardaxis, N. & Meanger, J. (2002). Respiratory syncytial virus matrix protein associates with nucleocapsids in infected cells. J Gen Virol 83, 753757.
Heminway, B. R., Yu, Y., Tanaka, Y., Perrine, K. G., Gustafson, E., Bernstein, J. M. & Galinski, M. S. (1994). Analysis of respiratory syncytial virus F, G, and SH proteins in cell fusion. Virology 200, 801805.[CrossRef][Medline]
Henderson, G., Murray, J. & Yeo, R. P. (2002). Sorting of the respiratory syncytial virus matrix protein into detergent-resistant structures is dependent on cell-surface expression of the glycoproteins. Virology 300, 244254.[CrossRef][Medline]
Henkel, J. R. & Weisz, O. A. (1998). Influenza virus M2 protein slows traffic along the secretory pathway. pH perturbation of acidified compartments affects early Golgi transport steps. J Biol Chem 273, 65186524.
Henkel, J. R., Apodaca, G., Altschuler, Y., Hardy, S. & Weisz, O. A. (1998). Selective perturbation of apical membrane traffic by expression of influenza M2, an acid-activated ion channel, in polarized MadinDarby canine kidney cells. Mol Biol Cell 9, 24772490.
Henkel, J. R., Gibson, G. A., Poland, P. A., Ellis, M. A., Hughey, R. P. & Weisz, O. A. (2000). Influenza M2 proton channel activity selectively inhibits trans-Golgi network release of apical membrane and secreted proteins in polarized MadinDarby canine cells. J Cell Biol 148, 495504.
Ikonen, E. (2001). Roles of lipid rafts in membrane transport. Curr Opin Cell Biol 13, 470477.[CrossRef][Medline]
Jeffree, C. E., Rixon, H. W. McL., Brown, G., Aitken, J. & Sugrue, R. J. (2003). Distribution of the attachment (G) glycoprotein and GM1 within the envelope of mature respiratory syncytial virus filaments revealed using field emission scanning electron microscopy. Virology 306, 254267.[CrossRef][Medline]
Le, P. U. & Nabi, I. R. (2003). Distinct caveolae-mediated endocytic pathways target the Golgi apparatus and the endoplasmic reticulum. J Cell Sci 116, 10591071.
Lencer, W. I., Delp, C., Neutra, M. R. & Madara, J. L. (1992). Mechanism of cholera toxin action on a polarized human intestinal epithelial cell line: role of vesicular traffic. J Cell Biol 117, 11971209.[Abstract]
Lencer, W. I., Constable, C., Moe, S., Jobling, M. G., Webb, H. M., Ruston, S., Madara, J. L., Hirst, T. R. & Holmes, R. K. (1995). Targeting of cholera toxin and Escherichia coli heat labile toxin in polarized epithelia: role of COOH-terminal KDEL. J Cell Biol 131, 951962.[Abstract]
Lencer, W. I., Hirst, T. R. & Holmes, R. K. (1999). Membrane traffic and the cellular uptake of cholera toxin. Biochim Biophys Acta 1450, 177190.[Medline]
McCurdy, L. H. & Graham, B. S. (2003). Role of plasma membrane lipid microdomains in respiratory syncytial virus filament formation. J Virol 77, 17471756.
Mould, J. A., Paterson, R. G., Takeda, M., Ohigashi, Y., Venkataraman, P., Lamb, R. A. & Pinto, L. H. (2003). Influenza B virus BM2 protein has ion channel activity that conducts protons across membranes. Dev Cell 5, 175184.[Medline]
Olmsted, R. A. & Collins, P. L. (1989). The 1A protein of respiratory syncytial virus is an integral membrane protein present as multiple, structurally distinct species. J Virol 63, 20192029.[Medline]
Parry, J. E., Shirodaria, P. V. & Pringle, C. R. (1979). Pneumoviruses: the cell surface of lytically and persistently infected cells. J Gen Virol 44, 479491.[Abstract]
Perez, M., Garcia-Barreno, B., Melero, J. A., Carrasco, L. & Guinea, R. (1997). Membrane permeability changes induced in Escherichia coli by the SH protein of human respiratory syncytial virus. Virology 235, 342351.[CrossRef][Medline]
Rixon, H. W., Brown, C., Brown, G. & Sugrue, R. J. (2002). Multiple glycosylated forms of the respiratory syncytial virus fusion protein are expressed in virus-infected cells. J Gen Virol 83, 6166.
Roberts, S. R., Compans, R. W. & Wertz, G. W. (1995). Respiratory syncytial virus matures at the apical surfaces of polarized epithelial cells. J Virol 69, 26672673.[Abstract]
Sakaguchi, T., Leser, G. P. & Lamb, R. A. (1996). The ion channel activity of the influenza virus M2 protein affects transport through the Golgi apparatus. J Cell Biol 133, 733747.[Abstract]
Sugrue, R. J., Bahadur, G., Zambon, M. C., Hall-Smith, M., Douglas, A. R. & Hay, A. J. (1990). Specific structural alteration of the influenza haemagglutinin by amantadine. EMBO J 9, 34693476.[Abstract]
Taylor, G., Stott, E. J., Furze, J., Ford, J. & Sopp, P. (1992). Protective epitopes on the fusion protein of respiratory syncytial virus recognized by murine and bovine monoclonal antibodies. J Gen Virol 73, 22172223.[Abstract]
Techaarpornkul, S., Barretto, N. & Peeples, M. E. (2001). Functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene. J Virol 75, 68256834.
van Meer, G. & Simons, K. (1988). Lipid polarity and sorting in epithelial cells. J Cell Biochem 36, 5158.[Medline]
Received 30 October 2003;
accepted 19 January 2004.