1 MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK
2 MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, UK
Correspondence
R. J. Sugrue
r.sugrue{at}vir.gla.ac.uk
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ABSTRACT |
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INTRODUCTION |
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During RSV infection, the bulk of the expressed SH protein remains cell-associated and a large proportion of the protein appears to localize in the early compartments of the secretory pathway, such as the endoplasmic reticulum (ER) and Golgi complex (Olmsted & Collins, 1989; Rixon et al., 2004
). Although the SH protein is expressed on the surface of infected cells, only very low levels are detected within virions (Collins et al. 1990
; Anderson et al., 1992
; Rixon et al., 2004
). The function of the SH protein during virus infection is currently unknown, although studies have suggested a function either in evading the host's immune system (Bukreyev et al., 1997
) or in providing an ancillary role in virus-mediated cell fusion (Heminway et al., 1994
; Perez et al., 1997
; Techaarpornkul et al., 2001
). A greater understanding of the biochemical properties of the SH protein is a prerequisite to understanding its function during virus infection.
Several RSV proteins undergo post-translational modification leading to their glycosylation (Lambert & Pons, 1983; Gruber & Levine, 1985
), acylation (Arumugham et al., 1989
) and phosphorylation (Lambert et al., 1988
). In the case of the SH protein, glycosylation is the only modification that has been proved experimentally (Olmsted & Collins, 1989
; Collins et al., 1990
; Anderson et al., 1992
). There is no published experimental evidence that the SH protein is either acylated or phosphorylated. However, mammalian cells elicit biological responses to different stimuli, including virus infection, through the activation of the mitogen-activated protein kinase (MAPK) signalling pathways. These pathways both induce the expression of specific cellular proteins and lead to the activation of specific host proteins, including kinases, by phosphorylation. The best-characterized of these are the extracellular signal-regulated kinase (ERK) and the p38 kinase signalling pathways (reviewed by Roux & Blenis, 2004
). Several host-cell proteins are subsequently phosphorylated by the phosphorylated, activated forms of ERK1/2 (e.g. CD120a; Cottin et al., 1999
; Van Linden et al., 2000
) and p38 kinase (e.g. hsp27; Stokoe et al., 1992
). The work presented here shows for the first time that the SH protein is modified by tyrosine phosphorylation via an MAPK p38-dependent pathway during RSV infection.
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METHODS |
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Antibodies and inhibitors.
The SH protein monoclonal antibody (mAbSH) was prepared as described previously (Rixon et al., 2004); anti-RSV antibody (RCL-3) was purchased from Novacastra and mAb30 was a gift from Geraldine Taylor (IAH, Compton, UK). The Golgi-specific marker GM130 was provided by Martin Lowe (School of Biological Sciences, University of Manchester, UK). The antibodies PY20, anti-phospho-p38 and anti-phospho-ERK1/2 were purchased from New England Biolabs. The kinase inhibitors SB203580, PD98059 and genistein were purchased from Calbiochem and stock solutions prepared in DMSO.
Radiolabelling.
Cell monolayers were either mock- or RSV-infected in DMEM plus 2 % FCS at 33 °C. Between 8 and 20 h post-infection (p.i.), cells were incubated in DMEM minus methionine, glucose or phosphate containing 100 µCi (3·7 MBq) [35S]methionine, D-[6-3H]glucosamine hydrochloride or [33P]orthophosphate ml1, respectively.
Radioimmunoprecipitation (RIP).
Cell monolayers were extracted at 4 °C for 10 min with lysis buffer (1 % NP-40, 0·1 % SDS, 150 mM NaCl, 1 mM EDTA, 2 mM PMSF, 20 mM Tris/HCl, pH 7·5) and clarified by centrifugation. The clarified lysate was incubated with mAbSH in binding buffer (0·5 % NP-40, 150 mM NaCl, 1 mM EDTA, 0·25 % BSA, 20 mM Tris/HCl, pH 8·0) overnight at 4 °C. Immune complexes were isolated by adding protein ASepharose for 2 h at 4 °C. The protein ASepharose was washed four times with high-salt buffer (1 % Triton X-100, 650 mM NaCl, 1 mM EDTA, 10 mM sodium phosphate, pH 7·0) and once with low-salt buffer (1 % Triton X-100, 150 mM NaCl, 1 mM EDTA, 10 mM sodium phosphate, pH 7·0). The protein ASepharose-bound immune complexes were resuspended in boiling mix (1 % SDS, 15 % glycerol, 60 mM sodium phosphate, pH 6·8) with or without 5 % -mercaptoethanol and heated at 100 °C. Samples were then analysed by 15 % SDS-PAGE, unless otherwise stated. The [35S]methionine- or [33P]orthophosphate-radiolabelled protein bands were detected using a Bio-Rad personal Fx phosphorimager and analysed using Quantity One software (v. 4; Bio-Rad). D-[6-3H]Glucosamine-labelled proteins were detected by fluorography. Apparent molecular masses were estimated using 14C-methylated Rainbow molecular mass markers (Amersham) in the range 14·3220 kDa: lysozyme (14·3 kDa), soybean trypsin inhibitor (21·5 kDa), carbonic anhydrase (30 kDa), ovalbumin (46 kDa), serum albumin (66 kDa), phosphorylase b (97·5 kDa) and myosin (220 kDa).
Western blotting.
Protein samples were separated by SDS-PAGE and transferred by Western blotting on to a PVDF membrane. After transfer, the membrane was washed with Tris-buffered saline containing 0·05 % Tween 20 (TBS/Tween) and blocked for 18 h at 4 °C in TBS/Tween containing 1 % BSA. It was then washed twice in TBS/Tween prior to incubation with the primary antibody for 60 min. The membrane was washed four times in TBS/Tween and probed using either goat anti-mouse or anti-rabbit IgG (whole molecule) conjugated to peroxidase (Sigma) as appropriate. Protein bands were visualized using the ECL protein detection system (Amersham). Apparent molecular masses were estimated using Rainbow molecular mass markers in the range 14·3220 kDa (Amersham).
Phosphoamino acid (PAA) analysis.
The SH protein was isolated from [33P]orthophosphate-labelled cells by RIP using mAbSH and transferred by Western blotting on to PVDF membranes. The labelled SH protein band was visualized by phosphorimaging and excised from the membrane. Amino acids were hydrolysed from the membrane slice by incubating it in 6 M HCl and heating at 110 °C for 90 min. The released amino acids were dried under vacuum and then resuspended in pyridine acetate, pH 3·5. Each sample was spotted on to a cellulose-coated thin layer chromatography (TLC) plate (Merck) and electrophoresed at 1000 V for 75 min in a Multiphor II electrophoresis tank (Amersham Biosciences), with pyridine acetate (pH 3·5) as the solvent. The TLC plate was air dried and the positions of the separated PAAs visualized by incubating the plate for 1 min in 0·2 % (w/v) ninhydrin in acetone and air drying for a further 10 min. The positions of the 33P-labelled PAAs was detected by phosphorimager analysis and compared with the position of phosphothreonine, phosphoserine and phosphotyrosine standards (Sigma-Aldrich).
Immunofluorescence.
RSV-infected cells were fixed with 3 % paraformaldehyde for 30 min at 4 °C. The fixative was removed and the cells washed once with PBS plus 1 mM glycine and four times with PBS. Cells were incubated at 25 °C for 1 h with primary antibody after which they were washed and incubated for a further 1 h with either anti-mouse or anti-rabbit IgG (whole molecule) conjugated to FITC or cy5 (1 : 100 dilution). Stained cells were mounted on slides using Citifluor and visualized using a Zeiss Axioplan 2 confocal microscope. The images were processed using LSM 510 v. 2.01 software.
Protein cross-linking.
This was performed as described previously (Sugrue & Hay, 1991). Briefly, a stock solution of dithiobis(succinimidyl) propionate (DSP; Pierce) in DMSO (100 mM) was added to PBS, pH 8·0, to give the final concentration required. This solution was then added to the cell monolayers and incubated at 4 °C for 1 h. The cross-linking solution was then removed and the reactions quenched by washing the cells extensively with PBSA plus 20 mM lysine. Cell extracts were prepared and analysed either by Western blotting or by RIP.
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RESULTS AND DISCUSSION |
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Identification of the phosphorylated amino acid residue present in the SH protein was carried out by a standard PAA analysis (Fig. 2a), performed as described in Methods. Migration of the [33P]phosphate-labelled amino acid derived from the SH protein was compared with that of the PAA markers phosphoserine, phosphothreonine and phosphotyrosine (Fig. 2a
). This revealed that the only labelled amino acid detected in the SH protein hydrolysate migrated similarly to phosphotyrosine, and no evidence for the presence of either phosphoserine or phosphothreonine in this protein was obtained.
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Further evidence for the role of tyrosine kinase activity in SH protein phosphorylation was obtained using the broad-spectrum tyrosine kinase inhibitor genistein (Fig. 2c). This reagent inhibits phosphorylation by blocking the binding of ATP to the ATP-binding site of tyrosine kinases, which is an essential step during this enzymic process. Virus-infected cells were exposed to varying concentrations of genistein between 8 and 20 h p.i., after which the SH protein was transferred on to a PVDF membrane by Western blotting and probed with PY20 as described above. A comparison of the non-treated and gentistein-treated samples showed that the PY20 reactivity of the SH protein was significantly reduced in the presence of concentrations of genistein as low as 25 µM, whereas SH protein levels remained unchanged at these concentrations.
The SH protein forms a phosphorylated pentameric structure that interacts with the RSV attachment protein
Previous studies have suggested that the SH protein is able to assemble into a homo-pentameric structure (Collins & Mottet, 1993; Kochva et al., 2003
). We used protein cross-linking reagents to determine whether the phosphorylated SH protein could also form similar oligomeric structures. Several different cross-linking reagents were used in this analysis, but due to the similarity of the results obtained, only the data obtained with one of these, DSP, are presented. DSP is a reversible cross-linking reagent; the intermolecular covalent bonds introduced by DSP can be removed by treatment with a reducing agent such as
-mercaptoethanol.
Virus-infected cells were mock-treated or treated with DSP and analysed directly by Western blotting using mAbSH (Fig. 3a). Under non-reducing conditions and in the presence of DSP, the appearance of at least four higher molecular mass species, in addition to SH0, of approximately 17, 24, 30 and 38 kDa, was noted. These were similar in size to the SH0 cross-linked species reported in a previous study (Collins & Mottet, 1993
). Under reducing conditions, only a single protein species corresponding in size to SH0 was observed. In a further analysis, [35S]methionine- or [33P]phosphate-labelled RSV-infected cell monolayers were mock-treated or treated in situ with 0·52·0 mM DSP (Fig. 3b and c
). The SH protein was isolated by RIP, separated by 15 % SDS-PAGE and the protein profile of the SH protein from non-treated and treated cells was compared. Following DSP treatment, four higher molecular mass species were detected in both [35S]methionine- and [33P]phosphate-labelling experiments; these were similar in size to the SH products detected by Western blotting (Fig. 3a
). These data were consistent with the pentameric structure that has been reported for the SH protein (Collins & Mottet, 1993
; Kochva et al., 2003
). Treatment with reducing agent prior to analysis by SDS-PAGE revealed [35S]methionine- and [33P]phosphate-labelled products of the expected sizes for SH0, SHg and SHp, although in each case SH0 accounted for approximately 95 % of the total detectable labelled protein.
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Under non-reducing conditions, a large amount of an additional, much higher molecular mass protein complex was apparent whose size approached 200 kDa. This material, which was not present in the non-DSP-treated samples, accounted for most of the radiolabelled protein recovered by RIP and could not be resolved, even on low-percentage polyacrylamide gels, which can resolve proteins up to 200 kDa [Fig. 3b(ii) and c(ii)]. A similar-sized product was reported in earlier studies (Collins & Mottet, 1993
) but remained uncharacterized. After treatment with reducing agent, this high molecular mass protein complex disappeared and correlated with the appearance of an 80 kDa protein species together with a large amount of SH0. The 80 kDa protein was only detected in [35S]methionine-labelled samples suggesting that it was not phosphorylated, and it was not detected by Western blotting using mAbSH, suggesting that it was not the SH protein.
In a similar study, mock- and RSV-infected cells were [3H]glucosamine labelled and treated with DSP prior to analysis by SDS-PAGE (Fig. 4a). This labelling procedure enhances the visualization of the glycosylated SH protein forms, which are expressed at relatively low levels and are difficult to detect by [35S]methionine labelling. Under non-reducing conditions, a labelled smear was observed that extended from 40 kDa up to a molecular mass that exceeded 200 kDa, with the majority of the recovered labelled protein being >200 kDa. Treatment of the samples with reducing agent revealed the presence of a smear from 21 to 40 kDa, which is consistent with the molecular mass of SHp (Anderson et al., 1992
), and a strongly labelled protein product of 80 kDa (Fig. 4a
, filled arrowhead). This was identical in migration to the 80 kDa protein detected by [35S]methionine labelling (Fig. 3b
) and suggested that this protein was a glycoprotein.
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The SH protein is phosphorylated via an MAPK p38-dependent pathway
Several reports have demonstrated that cells respond to RSV infection by the induction of cytokine expression via the ERK and p38-dependent pathways (Pazdrak et al., 2002; Meusel & Imani, 2003
; Kong et al., 2004
; Monick et al., 2004
). Mock- and virus-infected Vero C1008 cells were analysed by Western blotting using two antibodies, anti-phospho-p38 and anti-phospho-ERK1/2, which specifically recognize the phosphorylated forms of p38 and ERK1/2, respectively. This analysis showed the presence of similar levels of phosphorylated ERK1/2 and p38 in mock- and virus-infected cells (Fig. 5
), suggesting that in this cell line these kinases are phosphorylated prior to infection. We were therefore interested to determine whether or not the phosphorylation of the SH protein was dependent upon either or both of these different signalling pathways, since they have been implicated in RSV infection. In this analysis, two standard MAPK inhibitors that specifically inhibit p38 kinase and ERK were used, thus allowing determination of the effect that these different pathways have on SH protein phosphorylation. SB203580 is a specific inhibitor of MAPK p38 kinase (Gallagher et al., 1997
) and hence inhibits phosphorylation of p38 protein substrates. ERK1/2 is phosphorylated by MAP kinase kinase 1 (MEK1), and PD98059, which inhibits phosphorylation of MEK1 (Dudley et al., 1995
), thus blocks phosphorylation via the ERK1/2 pathway.
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Although SH protein phosphorylation occurs via a MAPK p38-dependent pathway, RSV infection appears to be required for efficient phosphorylation of the protein. We have made several attempts to identify a phosphorylated form of the SH protein using recombinant protein expression in Vero C1008 cells. In these studies, efficient expression of the SH protein was obtained, but we failed to detect SH phosphorylation either by radiolabelling or by the use of phosphorylation-specific antibodies (H. W. McL. Rixon and R. J. Sugrue, unpublished observations). It is therefore possible that host-cell changes induced by RSV infection are required for SH protein phosphorylation.
At present we have not identified the tyrosine residue(s) that are phosphorylated within the SH protein sequence. However, examination of the RSV A2 strain SH protein sequence revealed that both tyrosine residues are located within a tyrosine-based sorting signal consensus sequence. These motifs have been shown to be involved in the interaction between specific cellular proteins and adapter protein (AP) complexes that allow their sorting into specific cellular compartments such as lysosomes (e.g. LAMP-1), the Golgi complex (e.g. TGN38 and furin) and specialized endosomal compartments such as antigen-processing compartments (e.g. HLA-DEM) (reviewed by Bonifacino & Traub, 2003). In addition, the Golgi localization of several virus proteins has been shown to be mediated via similar tyrosine-containing sorting signals, a list that includes the gB and gE proteins of varicella-zoster virus (Alconada et al., 1996
; Heineman & Hall, 2001
), the gE protein of herpes simplex virus (Alconada et al., 1999
) and the B5R protein of vaccinia virus (Ward & Moss, 2000
). In all cases, these sorting signals are orientated on the cytoplasmic side of the particular membrane-associated protein in question. Analysis of the SH protein has shown that the carboxyl terminus of the protein is orientated extracellularly, while the amino terminus is located on the cytoplasmic side of the membrane (Olmsted & Collins, 1989
; Collins & Mottet, 1993
). Although it has not been possible to demonstrate that these tyrosine-containing sequences function as sorting signals during infection, this suggests that the sequence 17YFTL20 would be located at a position in the protein that would favour this as a sorting signal. This sequence is conserved in all known SH protein sequences (Chen et al., 2000
), whereas the putative sorting sequence present within the extracellular domain (47YNFL50) is specific only to the RSV A2 strain.
The tyrosine residue within this type of sorting signal is critical for recognition by the AP complex and several reports have suggested that tyrosine phosphorylation may regulate the interaction between the sorting signal and the AP complex (Zhang & Allison, 1997; Shiratori et al., 1997
). We have observed that the inhibition of SH protein phosphorylation correlates with a large increase in the levels of the SH protein associated with the Golgi complex. If one, or both, of these tyrosine-containing sequences within the SH protein functions as a sorting signal, tyrosine phosphorylation may be one method by which its transport through the secretory pathway is controlled, although this possibility requires further investigation.
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ACKNOWLEDGEMENTS |
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Received 28 August 2004;
accepted 25 October 2004.
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