Caveolin-1 is incorporated into mature respiratory syncytial virus particles during virus assembly on the surface of virus-infected cells

Gaie Brown1, James Aitken2, Helen W. McL. Rixon1 and Richard J. Sugrue1

MRC Virology Unit1 and Division of Virology, University of Glasgow2, Institute of Virology, Church Street, Glasgow G11 5JR, UK

Author for correspondence: Richard Sugrue. Fax +44 141 337 2236. e-mail r.sugrue{at}vir.gla.ac.uk


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
We have employed immunofluorescence microscopy and transmission electron microscopy to examine the assembly and maturation of respiratory syncytial virus (RSV) in the Vero cell line C1008. RSV matures at the apical cell surface in a filamentous form that extends from the plasma membrane. We observed that inclusion bodies containing viral ribonucleoprotein (RNP) cores predominantly appeared immediately below the plasma membrane, from where RSV filaments form during maturation at the cell surface. A comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the pattern of caveolin-1 (cav-1) fluorescence staining. Analysis by immuno-electron microscopy showed that RSV filaments formed in close proximity to cav-1 clusters at the cell surface membrane. In addition, immuno-electron microscopy showed that cav-1 was closely associated with early budding RSV. Further analysis by confocal microscopy showed that cav-1 was subsequently incorporated into the envelope of RSV filaments maturing on the host cell membrane, but was not associated with other virus structures such as the viral RNPs. Although cav-1 was incorporated into the mature virus, it was localized in clusters rather than being uniformly distributed along the length of the viral filaments. Furthermore, when RSV particles in the tissue culture medium from infected cells were examined by immuno-negative staining, the presence of cav-1 on the viral envelope was clearly demonstrated. Collectively, these findings show that cav-1 is incorporated into the envelope of mature RSV particles during egress.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract disease in young children. It causes severe bronchiolitis in some young infants and milder respiratory tract infections in most adults. However, in certain specific groups (e.g. the elderly and the immunocompromised) disease progression is more severe. There is currently no effective vaccine to protect individuals within these high risk groups from RSV infection. A detailed understanding of the mechanisms involved in the RSV assembly process should provide the basis for rational vaccine strategies and specific treatments.

RSV formation occurs in association with host cell membranes to produce pleomorphic virus particles (Roberts et al., 1995 ). These RSV particles comprise a ribonucleoprotein (RNP) core that is surrounded by a viral envelope in which three different glycoproteins, the attachment (G), fusion (F) and small hydrophobic (SH) proteins are located. The RSV assembly process is still poorly understood and the factors involved in the formation of progeny virus are poorly defined. The involvement of lipid rafts in the assembly of several different members of the Paramyxoviridae has been demonstrated (Ali & Nayak, 2000 ; Manie et al., 2000 ; Vincent et al., 2000 ) and similar membrane platforms may be involved in RSV assembly and maturation.

A particular area of the RSV assembly process that is poorly understood is the role played by host cell proteins. The involvement of F-actin in RSV morphogenesis has been established (Ulloa et al., 1998 ; Burke et al., 1998 ) but its precise function in virus assembly has not been defined. In this respect, RSV is not unique since an involvement of the actin network has been demonstrated in several other enveloped viruses where budding occurs at the cell surface (Damsky et al., 1977 ; Bohn et al., 1986 ; Cudmore et al., 1995 ; Sasaki et al., 1995 ; Rey et al., 1996 ). We have sought to identify other host cell proteins that may be involved in the RSV assembly process. We have identified the integral membrane protein caveolin-1 (cav-1) as a host cell factor that is efficiently incorporated into the mature RSV virion. A number of functions in the host cell have been ascribed to cav-1, which range from providing a scaffolding protein in the formation of caveoli to acting as a negative regulator of several cellular activities (reviewed in Smart et al., 1999 ). In this report we have characterized the location of cav-1 within the mature virion and speculate as to the role played by this protein during virus replication.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cells and viruses.
The RSV A2 strain was used throughout this study. Vero C1008 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal calf serum and antibiotics.

{blacksquare} Antibodies.
The F protein monoclonal antibody mAb19 was provided by Geraldine Taylor (Institute of Animal Health, Compton, UK). The anti-RSV monoclonal antibody NCL-RSV3 was purchased from Novocastra Laboratories. The caveolin antibody N-20 was purchased from Santa Cruz Laboratories.

{blacksquare} Western blotting.
Cell monolayers were either mock-infected or infected with RSV and incubated at 33 °C for 24 h. These monolayers were extracted using 1% SDS, 15% glycerol, 1% {beta}-mercaptoethanol, 60 mM sodium phosphate, pH 6·8 and heated at 100 °C for 2 min prior to separation by SDS–PAGE and transfer by Western blotting onto a PVDF membrane. After transfer, the membrane was washed with PBSA and blocked for 18 h at 4 °C in PBSA containing 1% Marvel and 0·05% Tween 20. It was then washed twice in PBSA prior to incubation with anti-RSV (1/200 dilution), mAb19 (1/1000 dilution) or anti-cav-1 (1/500 dilution) for 60 min. The membrane was washed four times in PBSA containing 0·05% Tween 20 and probed using either goat anti-mouse or anti-rabbit IgG (whole molecule) peroxidase conjugate (Sigma). The protein bands were visualized using the ECL protein detection system (Amersham). Apparent molecular masses were estimated using Rainbow protein markers (Amersham) in the molecular mass range 14·3–220 kDa.

{blacksquare} Immunofluorescence.
Cells were seeded onto 13 mm glass coverslips and incubated overnight at 37 °C. Cells were mock- or RSV-infected, after which they were fixed with 3% paraformaldehyde in PBS for 30 min at 4 °C. The fixative was removed and the cells washed five times with PBS plus 1 mM glycine and once with PBS. The cells were either examined directly or permeabilized using 0·1% saponin in PBS. Following incubation at 25 °C for 1 h with the primary antibody, the cells were washed and incubated for a further 1 h with the secondary antibody, goat anti-mouse or anti-rabbit IgG (whole molecule) conjugated to either FITC or Cy5 conjugate (1/100 dilution) as appropriate. The F-actin network was detected using phalloidin–FITC (Sigma). The stained cells were mounted on slides using Citifluor and visualized using a Zeiss Axioplan 2 confocal microscope. The images were processed using LSM 510 v2.01 software.

{blacksquare} Scanning electron microscopy (SEM).
Cells were seeded onto 10 mm glass coverslips and incubated overnight at 37 °C. Following infection with RSV for 24 h, the samples were processed as described previously (Sugrue et al., 2001 ) and visualized using a JEOL T100 scanning electron microscope.

{blacksquare} Transmission electron microscopy (TEM)
Unicryl embedding.
Cell monolayers were pelleted in BEEM capsules and fixed for 5 h at 4 °C using 0·5% glutaraldehyde in PBS, after which the fixed pellet was washed with PBS. The pellet was 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. Ultra-thin sections were placed on nickel-coated grids and incubated with either anti-cav-1 (1/100 dilution), mAb19 (1/100 dilution) or anti-RSV (1/50 dilution) for 4 h at 25 °C. The grids were washed with PBS and incubated for 12 h in anti-rabbit IgG (whole molecule) 20 nm colloidal gold (British Biocell International), 10 nm colloidal gold conjugate (Sigma) or anti-mouse IgG (whole molecule) 10 nm colloidal gold conjugate (Sigma) as appropriate. They were then washed in PBS and fixed in osmium tetroxide vapour for 2 h. The samples were then stained using uranyl acetate (saturated in 50:50 ethanol/water), washed in PBS and counter-stained with lead citrate.

Epon 812 embedding.
Samples embedded in Epon 812 (TAAB Laboratories) were processed as follows. Cells were pelleted and fixed using 2·5% glutaraldehyde in PBS at 4 °C, post-fixed in 1% osmium tetroxide and washed with PBS. The fixed cells were dehydrated by transfer through a gradient of 30, 50, 70, 90 and 100% ethanol and infiltrated with Epon 812. The resin was polymerized at 70 °C for 48 h and subsequently sectioned. The sections were stained with uranyl acetate (saturated in 50:50 ethanol/water), washed in PBS and counter-stained with lead citrate.

Immuno-negative staining of RSV preparations.
RSV-infected cells were detached from a T75 flask together with the tissue culture medium, and harvested. The cell debris was removed by low speed centrifugation (1000 g, 5 min) and the clarified tissue culture medium analysed by TEM. A droplet of the infected tissue culture medium was placed on a Formvar-coated grid and the virus adsorbed for 10 min. Grids were inverted onto a droplet of primary antibody for 4 h, washed in PBS, and then incubated for a further 4 h either with anti-rabbit IgG (whole molecule) 10 nm colloidal gold conjugate (Sigma) or anti-mouse IgG (whole molecule) 5 nm colloidal gold conjugate (Sigma). The grids were washed in PBS and fixed in osmium tetroxide vapour for 2 h. The samples were then stained with uranyl acetate (saturated in 50:50 ethanol/water), washed in PBS and counter-stained with lead citrate.

Samples were examined using a JEOL 1200 EX2 transmission electron microscope.


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
1. RSV maturation on the apical surface of Vero C1008 cells
The data presented in this report were obtained using the polarized Vero C1008 cell line. Two different antibodies were employed to identify RSV antigen in RSV-infected cells. These were mAb19, which recognizes the F glycoprotein (Taylor et al., 1992 ), and NCL-RSV3, which comprises a mixture of three different monoclonal antibodies that recognize the N, P and F proteins (Wright et al., 1997 ). The N and P proteins, together with the polymerase and vRNA, constitute the RNP core. In this report, NCL-RSV3 will be referred to as anti-RSV. The specificities of mAb19 and anti-RSV were examined by Western blotting analysis (Fig. 1A). Probing with mAb19 showed a single 50 kDa protein product, which is the expected size for the F1 subunit. In contrast, probing with anti-RSV showed three proteins whose sizes were 35 kDa, 40 kDa and 50 kDa and are the expected sizes for the P, N and F1 proteins, respectively. We noted that when Western blots were probed with anti-RSV, the intensity of the F glycoprotein band was significantly less than that of either the P or the N protein, suggesting that this reagent has a greater affinity for the P and N proteins. As expected, none of these protein bands was detected in mock-infected cells, confirming the specificity of these reagents.



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Fig. 1. The location of RSV filaments on the surface of virus-infected cells. (A) Western blot analysis of mock-infected (M) and RSV-infected (I) cells using anti-RSV and mAb19. The positions of the specific RSV proteins are indicated. (B) Localization of RSV filaments on the apical surface of RSV-infected Vero C1008 cells using fluorescence microscopy. Mock (M) and RSV (I)-infected cells were permeabilized and labelled using mAb19 or anti-RSV. The RSV filaments (VF) and an inclusion body (IB) are indicated, as is the magnification of the objective lens used (x20, x40 or x64). (C) Analysis of RSV-infected cells using SEM. The cell surface of mock-infected (M) and RSV-infected (I) cells was examined by SEM. A cluster of viral filaments (VF) is indicated and the arrows show the limits of a single large RSV filament cluster. Magnification x15000. Bar indicates 1 µm.

 
Analysis of the surface of RSV-infected cells by immunofluorescence (IF) microscopy showed the appearance of long filamentous structures that correspond to budding virus (Fig. 1B) and indicate the sites of virus budding. The filamentous structures that we observe are similar to those previously described on RSV-infected cells (Parry et al., 1979 ; Roberts et al., 1995 ; Sugrue et al., 2001 ). These filaments formed both singly and in clusters on the surface of this cell line, suggesting that virus assembly occurs at specific sites on the cell surface (Roberts et al., 1995 ). Whereas the staining with mAb19 was almost exclusively located in the RSV filaments, staining using anti-RSV showed, in addition to filaments, the presence of large punctate structures which were of variable size (Fig. 1B). These inclusion bodies (IBs) have been observed previously and contain RNP-associated virus proteins (Garcia et al., 1993 ; Garcia-Barreno et al., 1996 ).

The surface of RSV-infected cells was examined in detail using SEM (Fig. 1C). Our data clearly show clusters of RSV filaments budding from the cell surface and extending up to 6 µm in length. As in the case of the IF analysis, the filaments appeared to extend across the plane of the apical cell surface. It is likely that many of these filaments that traverse the cell surface are randomly cross-sectioned in the samples prepared for TEM analysis (described below), giving the impression of spherical shaped RSV particles associated with the cell membrane. The surface of RSV-infected Vero cells was additionally examined using in situ sectioning and TEM (data not shown), which confirmed earlier observations that RSV filaments form preferentially at the apical surface of Vero C1008 cells (Roberts et al., 1995 ).

We have used TEM to provide a detailed analysis of the RSV maturation process on the cell surface. These studies employed two different embedding techniques. RSV morphology was examined in samples embedded using Epon 812 resin since this allows the best preservation of ultra-structural detail, while Unicryl embedding preserves the antigenicity of the epitopes in the thin sections and was employed for immuno-electron microscopy. Examination of cell sections using immuno-electron microscopy clearly showed the presence of two virus-related structures that were not detected in mock-infected cells and which could be readily identified on the basis of their morphology (Fig. 2A, plate 1). Firstly, there were RSV filaments (VF) protruding from the surface of infected cells and secondly, throughout the cytoplasm we observed the presence of electron-dense IBs, the latter structures being identical to the IBs visualized by confocal microscopy (Fig. 1B). These structures were of variable size, ranging from approximately 300 nm up to several µm in length. As expected, these structures could not be labelled with either mAb19 or antibodies against the G glycoprotein (data not shown), suggesting the absence of viral glycoprotein. The specific recognition of IBs by anti-RSV (Fig. 2A, plate 1) suggests that these structures contain RNP-associated proteins. Examination of IBs using TEM at high magnification (Fig. 2A, plate 2) clearly shows that they are structured rather than being amorphous and contain material whose morphology resembles the RNP structures that are observed by TEM in RSV preparations (see Fig. 5B). The data that we have obtained using TEM show that in the majority of cases the IBs are intimately associated with the RSV filaments that form at the cell surface, and the material which is present within IBs appears to enter the interior of the filaments. However, we have also observed that, in some instances, material from the IBs appears to be associated with the cell membrane in the absence of RSV filaments (Fig. 2A, plate 1, highlighted by open box). It is interesting to note that structures resembling IBs could be detected by confocal microscopy on the surface of some non-permeabilized cells using anti-RSV (data not shown). The significance of the latter observation is at present unclear but suggests that the RNP cores may make direct contact with the cell exterior. It is likely that the dimensions of the RSV filaments (approximately 0·15x6 µm) may make them fragile and prone to detachment from the cell surface, consequently leaving the remaining underlying IB-associated material on the cell surface. Alternatively, RSV filaments may be detached from the cell surface as a consequence of virus release following maturation. However, we have no evidence that free RNP cores were present in the tissue culture medium of RSV-infected cells.



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Fig. 2. Ultra-structural analysis of RSV maturing at the surface of infected cells. (A) The relationship between the RSV filaments forming at the cell surface and the IBs. Plate 1 shows ultra-thin sections from Unicryl-embedded RSV-infected cells immunolabelled with anti-RSV as described in Methods. An open box highlights the apparent interaction of the IB-associated material with the plasma membrane in the absence of RSV filaments. Plate 2 (inset) is a high magnification image (x100000) of a representative region of an IB showing its structured organization. A close inspection of this image reveals the presence of structures that resemble viral RNP cores, some of which are highlighted using black arrows. VF, virus filaments; IB, inclusion bodies; PM, plasma membrane. (B) Early stages in RSV maturation on the surface of infected cells. RSV-infected BHK (plate 1) or Vero C1008 (plate 2) cells were embedded using Epon 812 and processed as described in Methods. (a)–(d) The probable order of RSV budding particle formation from the initial structure that forms (a) to the final mature RSV particle (d). Black arrows highlight the extended spike proteins on the surface of an early budding RSV particle. Plate 3 is a section through the mature virus particle from infected Vero C1008 cells exhibiting the major morphological features: a, surface proteins showing the electron-dense fuzzy coat appearance as described in the text; b, the viral envelope; c, the internal components, e.g. RNP. Plate 4 shows immuno-electron microscopy of RSV-infected cell sections prepared in Unicryl, stained using anti-RSV and decorated with 10 nm colloidal gold particles. RSV budding (VB) from the plasma membrane (PM) is indicated.

 
A detailed examination of the surface of Epon-embedded RSV-infected cells has allowed us to identify early budding structures that appeared to be at different stages of RSV maturation (Fig. 2B). These results show that budding from the cell surface is accompanied by morphological changes, both on the cell surface and within the internal structures of RSV particles. The RSV virion initially forms as a swelling on the surface membrane (Fig. 2B, plates 1 and 2). As this proceeds, the budding virus acquires an electron-dense core, presumably the viral RNP. In addition, these data suggest morphological changes within the viral envelope as budding proceeds. The viral spike proteins initially appear to be in an extended configuration, which then acquires the typical electron-dense fuzzy coat morphology (Fig. 2B, plates 1 and 3) that was described previously (Roberts et al., 1995 ). These early budding structures were efficiently labelled using anti-RSV in immuno-electron microscopy (Fig. 2B, plate 4).

2. The cell distribution of cav-1 is altered by RSV infection
A relatively unexplored aspect of RSV assembly concerns the role of host cell factors in the assembly process. Since the filaments are clearly visible by light microscopy (Parry et al., 1979 ), IF microscopy affords a relatively simple assay to determine which, if any, host cell proteins associate with mature RSV. In our assay, the host cell protein cav-1 showed a significant change in fluorescence staining upon RSV infection. In mock-infected cells, the observed staining pattern for cav-1 consisted of small red spots (Fig. 3A, plate 1). However, in RSV-infected cells, the cav-1 adopted a staining pattern that was composed of the small spots, similar to that observed in mock-infected cells, and a filamentous staining pattern (Fig. 3A, plates 2 and 3) that was not seen in mock-infected cells. The filamentous staining pattern appeared to resemble the RSV filaments that were visualized in infected cells using mAb19 and anti-RSV (Fig. 1B). This filamentous form of cav-1 appeared to align with the direction of the green-stained actin filaments (F-actin) when the latter were visualized following phalloidin–FITC staining of F-actin. We also noted that the number of visible actin filaments appeared to increase in the RSV-infected cells when compared with those observed in mock-infected cells, which agrees with recent observations (Gower et al., 2001 ). However, the filamentous cav-1 that we observed in RSV-infected cells showed no significant co-localization with F-actin. Analysis of mock- and RSV-infected monolayers by Western blotting using anti-cav-1 (Fig. 3A, plate 4) showed the presence of a single 23 kDa protein band, which is the expected size for cav-1. In addition Western blotting showed that the detectable levels of cav-1 remained unchanged following introduction of the virus, suggesting that RSV does not stimulate cav-1 expression, but rather, the cell surface distribution of cav-1 is altered during infection.



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Fig. 3. Alteration of cav-1 cellular distribution in RSV-infected cells. Vero C1008 cells were either mock-infected (plate 1) or RSV-infected (plates 2 and 3) and the distribution of F-actin (green) and cav-1 (red) was determined by fluorescence microscopy as described in Methods. A more detailed analysis of RSV-infected cells is shown in plate 3. The white arrow highlights a cav-1 filament. Plate 4 shows Western blot analysis of mock (M) and RSV (I)-infected cells using anti-cav-1. The position of cav-1 is indicated.

 
3. Cav-1 is incorporated into mature RSV
We compared the staining pattern of cav-1 and the RSV antigens during the course of infection, from 4 h up to 16 h post-infection (Fig. 4A). Early in the infection process (up to 12 h) we were able to detect both the presence of RSV antigen (red) and cav-1 (green), but no RSV filaments were visible. In each case, a distinct pattern of staining was observed with no co-localization of cav-1 with the RSV proteins. In the case of cav-1, a speckled pattern of staining typical of cav-1 in mock-infected cells was observed, which contrasted with the larger, speckled pattern observed for the RSV antigen staining. However, at longer incubation periods (16 h post-infection), we noted the appearance of both the RSV filaments and the cav-1 filamentous structures. The appearance of these structures by fluorescence microscopy coincided with co-localization of the RSV filaments with cav-1 (Fig. 4A, yellow colour) and suggests that a significant proportion of cav-1 re-locates into the filaments during virus egress. Although the data presented in this report have utilized Vero C1008 cells, the incorporation of cav-1 into RSV filaments was observed in several other cell lines, including BHK and HEp2 cells (data not shown). Therefore, this phenomenon is not related to the use of a single, specific cell line. Collectively, these data provide strong evidence that cav-1 is recruited into newly formed virus during the assembly process.



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Fig. 4. Fluorescence microscopy reveals the co-localization of cav-1 and RSV filaments. (A) Co-localization of cav-1 and RSV antigen is dependent upon the formation of RSV filaments. Cell monolayers were infected with RSV, fixed and permeabilized as described in Methods at 4, 8, 12 and 16 h post-infection. The cells were double-stained using anti-cav-1 (green) and mAb19 (red) and examined by confocal microscopy. Co-localization of anti-cav-1 and mAb19 antibodies is indicated by the yellow staining pattern. The open white box highlights RSV filaments. (B) Co-localization of anti-cav-1 and RSV filaments at 24 h post-infection. RSV-infected cells were either non-permeabilized and stained using anti-cav-1 and mAb19 (plate 1) or permeabilized and stained using anti-cav-1 and anti-RSV (plate 2). Cells stained with anti-cav-1 (green), mAb19 (red) and anti-RSV (red) are shown. Co-localization is indicated in the merged image by yellow staining. VF, RSV filaments; IB, inclusion bodies.

 
In a subsequent assay, RSV-infected cells were analysed by confocal microscopy at 24 h post-infection (Fig. 4B). Examination of non-permeabilized Vero C1008 cells showed that the filamentous pattern of cav-1, which is induced by virus infection, co-localizes with the vast majority of RSV surface filaments that are stained using mAb19 (Fig. 4B, plate 1, yellow colour). In virus-infected cells that were permeabilized prior to staining with anti-RSV and cav-1, co-localization was only observed on the RSV filaments. The IB structures showed no staining with anti-cav-1 and hence no co-localization (Fig. 4B, plate 2). Although labelling of the RSV filaments using the RSV-specific antibodies showed solid staining, we noted that labelling with anti-cav-1 failed to stain the filaments in their entirety. Co-localization of the cav-1 and RSV filaments occurred within easily visible patches, suggesting that cav-1 is located at specific sites along the RSV filaments rather than being uniformly distributed along the length of the filaments. However, the clear co-localization between anti-cav-1 and the two RSV antibodies suggests that cav-1 is a component of mature filaments, but is not associated with other virus-related structures that are within the infected cell, e.g. IBs.

The association of cav-1 with RSV filaments was further examined by immuno-electron microscopy (Fig. 5). In this study, two strategies were employed to prepare samples for analysis, namely thin sections obtained from RSV-infected cells (Fig. 5A) and immuno-negative staining of RSV particles that were present in the tissue culture medium (Fig. 5B). Examination of RSV-infected cell sections showed the labelling of RSV with anti-cav-1 (Fig. 5A, plates 1 and 2), as evidenced by the presence of the 10 nm gold probe on the morphologically distinct RSV filaments. In addition, we observed that the pattern of labelling was localized in specific areas of the viral filaments, visualized as between six and 12 tightly clustered 10 nm gold particles. We also observed a clustering of gold particles in specific regions of the infected cell plasma membrane, which may represent regions of cav-1 enrichment such as those present within the caveoli or lipid rafts. In this respect it is interesting to note that clusters of gold particles (i.e. cav-1 enrichment) generally appeared in regions of the plasma membrane that were close to RSV filament formation. Further analysis of mature cell-associated RSV filaments by double immuno-gold labelling showed co-localization of anti-RSV and anti-cav-1 (Fig. 5A, plate 3). Additionally, cav-1 appeared to be associated with the RSV filaments early in the assembly process, since early budding structures were labelled with anti-cav-1 (Fig. 5A, plate 4). This localized labelling of RSV filaments by anti-cav-1 using TEM is consistent with the observed localized staining pattern of cav-1 on RSV filaments by confocal microscopy.



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Fig. 5. TEM analysis of RSV-associated cav-1 by immuno-gold labelling with anti-cav-1. (A) Thin sections from RSV-infected cells were labelled using anti-cav-1, the presence of which was observed by the appearance of 10 nm colloidal gold. Plates 1 and 2 are different views of RSV filaments that have been decorated with 10 nm gold particles. Plate 3 shows double immuno-labelling with 20 nm (cav-1) and 10 nm (anti-RSV) colloidal gold. In this view, both a longitudinal and cross-sectioned filament are shown. Plate 4 shows that at the initial stages of RSV assembly, the early budding virus particles can be labelled using anti-cav-1 and decorated with 10 nm gold particles. Furthermore, the gradual acquisition of an electron-dense core in the early budding RSV particles can be seen in plate 3. (B) RSV particles were harvested from the tissue culture medium of virus-infected cells and the presence of cav-1 was detected by immuno-gold labelling. Plate 1 shows a mechanically disrupted RSV particle in which the viral envelope (VE) and RNP are clearly visible. The viral envelope is decorated at four places with 10 nm gold after labelling with anti-cav-1. Plate 2 shows immuno-negative staining of an intact RSV particle by double-labelling using 5 nm (mAb19) and 10 nm (cav-1) colloidal gold particles. The 10 nm gold particles are indicated by asterisks.

 
In a final analysis, we assayed the presence of cav-1 on the surface of RSV particles prepared from the tissue culture medium of RSV-infected cells (Fig. 5B). Analysis of the cell-free RSV particles by TEM, following immuno-negative staining, allowed us to visualize clearly the viral envelope and the associated spike proteins. RSV particles that had been mechanically disrupted were labelled with anti-cav-1. We observed that labelling with cav-1 was confined to the viral envelope, with no label being detected within the morphologically distinct viral RNP core that was liberated from the interior of the particle (Fig. 5B, plate 1). These observations are consistent with data obtained by confocal microscopy, using anti-cav-1, which showed specific labelling of the RSV filaments but not the viral IBs. In a similar experiment, we demonstrated that the surface of intact RSV particles could be labelled with both mAb19 and anti-cav-1 (Fig. 5B, plate 2). This provides clear evidence that cav-1 is present on the surface of mature RSV particles that have been released from the surface membrane of virus-infected cells.


   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The work presented in this report has provided us with an insight into some novel aspects of the RSV maturation process. The images that we have obtained, both by TEM and confocal microscopy, clearly show that RSV matures in the form of filamentous structures and that this maturation process is accompanied by visible morphological changes both within the spike proteins on the viral envelope, and within the interior of the virus, the latter presumably arising from the acquisition of the viral RNP and matrix protein. Our results show that during maturation, viral RNPs accumulate underneath the RSV filaments as the latter form at the cell surface. However, the precise biochemical mechanism by which RNP cores are transported into budding virus is not clearly defined, but may involve elements of the cyto-skeletal network (Ulloa et al., 1998 ; Burke et al., 1998 ).

Caveoli have been shown to provide a mechanism by which RSV can enter the host cell following cell attachment (Werling et al., 1999 ). However, our data suggest that the major component of caveoli structures, namely cav-1, is additionally involved in the subsequent steps of virus assembly. The results of immuno-electron microscopy showed that RSV filaments generally form at, or in close proximity to, regions on the cell surface that are strongly labelled by anti-cav-1. Furthermore, we observed that the maturing RSV could be labelled with anti-cav-1 soon after the budding process was initiated. This suggests that cav-1 may be an important host cell factor which is involved either in the targeting of RSV envelope proteins to the cell surface or as a focus point for the initiation of RSV assembly on the cell surface membrane. In addition, our results clearly show that cav-1 is subsequently incorporated into the envelope of maturing RSV in clusters along the length of the RSV filaments. The route by which cav-1 is incorporated into maturing RSV particles remains to be established. It is not clear if cav-1 incorporation occurs passively during virus maturation or actively by association with either host cell- or virus-specific proteins. However, the incorporation of host cell proteins into mature virus is not unique to RSV, since this phenomenon has been documented both in vesicular stomatitis virus (Lodish & Porter, 1980 ; Calafat et al., 1983 ) and human immunodeficiency virus 1 (Saifuddin et al., 1994 ; Marschang et al., 1995 ). In addition, a recent study has shown that several intracellular signalling molecules can be incorporated into virus particles (Pickl et al., 2001 ). These previous reports provide a precedent for our observation that cav-1 can be incorporated into the envelope of mature RSV.

We have noted that in RSV-infected cells, the cav-1 filaments appear to align with F-actin, although no direct interaction was identified, but it is possible that such an interaction may involve other actin-associated proteins. Recent studies have shown that Rho activation leads to the association of cav-1 with actin stress fibres via the actin-associated protein, filamin (Stahlhut & van Deurs, 2000 ). It is not clear if a similar mechanism operates during RSV assembly since we have so far failed to identify any stable interactions between filamin and either RSV filaments or cav-1 in RSV-infected cells (data not shown). However, the actin network may provide a mechanism, either directly or indirectly, whereby specific virus and host cell factors are recruited into maturing RSV.

The nature of the interaction between cav-1 and the mature RSV filaments is currently not defined. It has been established that cav-1 is an integral membrane protein (Kurzchalia et al., 1994 ) and as such has the potential to be inserted directly into the RSV envelope. In addition, we have failed to detect any stable interaction between cav-1 and either the F or G glycoproteins (data not shown). However, if such an interaction exists, it may be mediated indirectly by one or more host cell factor. Previous reports have shown that cav-1 is able to interact with RhoA GTPase (Gingras et al., 1998 ), a molecule which is activated during RSV infection (Gower et al., 2001 ) and which is able to interact with the F1 subunit of the F glycoprotein (Pastey et al., 1999 ).

A final question concerns the biological consequences of cav-1 incorporation into mature RSV particles. The role that RSV-associated cav-1 plays in the virus replication cycle and the effect of its presence on RSV pathogenesis is currently undefined. Although cav-1 is the major structural component of caveoli, several other specific functions have been ascribed for cav-1, such as regulating signalling pathways in the cell. Evidence has suggested that cav-1 can down-regulate steps in the p42/44 MAPK signal transduction pathway (Engelman et al., 1998 ; Galbiati et al., 1998 ) and the negative regulation of nitric oxide synthase by cav-1 has been demonstrated (Garcia-Cardena et al., 1997 ; Ju et al., 1997 ; Ghosh et al., 1998 ). It may be speculated that down-regulating specific host cell activities and signalling pathways could be advantageous to the virus either during the initial stages of virus entry or during the assembly process.

Taken as a whole, the data presented in this report suggest that the RSV assembly process is complicated, involving virus structural proteins as well as at least one specific host cell membrane-associated factor. Additionally, this is the first report of a cellular protein that is incorporated into the RSV envelope during the assembly process. Although we have shown that cav-1 is a host cell-derived factor that is incorporated into mature RSV, its role during virus infection remains to be established. In addition, it is not clear if cav-1 is a component of other paramyxoviruses, particularly those that mature as filaments on the infected cell surface.


   Acknowledgments
 
We thank Duncan McGeoch for critical review of the manuscript. We are grateful to Geraldine Taylor at the Institute of Animal Health, Compton, UK for providing mAb19.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 29 August 2001; accepted 16 November 2001.