Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK1
Author for correspondence: Geoffrey Smith. Present address: The WrightFleming Institute, Faculty of Medicine, Imperial College, St Marys Campus, Norfolk Place, London W2 1PG, UK. Fax +44 207 594 3973. e-mail glsmith{at}ic.ac.uk
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Abstract |
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Introduction |
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Five proteins have been identified as specific to the EEV particles and absent from IMV. These are F13L (Hirt et al., 1986 ), B5R (Engelstad et al., 1992
; Isaacs et al., 1992
), A33R (Roper et al., 1996
), A34R (Duncan & Smith, 1992
) and A56R (the virus haemagglutinin) (Payne & Norrby, 1976
). In addition, the A36R protein (Parkinson & Smith, 1994
) is present in IEV but not IMV, CEV or EEV particles (van Eijl et al., 2000
). Lastly, the F12L protein was identified recently as a 65 kDa protein that is synthesized predominantly late during infection and is conserved in chordopoxviruses. A deletion mutant lacking F12L did not produce actin tails, had a small plaque size, produced 7-fold reduced levels of EEV and was highly attenuated in vivo (Zhang et al., 2000
). Although low amounts of the F12L protein co-purified with EEV in density gradients (Zhang et al., 2000
), a direct physical interaction with these enveloped virions has not been demonstrated.
None of the EEV- and IEV-specific proteins are required for the formation of IMV, but they have differing roles thereafter. In the absence of F13L (Blasco & Moss, 1991 ) or B5R (Engelstad & Smith, 1993
; Wolffe et al., 1993
), the wrapping of IMV by intracellular membranes to form IEV is reduced or abolished and therefore subsequent stages of morphogenesis are inhibited. Deletion or repression of A33R (Roper et al., 1998
), A34R (Duncan & Smith, 1992
; McIntosh & Smith, 1996
; Wolffe et al., 1997
; Sanderson et al., 1998
) or A36R (Parkinson & Smith, 1994
; Sanderson et al., 1998
; Wolffe et al., 1998
) enables IEV, CEV and EEV to be formed, but cell-to-cell virus spread is reduced and these mutants form a small plaque. Enhanced levels of EEV are released when gene A33R (Roper et al., 1998
) or A34R (McIntosh & Smith, 1996
) is deleted, but without A34R the EEV has a reduced infectivity. Enhanced levels of EEV are also formed when the A34R (Blasco et al., 1993
) or B5R proteins (Herrera et al., 1998
; Mathew et al., 1998
, 2001
) are mutated. Loss of the A36R (Parkinson & Smith, 1994
) or F12L (Zhang et al., 2000
) genes caused a reduction in EEV formation. For all these mutants the production of intracellular actin tails is reduced or abolished, providing a direct correlation between actin tail formation and virus cell-to-cell spread. The only IEV or EEV-specific protein not required for normal morphogenesis is A56R (Ichihashi et al., 1971
; Sanderson et al., 1998
) (G. L. Smith, unpublished data).
In this paper, we have characterized the F12L protein further by determining its location in infected cells and analysing virus morphogenesis with the F12L deletion mutant. Data presented show that the F12L protein, like the A36R protein, is present only on IEV particles and absent from IMV, CEV and EEV. In the absence of F12L, IEV particles are made, but these do not move to the cell surface, implying a role for F12L in this transport process.
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Methods |
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Fluorescent microscopy.
Cells growing on glass microscope slides (Chance Proper) were infected at 1 p.f.u. per cell with the indicated virus. At the stated times post-infection (p.i.), cells were washed in PBS and fixed in 4% paraformaldehyde (PFA) as described (van Eijl et al., 2000 ). Cells were blocked and permeabilized for 5 min at room temperature (RT) in PBS containing 0·1% saponin and 10% FBS. Alternatively, infected cells were fixed by incubation for 1 min at -20 °C in acetone and dried in air for 30 min. Cells were stained with rat mAb 19C2 (Schmelz et al., 1994
) (diluted 1/50 in PBS containing 0·1% saponin and 10% FBS), which recognizes the VV B5R protein, mouse mAb HA.11 (BAbCo, Berkley, California) (diluted 1/400), which recognizes the HA epitope at the C terminus of F12LHA, mouse mAb AB1.1 (Parkinson & Smith, 1994
), which recognizes VV protein D8L (diluted 1/50), rat mAb alpha tubulin (Serotec) (diluted 1/10) or rhodamine-conjugated mouse mAb against the transferrin receptor (Boehringer Mannheim) (diluted 1/50). Bound mAbs were detected by a goat anti-rat IgG fluorescein isothiocyanate (FITC)- or goat anti-mouse IgG tetramethylrhodamine B isothiocyanate (TRITC)-conjugated secondary antibody (Stratech Scientific) (diluted 1/100). F-actin was visualized with TRITC-conjugated phalloidin (Sigma) (diluted 1/100). Cells were washed twice in PBS and once in water, and then mounted in MowiolDAPI mounting medium (Sanderson et al., 1996
). Images were viewed using a Bio-Rad MRC 1024 confocal laser-scanning microscope, collected using Lasersharp software and processed using Adobe Photoshop.
Live cells were stained with mAb 19C2 and mAb HA.11 (diluted 1/50 and 1/200, respectively in cell medium) as described (van Eijl et al., 2000 ). Cells treated with the microtubule depolymerizing drug nocodazole were lysed for 15 s with 0·1% Triton X-100 in PBS before fixation.
Preparation of cells for electron microscopy.
BS-C-1 cells were infected at 1 p.f.u. per cell. After 1 h the inoculum was replaced with MEM2·5% FBS and at the indicated times p.i. the cells were washed with ice-cold PBS, fixed in 0·5% glutaraldehyde in 200 mM sodium cacodylate (pH 7·4) for 30 min at room temperature. Where indicated, infected cells were incubated with horseradish peroxidase (HRP) (10 mg/ml) for 60 min before fixation and subsequently were reacted with 1 mg/ml diaminobenzidine for 30 min at room temperature. All cells were washed in water, and post-fixed in 1% osmium tetroxide and 1·5% potassium ferrocyanide for 60 min at room temperature. After being washed in water and then incubated in Mg2+ uranyl acetate overnight at 4 °C, the cells were washed again in water and then dehydrated in graded ethanol and flat-embedded in Epon. Sections were cut parallel to the surface of the dish and collected onto slot grids; lead citrate was added as a contrast agent.
Image collection and data processing.
All sections were examined in a Zeiss Omega 912 electron microscope equipped with a Proscan cooled slow-scan charge-coupled device camera (1024 by 1024 pixels). Digital images were captured with the integrated Soft Imaging Software (SIS) image analysis package and processed using Adobe Photoshop.
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Results |
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Cells infected with vF12LHA were stained with mAbs HA.11 (Fig. 1a) and 19C2 (Fig. 1b
) against the F12LHA and B5R proteins, respectively, and analysed by confocal microscopy. The F12LHA protein was present on particulate structures that were of a size (600700 nm) and distribution consistent with enveloped virions. An IEV particle of 350400 nm would appear larger due to the presence of primary and secondary antibodies and fluorescent flare. The proposal that F12LHA was associated with enveloped virions was supported by co-localization of these structures with B5R, a protein present on IEV and CEV particles as well as the membranes used to wrap IMV particles. Merging these images (Fig. 1c
) showed that a proportion of the enveloped virions were labelled by both F12L and B5R, but others were B5R-positive only. Another difference in the distribution of the F12L and B5R proteins was the presence of B5R but not F12L in the perinuclear region that represents Golgi.
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Taken together, these data are consistent with data obtained by confocal microscopy and indicated that F12LHA was not present on the cell surface nor was it associated with CEV particles.
Co-localization of F12LHA with microtubules
The subcellular localization of F12LHA was examined further by staining infected cells with mAb HA.11 followed by confocal microscopy. In some cases it was noticed that F12LHA-positive intracellular structures seemed to have a linear array (Fig. 4a). This was consistent with these structures being IEV particles that are moving along microtubules to reach the cell surface as shown recently (Hollinshead et al., 2001
; Ward & Moss, 2001
). The apparent co-localization of F12LHA and microtubules was investigated further by preparing infected cells that had been treated with nocodazole, a microtubule depolymerizing drug, and then extracted with Triton X-100. Under these conditions the microtubules are largely depolymerized and soluble tubulin has been removed, but some filaments remain (Fig. 4b
). When these extracted cells were stained with mAbs against F12LHA and
-tubulin, co-localization of microtubules and F12LHA (IEV particles) was seen (Fig. 4c
).
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The relative locations of B5R and F12L were studied further by treating cells with brefeldin A for 15 min, which causes the coalescence of endosomes into a tubular network (Tooze et al., 1993 ) and is illustrated here by labelling cells with either HRP (Fig. 5b1
) or rhodamine-conjugated mAb to the transferrin receptor (Fig. 5b2
). Note the reticular pattern of fused endosomes. In vF12LHA-infected cells, B5R was present in Golgi aggregates (bright staining) but also in endosome compartments as for the transferrin receptor (Fig. 5b3
). These were co-incident with F12L staining (Fig. 5b4
, 5
), confirming co-localization of F12L and endosomes.
Loss of F12L prevents migration of IEV to cell periphery
Electron microscopy showed that the F12L protein is associated with IEV particles (Fig. 3), but that when the F12L gene is deleted, actin tails are not made and there is a small plaque phenotype (Zhang et al., 2000
). The failure to make actin tails might have been due to an inhibition of IEV formation (like v
F13L), blockage of IEV transport to the cell surface, or inability of the CEV at the surface to induce actin tail formation (like v
A36R). To investigate these possibilities, v
F12L-infected cells were stained with B5R and phalloidin (Fig. 7a
). In these cells, the B5R (green signal) remained predominantly in the perinuclear regions and was not disseminated to the cell surface. Consistent with the lack of actin tails, the cell actin stress fibres remained prominent. In contrast, in cells infected with vF12LHA (or WR, not shown) B5R was dispersed from the central region and there were aggregates of virions in the cell periphery (Fig. 7b
). These cells produced actin tails (Fig. 7b
insert). To investigate if B5R reached the cell surface in the absence of F12L, live cells infected with v
F12L were stained with mAb 19C2, and after fixation and permeabilization the cells were stained for D8L (red signal). Under these conditions no B5R staining was observed (Fig. 7c
) showing that CEV particles were not made, and the D8L protein was located close to virus factories with only a few dispersed virions. In comparison, cells infected with v
A36R produced cell surface B5R and CEV particles, which also contained D8L (Fig. 7d
). Therefore, although both v
F12L and v
A36R do not make actin tails, these viruses arrest at different stages of the morphogenic pathway.
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Discussion |
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The location of the F12L protein in infected cells was examined using a recombinant virus in which the F12L protein was tagged at the C terminus with a 9-amino-acid epitope that is recognized by a mAb. Immunoelectron microscopy showed that the F12L protein was present on IEV particles, predominantly near the outer membrane, but it was absent from IV, IMV and CEV (Fig. 3). F12L is therefore the second IEV-specific protein that has been identified. The presence of low amounts of the protein associated with EEV that had been purified on density gradients (Zhang et al., 2000
) is reminiscent of the A36R protein that also co-purified with EEV, but which was associated with fragments of cell membranes that remained attached to EEV particles after their detachment from the cell. Within the infected cells, one difference between F12L and A36R was the presence of A36R but not F12L on the cytosolic face of the plasma membrane beneath CEV particles. The position of A36R at this site is consistent with the role of A36R in inducing the polymerization of actin tails to drive CEV particles away from the cell. How F12L is absent from this site while having a similar distribution on IEV particles remains to be determined.
Immunofluorescence showed that the F12LHA protein was not detected on the cell surface, consistent with its absence from CEV particles. However, since the mAb detects only the C terminus of F12LHA, it was uncertain if all the F12L protein was within the cell. The F12L protein has several potential sites for the attachment of N-linked carbohydrate that apparently are not used because the protein has a similar electrophoretic mobility after synthesis in the presence or absence of tunicamycin (Zhang et al., 2000 ). This would be consistent with the majority of the F12L polypeptide being within the cytosol, rather than within the lumen of membrane vesicles of the exocytic pathway. The mechanism of association of the F12L protein with IEV particles is unknown. Other virus-encoded proteins that are present in the IEV membranes either have a transmembrane hydrophobic domain (A33R, A34R, A36R, A56R, B5R) or have fatty acids that mediate membrane interaction (F13L). F12L does not have a predicted transmembrane domain and it is unknown if the protein is modified by addition of fatty acids. Another possibility is that F12L associates with IEV via proteinprotein interactions with other virus or cell proteins present in the IEV outer membrane. The A36R and F13L proteins have the majority of their amino acids in the cytosol and so might have the greatest chance of interacting with F12L; however, it is known that the A36R deletion mutant is able to move to the cell surface (van Eijl et al., 2000
), implying that F12L can function in the absence of the A36R protein.
The F12L protein showed an interesting distribution within infected cells and co-localized with both microtubules and endosomes. The co-localization with microtubules is consistent with their role in the egress of IEV to the cell surface (Hollinshead et al., 2001 ; Ward & Moss, 2001
), and this co-localization, together with the failure of IEV made by the deletion mutant to disseminate to the cell surface, implies that F12L has a role in this microtubule-dependent IEV egress. Computational analyses of the F12L protein with known microtubule-binding proteins did not detect significant amino acid similarity. However, a microtubule-binding domain may exist. Alternatively, F12L might co-localize with microtubules indirectly via interactions with other proteins.
The co-localization of F12L with endosomes was shown by the presence of F12L in areas of the cells that contain endosomes (defined by the uptake of a fluid phase marker) and by its behaviour in infected cells treated with drugs that alter endosome location and appearance. The localization of F12L with endosomes did not require the formation of IEV particles, since when IEV particle formation was blocked by addition of IMCBH the F12L protein was localized to peripheral areas of the cell where no virions were present (Fig. 5a), but which were rich in endosomes (Figs 5
and 6
). The membranes used to wrap IMV particles to form IEV particles were shown to be of endosomal origin since they were labelled with a fluid phase marker that was efficiently taken up into endosomes. This observation is consistent with an earlier study (Tooze et al., 1993
) and inconsistent with the wrapping membranes deriving from the trans-Golgi network (Schmelz et al., 1994
). This discrepancy might be explained by use of membranes from both compartments, or due to increased traffic between these intracellular membrane compartments during VV infection such that their distinction is blurred.
The role of microtubules in intracellular transport of virus particles following infection has been studied with other viruses such as herpes simplex virus (Sodeik et al., 1997 ), but less attention has been paid to the role of microtubules during virus egress. The use of microtubules by vaccinia virus during egress is therefore unusual and we have identified the F12L protein as necessary for this process.
In summary, the F12L protein is shown to be a component of IEV particles, but is absent from other virions. It is required for the egress of IEV particles to the cell surface and the deletion mutant defines a new stage in VV morphogenesis. Preliminary data indicate a co-localization with microtubules, which would be consistent with a unique role for F12L in mediating the transport of IEV particles to the cell surface on microtubules.
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Acknowledgments |
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Footnotes |
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References |
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Received 25 June 2001;
accepted 27 September 2001.