African swine fever virus infection disrupts centrosome assembly and function

Nolwenn Jouvenet and Thomas Wileman

Department of Immunology and Pathology, Pirbright Laboratories, Institute for Animal Health, Ash Road, Woking, Surrey GU24 0NF, UK

Correspondence
Thomas Wileman
thomas.wileman{at}bbsrc.ac.uk


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African swine fever virus (ASFV) is a large, enveloped DNA virus that assembles in perinuclear sites located close to the centrosome. It is reported here that the microtubule network becomes disorganized soon after the onset of viral DNA replication and formation of assembly sites. ASFV infection resulted in loss of {gamma}-tubulin and pericentrin at the centrosome; this was due to protein relocalization, but not degradation. ASFV infection also inhibited the ability of the centrosome to nucleate microtubules. The reorganization of microtubules seen in ASFV-infected cells may therefore be mediated by {gamma}-tubulin and pericentrin redistribution, and consequent disruption of centrosome assembly and function.

A figure showing the levels of expression of {gamma}-tubulin and pericentrin in ASFV-infected cells is available as supplementary material in JGV Online.


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Microtubules are essential for the movement of cellular organelles in the cytoplasm during interphase and for chromosome segregation during cell division (Kirschner & Mitchison, 1986). Microtubules are nucleated, stabilized and organized by the centrosome (Zimmerman et al., 1999; Dammermann et al., 2003). The centrosome is formed by a matrix of filaments made of several {alpha}-helical coiled-coil proteins, such as pericentrin, together with hundreds of ring-shaped structures containing {gamma}-tubulin (Zimmerman et al., 1999). Each {gamma}-tubulin ring serves as a nucleation site for the growth of one microtubule filament. Functional abrogation or depletion of {gamma}-tubulin or pericentrin disrupts centrosome assembly and creates structural defects in microtubule asters (Zimmerman et al., 1999; Bornens, 2002).

Infection of cells by viruses often produces a cytopathic effect, resulting from reorganization of the microtubule network (Ploubidou & Way, 2001). Even though this is a common feature of viral infection, the molecular mechanisms underpinning reorganization of microtubule networks are not understood. In the small number of cases where this has been studied in detail, virus infection has been shown to alter centrosome composition. Structural modification of the centrosome has, for instance, been observed by electron microscopy in cells infected with human cytomegalovirus (Bystrevskaya et al., 1997; Gilloteaux & Nassiri, 2000). Infection with vaccinia virus (VV), the prototype of the family Poxviridae, causes reduction of proteins at the centrosome and loss of centrosome-nucleation efficiency, as early as 2 h post-infection (p.i.) (Ploubidou et al., 2000).

African swine fever virus (ASFV) is a large, cytoplasmic DNA virus that assembles in specialized sites, called virus factories, that form close to the centrosome (Heath et al., 2001). Early during infection, ASFV uses microtubules and the minus-end-directed motor dynein to establish assembly sites (Alonso et al., 2001; Heath et al., 2001) and, once viruses are assembled, they utilize the microtubule motor conventional kinesin to reach the cell surface (Jouvenet et al., 2004). Our previous work has shown that, once factories are established in the cell, microtubules are excluded from the core of the replication site, but some microtubule filaments remain in contact with the edge of the factory (Jouvenet et al., 2004). We have studied these rearrangements in more detail by immunofluorescence analysis, using a mouse mAb against {alpha}-tubulin (Sigma) (Piperno et al., 1987). Vero cells were grown as described previously (Jouvenet et al., 2004) and infected for 8, 12 or 16 h with the tissue culture-adapted Ba71V strain of ASFV (Carrascosa et al., 1985). The crude virus stock used in this study was obtained from infected cell lysates. Cells were infected at an m.o.i. of 1, fixed in methanol and processed as described previously (Jouvenet et al., 2004). Images were captured with a Leica SP2 confocal microscope. At 8 h, infected cells were identified with a mouse antibody specific for the ASFV non-structural protein p30, which is expressed as early as 2 h p.i. (Afonso et al., 1992). Antibody to p30 was labelled with biotin as described by Harlow & Lane (1988) and was detected with Streptavidin594 (Molecular Probes). Viral factories were located by extranuclear 4',6-diamidino-2-phenylindole (DAPI) staining of viral DNA. At 8 h p.i., no obvious changes in the astral organization of the microtubule cytoskeleton were noticeable in p30-positive cells compared to p30-negative cells (Fig. 1a) and microtubules radiated from a perinuclear aster to the cell periphery (Fig. 1a, arrows). At late times p.i., infected cells were visualized by positive staining for the late structural protein pE120R (Andrés et al., 2001) (Fig. 1b–d). The anti-pE120R antiserum was raised in rabbits as described previously (Jouvenet et al., 2004). At 12 h p.i., microtubule converging centres were no longer detectable in cells positive for pE120R. Microtubules were partially excluded from the viral factories and more condensed at the cell periphery (Fig. 1b). At 16 h p.i., microtubules were completely excluded from viral factories and often formed quasispherical rings around the cytoplasm (Fig. 1c). Cells with long projections containing microtubule bundles were also observed at 16 h p.i. (Fig. 1d, arrows). The normal aster configuration of microtubules was lost. These observations are in agreement with previous work showing microtubule rearrangements in Vero cells infected with the Lisbon-60 ASFV strain (Carvalho et al., 1988). Similar cytoskeleton rearrangements, such as microtubule rings and bundles, have been described in cells infected with VV, herpes simplex virus 1 and the iridovirus frog virus 3 (Murti & Goorha, 1983; Avitabile et al., 1995; Ploubidou et al., 2000; Kotsakis et al., 2001).



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Fig. 1. ASFV infection disrupts microtubule organization. Vero cells were fixed 8 (a), 12 (b) or 16 (c and d) h after infection with the Ba71V strain of ASFV. Samples were incubated with an antibody against {alpha}-tubulin (green signal) and with an antibody specific for early viral protein p30 (a) or with an antibody specific for the late ASFV structural protein pE120R (b–d) (red signal). Viral and cellular DNA was labelled with DAPI (blue signal). Arrows indicate microtubule-organizing centres in panel (a) and microtubule bundles in panel (d). Bars, 8 µm. The inset is an enlarged view of the merged panel of images shown in panel (d).

 
Pericentrin and {gamma}-tubulin are centrosome proteins that play a role in microtubule nucleation and organization (Dictenberg et al., 1998; Young et al., 2000). For nucleation to take place, pericentrin and {gamma}-tubulin must first be recruited to and assembled onto centrosomes. Given the loss of microtubule radiation observed at late stages of ASFV infection (Fig. 1), the effect of infection on the recruitment of nucleating proteins to the centrosome was studied. The location of {gamma}-tubulin was analysed by immunofluorescence at 8, 12 and 16 h p.i. using a mouse mAb against {gamma}-tubulin (Sigma) (Oakley, 1992). The biotinylated antibody against p30 was used as an early marker of infection and the rabbit anti-pE120R antiserum was used as a late marker. Preparations were viewed with a Nikon E800 microscope and images were captured with a Hamamatsu C-4746A CCD camera. At 8 h p.i., the {gamma}-tubulin signal had a similar intensity, regardless of expression of p30 (Fig. 2a). Fig. 2b shows four cells viewed at 12 h p.i.: three cells are pE120R-negative and do not exhibit extranuclear DAPI staining. They are either not infected or cells at an early stage of infection, prior to viral DNA replication. At 12 h p.i., the {gamma}-tubulin signal was clearly diminished in numerous cells that were positive for pE120R when compared to cells that were negative for viral antigen (Fig. 2b, thin arrow). In some extreme cases, the {gamma}-tubulin signal was lost completely. We looked at the appearance of the {gamma}-tubulin signal in 121 infected cells at 12 h p.i. and found that the {gamma}-tubulin signal was reduced in 58 % of the cells and lost in 13 % of the cells. At 16 h, the {gamma}-tubulin signal was reduced in approximately half of the cells that were positive for pE120R and no longer detectable in 40 % of these cells (Fig. 2c, thick arrow) (n=124). Loss of the {gamma}-tubulin signal did not arise because the centrosome was out of the plane of focus. Ten 0·2 µm thick optical sections of the same view were captured and merged by using Improvision Openlab software (Improvision). Very similar results were obtained when using a rabbit antibody for pericentrin (Covance Research Products) (Doxsey et al., 1994). At 16 h p.i., pericentrin staining was either reduced (47 %) or lost (45 %), whereas it was clearly visible in all cells that were negative for viral antigens (n=117) (data not shown). Reduced levels of {gamma}-tubulin and pericentrin at the centrosome were also observed in porcine aortic endothelial cells infected with the virulent Malawi strain of ASFV for 12 or 16 h (data not shown), demonstrating that the loss of nucleating proteins is not associated with adaptation of ASFV to cell culture.



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Fig. 2. The level of {gamma}-tubulin is reduced in ASFV-infected cells. Vero cells were fixed 8, 12 or 16 h after infection with the Ba71V strain of ASFV. Samples were incubated with an antibody against {gamma}-tubulin (green signal). Infected cells were visualized with an antibody specific for early viral protein p30 (a) or with antibodies against the late ASFV structural protein pE120R (b and c) (red signal). Viral and cellular DNA was labelled with DAPI (blue signal). The experiment was repeated and cells were incubated with Ara-C at 2 h p.i. (d). Cells were stained for p30 (red) and pericentrin (green). The right panels are merged images taken from ten 0·2 µm thick optical sections of the same view. Bar, 8 µm. The thin arrow indicates a cell with a reduced centrosome signal and the thick arrow indicates a cell that has lost the centrosome signal.

 
The time-course of loss of {gamma}-tubulin and pericentrin signals in infected cells suggested that late gene expression was required for the loss of anchoring proteins from the centrosome. To investigate this possibility, infected cells were incubated with 50 µg cytosine {alpha}-D-arabinofuranoside (Ara-C; Sigma) ml–1 at 2 h p.i. Ara-C is a selective inhibitor of DNA replication and can be used to prevent expression of intermediate and late viral DNA-dependent proteins (Rodríguez et al., 1996). Cells were incubated for a further 14 h and then processed for immunofluorescence analysis. The mouse anti-p30 antibody was used to identify infected cells and the rabbit antibody against pericentrin was used to visualize the centrosome. Cells positive for p30 lacked extranuclear DAPI staining, indicating that the drug had inhibited ASFV late DNA replication effectively (Fig. 2d). No reduction of centrosome markers was observed in p30-positive cells compared to p30-negative cells (Fig. 2d), demonstrating that loss of pericentrin from the centrosome was dependent on intermediate and/or late viral gene expression.

The reduction of {gamma}-tubulin and pericentrin at the centrosome observed from 12 h p.i. could result from protein degradation or redistribution. To distinguish between the two possibilities, the levels of expression of {gamma}-tubulin and pericentrin were analysed at different times p.i., at an m.o.i. of 10. Expression of {gamma}-tubulin was examined by Western blot and the level of pericentrin was assessed by immunoprecipitation (see Supplementary Fig. S1, available in JGV Online). Cells were lysed with RIPA buffer [1 % Nonidet P-40, 1 mM EDTA, 50 mM Tris/HCl (pH 7·4), 150 mM NaCl, 0·25 % sodium deoxycholate, 1 mM PMSF, 1 µg aprotinin ml–1, 1 µg leupeptin ml–1 and 1 µg pepstatin ml–1, pH 7·9] with 1 % Triton X-100. For Western blotting, proteins were resolved by SDS-PAGE (12·5 % gels) and transferred onto Protan BA85 nitrocellulose membranes as described previously (Rouiller et al., 1998). For immunoprecipitation, cells were labelled with [35S]methionine–cysteine Promix (Amersham Biosciences) for 60 min prior to lysis and proteins were immunoprecipitated and resolved as described previously (Cobbold & Wileman, 1998). As reported previously, the level of {alpha}-tubulin remained constant during infection (Jouvenet et al., 2004) and was used to ensure an equal loading. Immunodetection of p30 was used as a marker of infection. The level of {gamma}-tubulin increased at around 12 h p.i. and remained at the same level to 16 h p.i., whereas the level of pericentrin stayed constant throughout infection (see Supplementary Fig. S1, available in JGV Online). Loss of the {gamma}-tubulin and pericentrin signals at the centrosome in ASFV-infected cells is therefore due to a reduction in centrosomal recruitment of the nucleating proteins, rather than degradation of the proteins. The increase in the level of {gamma}-tubulin as infection progresses may mean that the protein becomes extracted more easily, possibly as a consequence of centrosome disruption.

Centrosomes with reduced levels of pericentrin and {gamma}-tubulin have a diminished capacity to nucleate microtubules (Young et al., 2000). The capacity of microtubules to renucleate following their depolymerization by the drug nocodazole (Sigma) in infected cells was therefore tested. Nocodazole (10 µg ml–1) was added to the culture medium at 12 h p.i. and cells were incubated for a further 4 h. The cells were then washed four times in warm medium to remove nocodazole and fixed at increasing time points to follow repolymerization of microtubules. Viral factories were located by extranuclear DAPI staining of viral DNA and the presence of viral particles was revealed by the anti-pE120R antiserum (Fig. 3). In uninfected cells, 1 min following nocodazole washout, new microtubules were observed growing out from the centrosome as small, star-like structures (thin arrows), which is in agreement with published work (Dictenberg et al., 1998; Quintyne et al., 1999; Young et al., 2000; Abal et al., 2002). In pE120R-positive cells, such star-like structures were not observed (Fig. 3). Three minutes following removal of nocodazole, microtubules started to elongate from the centrosome toward the cell periphery in cells negative for viral antigens (thick arrows). In infected cells, microtubules had still not repolymerized (Fig. 3). Fifteen minutes following removal of nocodazole, microtubules eventually started to repolymerize in infected cells. However, microtubules did not converge into a single, perinuclear aster, but were arranged randomly in the cell (Fig. 3). The data demonstrate that, in cells infected with ASFV for 16 h, the ability of the centrosome to nucleate microtubules is highly compromised. This could be a direct consequence of the reduced recruitment of nucleating proteins, such as pericentrin and {gamma}-tubulin, to the centrosome (Fig. 2), and could explain microtubule rearrangement in ASFV-infected cells (Fig. 1). Structural alterations of the centrosome have been observed in cells infected with human cytomegalovirus (Bystrevskaya et al., 1997; Gilloteaux & Nassiri, 2000) and VV infection causes reduction of proteins at the centrosome and loss of centrosome-nucleation efficiency (Ploubidou et al., 2000). The molecular basis of centrosome stability in general is poorly understood and understanding the event(s) that disrupt this organelle in human cytomegalovirus, VV and ASFV-infected cells will certainly provide interesting insights into the regulation of centrosome integrity. Further experiments would be needed to determine whether these viruses benefit from the decreased ability of the centrosome to nucleate microtubules.



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Fig. 3. The ability of the centrosome to nucleate microtubules is highly compromised in ASFV-infected cells. Nocodazole was added at 12 h p.i. and cells were incubated for a further 4 h. Prior to fixation, the drug was washed out to allow microtubules to repolymerize for the indicated times. Samples were incubated with a specific antibody for the late ASFV structural protein pE120R (right panels) and with an antibody against {alpha}-tubulin (middle panels). Viral and cellular DNA was labelled with DAPI (left panels). Arrows indicate the position of the centrosome. Bar, 8 µm.

 
We have previously reported that ASFV infection leads to scattering of the Golgi network (McCrossan et al., 2001). Interestingly, the Golgi complex is a potent microtubule-organizing organelle in interphase cells (Chabin-Brion et al., 2001). Therefore, ASFV infection disrupts the two microtubule-organizing organelles, i.e. the centrosome and the Golgi network. Similar fragmentation of the Golgi apparatus has been described in cells infected with herpes simplex virus 1, VV and foot-and-mouth disease virus (Avitabile et al., 1995; Ploubidou et al., 2000; Monaghan et al., 2004).


   ACKNOWLEDGEMENTS
 
We thank Michael Way (Cancer Research UK) for suggesting this study and for helpful discussions. We also thank Kyle Miller (Cancer Research UK) for critical reading of the manuscript and Paul Monaghan (IAH, Department of Immunology, Pirbright, UK) for confocal microscopy assistance. This work was supported by the Biotechnology and Biological Sciences Research Council.


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Received 20 September 2004; accepted 22 November 2004.



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