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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ABSTRACT |
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A figure showing the levels of expression of -tubulin and pericentrin in ASFV-infected cells is available as supplementary material in JGV Online.
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MAIN TEXT |
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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
-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. 1bd
). 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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The reduction of -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
-tubulin and pericentrin were analysed at different times p.i., at an m.o.i. of 10. Expression of
-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 ml1, 1 µg leupeptin ml1 and 1 µg pepstatin ml1, 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]methioninecysteine 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
-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
-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
-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
-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 -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 ml1) 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
-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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ACKNOWLEDGEMENTS |
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Received 20 September 2004;
accepted 22 November 2004.
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