1 Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK
2 Division of Cell and Molecular Biology, University of St Andrews, Irvine Building, North Street, St Andrews, Fife KY16 9AL, UK
Correspondence
Paul Monaghan
paul.monaghan{at}bbsrc.ac.uk
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ABSTRACT |
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INTRODUCTION |
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FMDV is the type species of the genus Aphthovirus of the Picornaviridae (Belsham, 1993). Currently, eight other picornavirus genera are recognized including the cardioviruses [e.g. Encephalomyocarditis virus (EMCV)], enteroviruses [e.g. Poliovirus (PV), Bovine enterovirus (BEV) and echovirus 11 (EV11)], parechoviruses [e.g. parechovirus-1 (HpeV-1)] and rhinoviruses. Picornaviruses are small, single-stranded, positive-sense RNA viruses which have a non-enveloped icosahedral capsid (of approximately 25 nm diameter) formed from 60 copies each of four virally encoded proteins, VP1VP4.
Members of the Picornaviridae have a similar genome organization and are believed to follow a similar replication strategy (King et al., 2000). Infection is initiated by virus binding to a specific cell surface receptor and culminates in the translocation of the viral RNA (vRNA) across a cellular membrane into the cytosol (Belnap et al., 2000
; Miller et al., 2001
; Schober et al., 1998
). Inside the cytosol, the vRNA functions sequentially as a template for the synthesis of the viral proteins and the complementary negative-stranded genome copies that are, in turn, used as a template for the synthesis of new progeny genomes.
Infection by picornaviruses results in a major rearrangement of the host cell membranes to create vesicular structures upon which virus genome replication takes place (Bienz et al., 1983, 1987
; Egger et al., 2002
; Frankel et al., 1987
; Gosert et al., 2000
, 2002
). The precise origin of these vesicles is not clear and, for PV, a number of cytoplasmic organelles have been implicated in their derivation. At early times following infection, PV-induced vesicles are most likely formed at the endoplasmic reticulum (ER) (Dales et al., 1965
; Rust et al., 2001
; Schlegel et al., 1996
; Suhy et al., 2000
) whereas, at later times, membranes from other cellular organelles, including lysosomes and the Golgi, may also contribute to vesicle formation (Rust et al., 2001
; Schlegel et al., 1996
).
Despite having a similar replication strategy, several studies have revealed important differences in the replication of picornaviruses from different genera. These studies have suggested that virus genome replication may take place on vesicles derived from different cellular membranes and at a different site within the cell (Gazina et al., 2002; Rust et al., 2001
). The membrane vesicles induced, following infection by PV, EV11 and EMCV, appear to have a similar morphology consisting of heterogeneously sized vesicles arranged as tightly packed clusters. However, the vesicles induced by HpeV-1 (Gazina et al., 2002
) are distinct from the other picornaviruses studied in that they are homogeneously sized, less numerous and do not associate to form tight clusters. Another important difference in picornavirus replication is revealed by their sensitivity to brefeldin A (BfA). BfA inhibits membrane transport between the ER and the Golgi by preventing the formation of COPI-dependent secretory transport vesicles (Duden et al., 1994
; Rothman, 1994
). Replication of FMDV and EMCV are not affected by BfA (Gazina et al., 2002
; O'Donnell et al., 2001
) whereas replication of human rhinovirus and several enteroviruses, such as PV, EV11 and BEV are extremely sensitive to this agent. Replication of HpeV-1 is intermediate being partially resistant (Gazina et al., 2002
).
Whilst there is considerable information on the morphological changes that take place within picornavirus-infected cells (Bienz et al., 1987, 1994
; Dales et al., 1965
), little is known about the nature of any membrane rearrangement and the development of a specialized area of virus replication in FMDV-infected cells. Wool et al. (1982)
and Yilma et al. (1978)
reported FMDV in cytoplasmic blebs, and, more recently, cytoplasmic blebs were reported to be a feature of BHK-21 cells persistently infected with FMDV, although the authors were unable to correlate these structures with the presence of virions (Donn et al., 1995
).
In the absence of a definitive study of FMDV-infected cells, we have performed an ultrastructural study of BHK-38 cells infected with FMDV. This study has shown that FMDV infection results in a dramatic condensation of the cytoplasmic contents to a distinct region, located to one side of the cell nucleus, which contains both viral proteins and RNA. The cytoplasm between this region and the plasma membrane appears generally devoid of recognizable cellular structures apart from a ring of mitochondria which surrounds the accumulated organelles. Prominent within this region are densely packed free ribosomes and smooth membranous vesicles that have no counterpart in the uninfected cell. These vesicles are distinct from those induced by enteroviruses and more closely resemble those induced by HpeV1, since they are relatively few in number and do not aggregate into densely packed clusters (Gazina et al., 2002). It could be argued that the membrane rearrangements induced by FMDV were characteristic of the cell type (BHK-38) and not the virus (FMDV). However, when the same cell type was infected with BEV the resultant series of morphological changes did not resemble those seen with FMDV but were similar to those reported for cells infected with PV.
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METHODS |
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Cell infections
One-step growth curves.
Virus (m.o.i.=5 p.f.u. per cell) was adsorbed to BHK-38 monolayers in 60 mm dishes at room temperature for 0·5 h in the presence of 5 µg BfA ml-1 or DMSO (1 in 1000; mock-treated). The cells were washed with (PBS) pH 7·5 and incubated at 37 °C in the presence of BfA (5 µg ml-1) or DMSO in cell culture media supplemented with 1 % FCS or pig serum for FMDV and BEV respectively. At various times, the cell culture medium was assayed for the presence of virus by plaque assay on BHK-38 cells as described previously (Jackson et al., 1996) except that for BEV pig serum was used in place of FCS.
Microscopy.
Cells were seeded onto 13 mm glass coverslips (Agar Scientific) for immunofluorescence and onto 13 mm thermanox coverslips (Science Services) for electron microscopy 16 h before use. Cells prepared on coverslips as above, were washed with cold PBS containing 1 mM CaCl2 and 1 mM MgCl2 and incubated with virus at a m.o.i. of between 10100 p.f.u. per cell for 0·5 h at 4 °C. The cells were washed with PBS to remove unattached virus and infection initiated by adding cell culture medium at 37 °C. Infected cells were then cultured at 37 °C.
Fluorescence immunolabelling.
Coverslips of infected and uninfected cells (control) were fixed at 1·5, 2·0, 2·5 and 3·0 h post-infection (h p.i.) for 0·5 h in 4 % paraformaldehyde in PBS, washed in PBS and stored at 4 °C. For immunolabelling, cells were permeabilized in 0·05 % Triton X-100 in PBS for 15 min and washed in PBS. Non-specific binding of antibody was blocked by 0·5 h incubation in PBS containing 0·5 % bovine serum albumin (Sigma) (PBS/BSA). Cells were immunolabelled by incubation in rabbit antiserum raised against whole FMDV diluted 1 : 600 in PBS/BSA or mouse anti-tubulin antibody (Sigma) at 1 : 1000 for 1 h at room temperature followed by PBS washes (3x5 min). Primary antibody was detected with Alexa 488- or Alexa 568-conjugated species-specific immunoglobulins (Molecular Probes) diluted 1 : 200 in PBS/BSA. Incubation was for 1 h at room temperature followed by PBS washes (3x5 min). Nuclei were labelled with ToPro 3 (Molecular Probes) diluted 1 : 5000 in PBS for 5 min. Coverslips were mounted in Vectashield (Vector Laboratories), sealed with nail varnish and were imaged in a Leica TCS SP2 confocal microscope. Control uninfected cells were labelled as for infected cells.
RNA labelling and localization.
Newly synthesized FMDV RNA was detected by bromouridine (BrU) incorporation. Cells on coverslips were infected with FMDV and then transfected with BromoUTP (BrUTP) using Lipofectin (Gibco-BRL) according to the manufacturer's instructions. Briefly, 10 µl of Lipofectin were added to 90 µl of OptiMEM (Gibco-BRL). After 10 min at room temperature, 100 µl of 10 mM BrUTP, diluted in OptiMEM, was added and the mixture incubated at room temperature for a further 45 min. The mixture was further diluted with 1 ml OptiMEM that contained 20 µg actinomycin D and added to cells immediately following FMDV infection. Control cells were processed in parallel. Control cells were transfected either (1) in the absence of infection; (2) in the absence of actinomycin D or (3) in the absence of BrUTP in the transfectant medium.
Transfected cells were incubated at 37 °C in 5 % CO2. At 2 h p.i., the cells were fixed for 1 h in 4 % paraformaldehyde in PBS and washed in PBS. Cells were permeabilized in 0·05 % Triton X-100 in PBS for 15 min. FMDV proteins were identified using a rabbit anti-FMDV antiserum (as above) and newly synthesized viral RNA was detected using a mouse anti-BrdU (Boehringer Mannheim Biochemica), diluted 1 : 10 and incubated as described by the manufacturer. Primary antibodies were detected with anti-mouse Alexa 568- and anti-rabbit Alexa 488-conjugated species-specific immunoglobulins (Molecular Probes) diluted 1 : 200 in PBS/BSA. The nuclei were labelled with 4',6-diamidino-2-phenylindole (DAPI, Molecular Probes).
Routine electron microscopy of adherent cells.
At 1·5, 2·0, 2·5 and 3·0 h p.i. control uninfected and FMDV-infected cells on Thermanox coverslips were fixed in situ in 2 % glutaraldehyde in 0·05 M phosphate buffer (pH 7·27·4, osmotic pressure adjusted to 350 mosmol with sucrose) for a minimum of 2 h. BHK-38 cells infected with BEV were fixed at 2·0, 4·0, 6·0 and 8·0 h p.i. and processed as for FMDV-infected cells. Samples were post-fixed in 1 % osmium tetroxide in phosphate buffer for a minimum of 4 h, dehydrated through a graded series of ethanols and embedded in Agar 100 resin (Agar Scientific). After overnight polymerization at 60 °C the thermanox coverslips were peeled from the resin block leaving the cells embedded in the resin. Sections were cut parallel to the coverslip surface on a Diatome diamond knife (Leica Microsystems) and contrasted with uranyl acetate and lead citrate with an EMStain (Leica). They were imaged in a JEOL 1200EX or FEI Tecnai 12 at 100 kV.
Routine electron microscopy of non-adherent cells.
For cells fixed at 2·0, 2·5 and 3·0 h p.i., the tissue culture medium was removed prior to fixation and centrifuged at 1000 g for 5 min. The medium was removed and the cell pellet consisting of infected cells that had rounded up and detached from the coverslip was fixed in 2 % glutaraldehyde, washed in phosphate buffer and embedded as a pellet in 8 % gelatin. Subsequent processing was as for adherent cells.
High-pressure freezing and freeze-substitution.
Twenty-five cm2 flasks of BHK-38 cells were harvested at 2·0, 2·5 and 3 h p.i. Flasks were firmly shaken and all detached cells were collected by removal of the medium and centrifugation at 1000 g for 5 min. The supernatant was removed and the cells mixed with a small residual quantity of medium to form a thick suspension. This was introduced into 200 µm diameter cellulose tubing (Hohenberg et al., 1994) placed in hexadecene within 200 µm planchettes and frozen in a Bal-Tec HPM 010 high-pressure freezer (Bal-Tec, Liechtenstein). After freezing, the planchettes were separated under liquid nitrogen and the planchettes containing the cellulose tubes were transferred to a Leica AFS for freeze-substitution. This was done essentially as described previously (Monaghan et al., 1998
). Samples were freeze-substituted in acetone containing 1 % osmium tetroxide. Freeze-substitution was undertaken at -90 °C for 48 h, -60 °C for 8 h, and -30 °C for 8 h. The samples were then warmed to 0 °C and the acetone/osmium tetroxide replaced with fresh acetone. Finally the samples were warmed to room temperature and embedded in Agar 100 resin.
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RESULTS |
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Localization of RNA
We followed incorporation of BrUTP into newly synthesized viral RNA molecules using an anti-BrdU antibody, which is cross-reactive for BrUTP. BHK-38 cells were infected with FMDV and transfected with BrUTP in the presence of actinomycin D. Approximately 510 % of the infected cells were positive for BrUTP staining. Fig. 3 shows a number of FMDV-infected cells, one of which is showing positive labelling for BrUTP incorporation. In these double-labelled cells, the subcellular patterns of labelling for the FMDV protein (Fig. 3a
) and BrUTP-labelled viral RNA (Fig. 3b
) were similar (Fig. 3c
). The antiserum used in these studies predominantly recognizes structural proteins, but similar results have been obtained with antibodies detecting non-structural proteins (data not shown). Thus the region of the cell containing both viral proteins and virus-specific RNA is likely to be the site of virus replication and will be referred to as the replication site.
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Electron microscopy of FMDV-infected BHK-38 cells
Next we performed a detailed analysis of FMDV-infected BHK-38 cells by electron microscopy. The methods used to fix, embed and section the infected cells in situ allowed the changes taking place during virus infection to be studied without the risk of mechanically induced artefacts. Uninfected BHK-38 cells exhibit a standard mammalian cell complement of organelles with, for example, abundant Golgi, rough ER (rER), free ribosomes and mitochondria (see Fig. 5b).
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Early phase: 1·52·0 h p.i.
The first morphological indication of virus infection that can be unequivocally identified by electron microscopy is the accumulation of cytoplasmic organelles in a region to one side of the nucleus (Fig. 4). This region has the same location within the cell as the early replication site identified by immunofluorescence (Figs 2c, 3c
).
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Mid-phase: 2·02·5 h p.i.
The majority of cells in cultures 2·02·5 h p.i. had a well-developed replication site. The rER was almost devoid of ribosomes and had fragmented considerably, although larger fragments remained at the outer region of the replication site (Fig. 5a). In addition, the nuclear membrane showed blebbing similar to that seen in the cytoplasmic rER (supplementary images: www.iah.bbsrc.ac.uk/bioimaging/jgv). In comparison, Fig. 5(b)
shows the region of cytoplasm adjacent to the nucleus in an uninfected cell. The cytoplasm contains rER with attached ribosomes and intact nuclear membrane.
Within the replication site, the number of membrane-bound vesicles increased and they were often seen in close proximity to the remnants of the rER (Fig. 6a). Whilst in the early replication site these vesicles were almost always lined by a single membrane, from 2·0 h p.i. a small number were lined by a double membrane (Fig. 6b
). These double membrane vesicles formed less than 10 % of the total vesicle population.
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Late phase: 2·53·0 h p.i.
At this stage of infection the majority of infected cells were rounded (Fig. 2d) and the cytoplasm was composed, almost exclusively, of the replication site (Fig. 7
). Small remnants of the rER were scattered throughout the replication site. The ribosomes were no longer arranged in the ordered chains seen in the mid-phase of infection but were randomly dispersed throughout the replication site. Membrane vesicles were prominent and remain a feature of cells recovered from the medium at all later times post-infection (see below). As in the mid-phase of infection, the majority of these vesicles appeared to be bounded by a single membrane, although occasional double membrane structures were seen. These vesicles were dispersed throughout the replication site and did not form ordered clusters.
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The time course of the infection was longer for BEV than for FMDV-infected cells (see Fig. 1); nonetheless it was possible to identify similar time points in the progress of infection. The cytoplasmic changes in BEV-infected cells were different from those induced by FMDV and resembled more closely the structures reported for cells infected by poliovirus. In the early phase of infection, the initial changes were seen in a similar location to FMDV-infected cells but the accumulation of virtually all cellular organelles within this region was not as pronounced. At the mid-phase of infection the rER was disordered throughout the cytoplasm and within the region of condensed organelles was extensively fragmented. In contrast to FMDV-infected cells, the ribosomes remained associated with membrane fragments of the ER.
From the mid-phase of infection, membrane vesicles, similar to those induced by PV, were seen within BEV-infected cells. They did not contain the fibrillar material seen in the FMDV-infected cells and the majority were bound by a single membrane. In addition, like PV, the vesicles were of variable size and were seen to accumulate into rosette-like formations.
At the later stages of infection, BEV did not cause the total collapse of the cell organelles round the replication site seen in FMDV-infected cells (supplementary images: www.iah.bbsrc.ac.uk/bioimaging/jgv). However, unlike in the FMDV-infected cells, virus-like structures were readily apparent. In the later stages of infection, these structures accumulated into paracrystalline arrays (Fig. 8c), but were also often seen in association with the rosettes of vesicles (Fig. 8d
).
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Several membrane-bound vesicles were present in infected cells and these were characterized by contents with variable electron density. These vesicles were similar to those seen in the routine electron micrographs in that they did not form clusters, and the majority appeared to have only a single membrane. In addition, many of the cells contain clusters of particles with an approximate diameter of 25 nm which could correspond to FMD virions (Fig. 8a, b and see Discussion).
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DISCUSSION |
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It is clear from the results presented that the morphological changes that take place within FMDV-infected cells are strikingly different from those reported following infection by other picornaviruses. For example, the earliest ultrastructural change that can be reliably recognized in cells infected with FMDV (at around 1·52·0 h p.i.) is a dramatic accumulation of virtually all of the cytoplasmic organelles to a defined region of the cell, which we describe as the virus replication site. This difference was unlikely to be BHK-38 specific since the morphological changes that take place in these cells infected with BEV, a close relative of PV, were similar to those reported for PV in Hep-2 cells (Bienz et al., 1994) and EV11 in BS-C-1 cells (Gazina et al., 2002
).
The replication site became the main focus of the subsequent events that take place within the FMDV-infected cell. As this site developed, the rER became distorted and eventually fragmented into small remnants although larger areas of rER remained at the periphery of the replication site. Alterations were also seen in the nuclear membrane which was dilated and extended into the cytoplasm. The number of ribosomes associated with the rER was greatly reduced. At the same time, free ribosomes accumulated in the replication site. The loss of ribosomes from the rER membranes might indicate that at least some of these free ribosomes are derived from rER. At the mid-phase of infection, ribosomes were seen to associate into long straight chains. The significance of the appearance of chains of ribosomes is unclear. Certainly, this is a stage of rapid protein synthesis and it is possible that these structures represent actively translating viral RNA molecules. Taken together, these observations point to a major modification of the rER in the development of the replication site, notwithstanding the possible role for the rER in the genesis of the membrane-bound vesicles (see Introduction). However, rER redistribution was not noted as a feature of FMDV-infected cells when studied by immunofluorescence microscopy (O'Donnell et al., 2001), but this discrepancy may be explained by the increased resolution of the electron microscope.
The changes in the rER appeared to occur before any structural changes in the Golgi region, despite the fact that the first signs of de-novo FMDV protein synthesis was detected in the Golgi-rich area of the cells (data not shown). However, a later feature of the replication site is the loss of normal-appearing Golgi structures, and this is consistent with reports of the loss of Golgi organization in PV (Sandoval & Carrasco, 1997) and FMDV-infected cells (O'Donnell et al., 2001
).
As infection progressed, membrane-bound vesicles became more numerous within the replication site and were a prominent feature of cells in the late stage of infection. These vesicles appear distinct from those induced by enteroviruses such as poliovirus and EV11 and more closely resemble those induced by HpeV-1, since they were relatively few in number and did not appear to aggregate into the rosettes or clusters (Bienz et al., 1994; Gazina et al., 2002
). A second major difference is that the proportion of vesicles with double membranes is considerably less in FMDV-infected cells than in cells infected with PV. Again these differences are not explained by the use of BHK-38 cells, since the vesicles induced by BEV were similar in appearance to those seen in PV-infected cells. From their morphological characteristics it was not possible to determine the origins of the vesicles induced by FMDV but there was a close association between areas rich in these vesicles and the remnants of the rER, as illustrated in Fig. 6a
. This association of virus-induced vesicles and fragments of the ER is consistent with previous observations made with PV (Bienz et al., 1987
).
The precise origins of the membrane vesicles induced by picornavirus infection has been the subject of some debate although it is generally considered that, at least early in infection, they are derived from vesicles trafficking between the ER and the Golgi. Rust and colleagues (Rust et al., 2001) have shown that vesicles induced following infection by PV are formed at the ER by a budding mechanism that is similar to COPII-dependent formation of anterograde transport vesicles. Recently, Gazina et al. (2002)
have observed components of COPI-dependent anterograde transport vesicles associated with EV11, but not with EMCV-induced vesicles, leading the authors to suggest that the differential sensitivity of picornaviruses to BfA (see Introduction) may be explained if the vesicles they induce are formed at BfA-sensitive or Bfa-insensitive steps of the anterograde transport pathway. Based on these observations and the insensitivity of FMDV to BfA (Fig. 1
), it would be predicted that the vesicles induced by FMDV would be formed by a similar mechanism to that used by EMCV. However, further work will be required to determine if these vesicles retain a similar set of markers from the secretory pathway as described for other picornaviruses.
The morphology of the FMDV-induced vesicles appeared different depending upon the preparation method, although these vesicles are clearly the same structures. In conventionally fixed samples, vesicles contained a fine lattice-work of electron density, whereas in samples prepared by high-pressure freezing they have a dense core. The latter appearance corresponds to the vesicles seen in PV-infected cells (Schlegel et al., 1996) also prepared by high-pressure freezing; however, the prominent double membrane of these vesicles was not commonly seen in the FMDV-infected cells. When double membrane structures were detected in the FMDV-infected cells, it was almost exclusively in the mid to late stages of infection.
The issue of when the virus particles begin to form in the infected cell remains unresolved. Certainly, they must be present at later stages of infection. The failure to observe newly formed FMDV particles within any of the conventionally fixed cells is difficult to explain, given that virus-like particles were seen in BEV-infected cells prepared in an identical manner. However, FMDV is acid-labile, and when exposed to pH values below neutrality will dissociate. Although buffered, it may be that conventional fixatives for electron microscopy allow virions to be exposed to a brief period of low pH during the fixation process. This may explain the paucity of convincing electron micrographs of FMDV in infected cells using conventional processing. This explanation, that the virions are degraded during sample preparation, is supported by the presence of virus-like structures in samples prepared using high-pressure freezing and freeze-substitution.
In conclusion, we have shown that, like for other picornaviruses, infection of host cells by FMDV results in a major rearrangement of intracellular membranes. However, many of the cellular structural events resulting from FMDV infection differ markedly from the responses reported for other viruses within the Picornaviridae.
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ACKNOWLEDGEMENTS |
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Received 11 June 2003;
accepted 27 November 2003.