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1 Department of Biomedical and Biomolecular Sciences, Griffith University, Nathan, QLD 4111, Australia
2 Division of Immunology, Institute for Animal Health, Pirbright Laboratories, Woking, Surrey GU24 0NF, UK
Correspondence
Thomas Wileman
T.Wileman{at}uea.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: School of Medicine, University of East Anglia, Norwich NR4 7TJ, UK. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Kelly & Robertson, 1973; Carrascosa et al., 1984). ASFV has recently been classified as the sole member of the family Asfarviridae and may be the missing evolutionary link between the large cytoplasmic DNA iridovirus and poxvirus groups.
ASFV assembles in cytoplasmic inclusions called virus factories (Nunes et al., 1975), which locate close to the microtubule-organizing centre and are surrounded by vimentin filaments and mitochondria (Carvalho et al., 1988; Rojo et al., 1998). In this way, ASFV factories resemble cellular inclusions called aggresomes (Heath et al., 2001; Stefanovic et al., 2005; Wileman, 2006), which form in response to protein aggregation. Electron micrographs of sections taken through virus factories reveal virions with concentric layers of differing electron density (Breese & Hess, 1966; Carrascosa et al., 1986; Arzuza et al., 1992). The outer capsid layer contains the major capsid protein, p73. This surrounds inner lipid envelopes derived from the endoplasmic reticulum (ER) (Cobbold et al., 1996; Andres et al., 1998; Rouiller et al., 1998), a matrix protein layer called the core shell and a central core structure containing the nucleoid (Andres et al., 1997). p73 is encoded by the B646L gene (Yanez et al., 1995) and provides 35 % of the protein mass of the virus, whilst a further 25 % is produced by proteolytic cleavage of the pp220 polyprotein (Andres et al., 1997, 2002a, b; Heath et al., 2003), which is incorporated into the matrix.
Assays that follow the biosynthesis and subcellular distribution of p73 have shown that p73 is synthesized in the cytosol, where it binds to a specific, virally encoded chaperone (CAP80) encoded by the B602L gene (Cobbold et al., 2001). When p73 is expressed in the absence of CAP80, the protein is folded incompletely and forms insoluble aggregates. In the presence of CAP80, the conformation of p73 changes, allowing it to remain soluble. During infection, p73 is released from CAP80 into the cytoplasm and then binds rapidly to the cytoplasmic face of ER-derived membranes (Cobbold et al., 2001), a process that may depend on virus membrane protein p54 (Rodriguez et al., 2004). Approximately 60 % of this membrane-bound pool of p73 becomes resistant to trypsin within 2 h and forms large, oligomeric complexes, suggesting assembly into the virus capsid (Cobbold et al., 1996; Cobbold & Wileman, 1998). This later stage requires continual protein synthesis, ATP and an intact ER calcium store (Cobbold et al., 2000).
Release of ASFV from the cell involves transport from virus factories along microtubules to the cell surface (Jouvenet et al., 2004) and budding from the plasma membrane from projections that resemble filopodia (Jouvenet et al., 2006). At this point, ASFV leaves the reducing environment present in the cytosol and enters an extracellular space rich in oxygen. For small, enveloped viruses, preparation for entry into an oxidizing environment takes place in the lumen of the ER, where virus envelope proteins encounter an oxidizing environment and redox-sensitive chaperones that facilitate protein folding and disulphide-bond formation (Maggioni & Braakman, 2005). For large, cytoplasmic DNA viruses, such as poxviruses and ASFV, the situation is more complex because assembly takes place on the cytoplasmic face of membranes, which is reducing. In the case of poxviruses, a series of virally encoded thiol oxoreductases are able to oxidize virus proteins exposed to the cytosol (Locker & Griffiths, 1999; Senkevich et al., 2000, 2002). Two of the proteins involved, A25 and G4, are thought to transfer electrons from thiol groups on virus proteins bound to the cytoplasmic face of the vaccinia virus (VACV) membrane to FAD bound within the VACV E10 protein.
The VACV E10 protein is a member of the ERV1/ALR family of oxoreductases, and ERV1/ALR orthologues are also encoded by other cytoplasmic DNA viruses, including mimivirus (Raoult et al., 2004) and ASFV (Yanez et al., 1995). Disruption of the ERV1-ALR protein of ASFV results in slow replication in macrophages, small-plaque phenotype and defective morphogenesis (Lewis et al., 2000), suggesting that control of cytosolic redox potential may be important for ASFV assembly. Given these observations, we have asked whether agents that perturb the redox state of cells have an effect on the assembly of p73 into the virus capsid. The folding of p73 bound to CAP80 and the subsequent release and transfer of p73 to ER-derived membranes were inhibited when cells were oxidized, showing that a reduced cytosol was critical during early stages of ASFV assembly. Newly synthesized capsid precursors were disrupted by oxidizing conditions, suggesting that they would not survive in the extracellular milieu; however, they became resistant to oxidation as maturation progressed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Vero cells were infected at 10 p.f.u. per cell. After virus adsorption for 2 h, the supernatant was removed and cell cultures were incubated with fresh Eagle's medium at 37 °C for 16 h. Extracellular ASFV was purified as described by Black & Brown (1976). Monoclonal antibody (mAb) 4H3, to the major capsid protein, and a polyclonal serum raised to the N terminus of CAP80 have been described previously (Cobbold et al., 2001).
Metabolic labelling and immunoprecipitation.
Cells infected with ASFV were preincubated with cysteine- and methionine-free medium for 15 min, and then medium was replaced with 1–2 MBq [35S]methionine and cysteine (35S-Express label; New England Nuclear) ml–1 in cysteine- and methionine-free medium. Cells were washed and chased in Dulbecco's modified Eagle's medium. In all experiments where chemicals were used, they were added after pulse labelling in the absence of chemical. At the appropriate time intervals, cells were washed once in PBS and released from the flask with EDTA/trypsin. Cells were lysed in 1 % Brij : 35 immunoprecipitation buffer [10 mM Tris (pH 7.8), 150 mM NaCl, 10 mM iodoacetamide, 1 mM EDTA, 1 mM PMSF, 1 µg (each) of leupeptin, pepstatin, chymostatin and antipain ml–1 (Boehringer Mannheim)] and immunoprecipitated as described previously (Wileman et al., 1993).
Preparation of cellular membrane fractions.
ASFV-infected cells were resuspended in buffered sucrose [250 mM sucrose, 1 mM EDTA, 20 mM Tris (pH 7.5)] and homogenized by 20 passages through a 25-gauge needle. Whole cells and nuclei were removed by centrifugation at 6000 r.p.m. for 2 min in an Eppendorf 5415 centrifuge. Post-nuclear supernatants were pelleted at 14 000 r.p.m. for 20 min at 4 °C in an Eppendorf 5402 centrifuge to separate membranes (pellet) from cytosol (supernatant).
Trypsin-protection assays.
Membrane fractions prepared from metabolically labelled cells were incubated with trypsin (0.4 mg ml–1) for 30 min at 37 °C. Proteolysis was stopped by addition of 3 vols immunoprecipitation buffer containing 3 % fetal calf serum and 10 mg hen egg white trypsin inhibitor ml–1 (Boehringer Mannheim). The amount of p73 remaining was determined by immunoprecipitation. For analysis of membrane-bound p73, membrane fractions were incubated with reduced or oxidized glutathione for 15 min and then incubated with trypsin as described above.
Sucrose-density sedimentation.
Membrane fractions prepared from metabolically labelled cells infected with ASFV were solubilized in 1 % Brij : 35 immunoprecipitation buffer and applied to the top of sucrose-step gradients containing 2 ml layers of 10, 20, 30, 35 and 40 % sucrose with 1 ml of a 70 % sucrose cushion, and left to equilibrate overnight at 4 °C. After centrifugation at 40 000 r.p.m. in a Beckman SW40 rotor, gradients were separated into 1.2 ml fractions and immunoprecipitated with mAb 4H3.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). CAP80–p73 complexes were recovered from infected cells labelled for 2 min (lane 1). We have shown previously by quantitative immunoprecipitation using conformation-dependent and -independent antibodies that p73 is conformationally immature when bound to CAP80 at 2 min and is folded and then released from CAP80 during a 60 min chase (Cobbold et al., 2001). The loss of the lower band in lane 2 confirmed dissociation of p73 from the CAP80 protein after 60 min. In lanes 3 and 4, redox reagents were added after pulse labelling. Dissociation of p73 from CAP80 was unaffected by the reducing agent DTT (lane 4); however, the thiol-oxidizing agent diamide (O'Brien et al., 1970) (lane 3) inhibited CAP80–p73 dissociation.
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). Diamide was added after pulse labelling. Under control conditions (top), CAP80–p73 complexes were recovered from cells labelled for 2 min, and p73 dissociated from CAP80 30 min into the chase [top of panel (b)]. A slight fall in levels of CAP80 recovered by immunoprecipitation was observed at the 30 and 60 min chase times in the presence or absence of diamide [bottom of panel (b)]; even so, it was clear that, in cells incubated with diamide during the chase, CAP80 and p73 remained associated, even after an extended 1 h chase. A densitometric analysis of autoradiographs (not shown) indicated that, in the presence of diamide, 80 % of the bound p73 remained associated at 1 h, compared with 15 % in control cells.
Diamide slows the conformational maturation of p73
The folding of newly synthesized p73 can be studied by following the appearance of epitopes recognized by a conformation-dependent mAb (4H3) and by analysing the production of trypsin-resistant fragments (Cobbold et al., 2001). The results of this type of experiment are shown in Fig. 1(c). Infected cells were pulse-labelled for 2 min to label nascent p73 and then chased for increasing times. After the 2 min pulse, low levels of the epitope recognized by 4H3 were detected (left lane of each time point); however, the epitope increased with the chase time, indicating conformational maturation of the protein. The right-hand lane shows the level of protease-resistant fragments obtained at each time point. These mirrored the increase in levels of p73 immunoprecipitated by 4H3 and showed that conformational maturation was rapid and essentially complete within 15 min of synthesis.
The effect of diamide present during the chase is shown in Fig. 1(d). Diamide was added after pulse labelling. Low levels of p73 were recognized by mAb 4H3 after a 2 min chase, and these increased slowly during the chase; moreover, production of protease-resistant fragments was also slowed in the presence of diamide. Densitometric analysis of autoradiographs demonstrated that, under control conditions, the conformational maturation of p73 was rapid between 2 and 5 min and peaked at 15 min. However, in the presence of diamide during the chase, the rate of maturation slowed and only 60 % of p73 was recognized by 4H3 after 1 h (Cobbold et al., 2001). Addition of reducing agents such as DTT during the chase period had no effect on the rate of p73 folding (data not shown).
Diamide inhibits the transfer of p73 onto the ER-derived membranes
Once p73 molecules are released from CAP80, they are recruited rapidly onto ER-derived cisternae (Cobbold et al., 1996, 2001). We have shown previously that all of the p73 bound to ER-derived membranes is immunoprecipitatable with 4H3, showing that it undergoes conformational maturation before transfer to the membranes. The next experiments tested the effects of diamide on transfer of p73 to these ER-derived membranes. The pulse-labelling time was increased to 5 min to allow the initial folding of p73 into a conformation recognized by mAb 4H3, and diamide or DTT was added after pulse labelling. At this point, p73 is in the cytosol. Cells were then chased for 5, 15 and 30 min to follow transfer to ER-derived membranes (Cobbold et al., 2001). The levels of folded p73 present were determined by lysis and immunoprecipitation using the conformation-dependent antibody to p73. Fig. 2(a) shows that, under control conditions, p73 was detected in the membrane fraction (M) during the first 5 min of the chase, and levels reached a maximum at 30 min. This was largely unaffected when the cells were incubated with DTT during the chase. Less p73 was recovered during a chase in the presence of diamide. This is because diamide slows the folding of p73 into a conformation recognized by mAb 4H3 (Fig. 1d). The results also show that recruitment of p73 onto ER-derived membranes was inhibited when cells were incubated with diamide; densitometric analysis showed that only 5 % of the pulse-labelled p73 protein was recovered from membranes after 30 min, compared with between 30 and 40 % for control cells.
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). Diamide caused a marked increased in reactive oxygen species, as shown by elevated green fluorescence in the middle panel. Following diamide wash-out for 1 h (lower panel), cells lost the strong fluorescent signal and could therefore recover a reduced cytosol. The effects of washing diamide from cells on the membrane binding of p73 were also studied. Cells pulse-labelled for 5 min and incubated for 30 min in the presence of diamide were transferred to complete medium for a further 60 min in the absence of diamide. Fig. 2(c) shows that p73 was recruited onto membranes once diamide was removed, showing that the inhibitory effects of diamide on p73 membrane binding were reversible.
CAP80 undergoes a redox-sensitive conformational change, but does not form disulphide-linked complexes with p73
The CAP80 protein encoded by the BA71v ASFV isolate used in these studies has a central domain containing 15 repeats of a Cys–Ala–Ser–Thr (CAST) motif. The presence of a large number of cysteine residues in the centre of CAP80 prompted us to use non-reducing SDS-PAGE gels to search for disulphide bonds in the CAP80–p73 complex. CAP80 migrates at approximately 80 kDa on reducing SDS-PAGE gels. This is approximately 10 kDa larger than the size predicted from the B602L reading frame and is caused by the central cysteine-rich domain (Cobbold et al., 2001). Control cells or cells incubated with diamide after pulse labelling were chased for 30 min, the time taken for p73 to dissociate from CAP80. Fig. 3 shows that CAP80 migrated at approximately 70 kDa on non-reducing gels. The non-reduced protein therefore migrated faster than the reduced form, suggesting a more compact conformation, and implies that reduction of the cysteine-rich domain of CAP80 allowed CAP80 to adopt a more open conformation. The gel also shows that immunoprecipitates of control cells and cells incubated with diamide during the chase produced a single band for CAP80 at 70 kDa and did not show any bands migrating further up the gel, indicative of proteins interacting through disulphide bonds; this suggests that CAP80 does not form disulphide-linked complexes with itself, p73 or other proteins.
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). At the same time, p73 oligomerizes from a 150–200 kDa homodimer/trimer (Cobbold et al., 2001), seen in the centre of the gradient (fraction 6), into a large complex of approximately 50 000 kDa, which migrates in the bottom fractions of a 10–40 % sucrose gradient (Cobbold & Wileman, 1998). This is approximately 10 % of the size of ASFV particles (555 000 kDa; Andres et al., 1997). The complex may represent virus capsids partially dissociated by detergent or very large assembly intermediates. Protease-protection and sucrose-centrifugation assays were used to determine the effect of DTT and diamide on the assembly and conformational maturation of p73 within these structures. Infected cells were pulse-labelled for 20 min, which allowed binding of p73 to ER-derived cisternae, and then chased for 2 h in complete medium. Post-nuclear membrane fractions were divided into two aliquots; one was incubated with trypsin, whilst the other was solubilized and centrifuged on 10–40 % sucrose gradients. A comparison of the autoradiographs on the right of Fig. 4(a) showed that the majority of membrane-associated p73 was resistant to trypsin after 2 h, indicating conformational maturation of p73 on ER-derived membranes; during this period, the majority of the solubilized membrane-associated p73 [lanes 1–10 of Fig. 4(a)] migrated towards the bottom of the gradient, indicating incorporation into a large complex. Addition of DTT during the 2 h chase had no effect on the maturation of p73 (Fig. 4b). Fig. 4(c) shows cells incubated with diamide during the 2 h chase. This time period was sufficient for p73 to reach a conformation recognized by the p73 antibody (Fig. 1d) and to bind membranes. In the presence of diamide, the quantity of p73 protected from trypsin and the formation of large complexes containing p73 migrating at the bottom of the sucrose gradient were reduced substantially. Capsid maturation on membranes was therefore inhibited when cells were oxidized by diamide, indicating that the reduced redox potential maintained in the cytosol is important for both p73 recruitment onto membranes and subsequent assembly into capsid and capsid precursors.
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), approximately 70 % of the p73 complex present in the membrane fraction migrated towards the bottom of the sucrose gradient and was resistant to trypsin. In contrast, when membranes were treated with GSSG and trypsin, only a small proportion of p73 was observed in fractions 1–3 of the sucrose gradient (Fig. 4e). In addition, tryptic fragments of p73 were observed running in fractions 5–8 of the gradient (representing complexes of 150–250 kDa), suggesting that addition of GSSG to membranes caused disassembly of the virus capsid and/or capsid precursors and exposed them to trypsin.
The above experiment showed that GSSG destabilized the membrane-bound p73 complex; even so, some material resistant to GSSG was detected at the bottom of the sucrose gradient. To see whether the proportion of protected material would increase with time, post-nuclear membrane fractions were taken from infected cells at increasing times and incubated in the absence or presence of trypsin and/or GSSG and then immunoprecipitated by using mAb 4H3 (Fig. 5a). A comparison of lanes 1 and 2 at each time point shows that, following the 10 min pulse, very little p73 was resistant to protease digestion; however, trypsin resistance increased during the chase. Interestingly, the proportion of p73 protected from trypsin at each time point decreased following addition of GSSG (lane 3), showing that GSSG increased sensitivity to the protease. Interestingly, after an extended 3 h chase, almost 70 % of p73 molecules remained resistant to protease in the presence of GSSG, showing that complexes containing the p73 protein became progressively more resistant to oxidation with time (Fig. 5b). This may be important to prepare the virus for release from the cells. When the experiment was repeated for virions purified from medium taken from labelled cells, virus particles were completely resistant to the combined effects of trypsin and GSSG (Fig. 5c). It is important to note that increased resistance of viruses released from cells to GSSG may also be due to the fact that extracellular viruses gain an additional outer envelope when they are released from the plasma membrane, and that this envelope could be responsible for a proportion of the increased resistance observed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), and non-reducing SDS-PAGE gels failed to detect p73–CAP80 complexes in control cells or cells incubated with diamide.
Having shown that capsid assembly required reducing conditions, the assay was modified to determine whether the stability of the pre-assembled capsid and/or large capsid precursors was also dependent on reducing conditions. Interestingly, large complexes containing p73 isolated from membrane fractions were unstable in the presence of GSSG, despite being resistant to protease digestion (Fig. 5a, 120 min chase). This result was unexpected because it implied that the large capsid complexes, which we believe are late assembly intermediates, would be unstable once they reached an oxidizing environment following release from the cell. The capsid precursors became progressively more resistant to GSSG with time and, 3 h following transfer of p73 to the ER-derived membranes, the capsid and/or capsid precursors were resistant to GSSG. This time course was consistent with our previous results showing that ASFV is released from cells between 3 and 4 h following synthesis of p73 (Cobbold et al., 1996) and the observation that viruses isolated from culture supernatants were also resistant to GSSG. The mechanism that provides a progressive increase in stability to oxidizing conditions is unknown, but may involve structural protein pE248R, which has two intramolecular disulphide bonds. pE248R is associated with the inner virus envelope and exposed to the cytosol, where it binds to the ASFV-encoded Erv1p/Alrp family thiol oxidase (pB119L) (Rodriguez et al., 2006). pB119L is a late protein that locates to virus factories, making it possible that pB119L oxidizes pE248R during assembly; this may stabilize ASFV during capsid maturation.
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ACKNOWLEDGEMENTS |
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Received 1 June 2006;
accepted 25 September 2006.
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-amylase), 473 kDa (apoferritin) and 50 000 kDa (ASFV capsid) on the same gradient are shown. (d, e) Newly synthesized capsids are destabilized by oxidized glutathione. Vero cells infected with ASFV for 16 h were labelled metabolically for 20 min with [35S]methionine and cysteine in the absence of redox reagents. Medium containing diamide (0.5 mM), DTT (1.0 mM) or control medium was added immediately after pulse labelling and then cells were chased for 2 h. Cells were homogenized and post-nuclear membranes were incubated in the absence or presence of oxidized glutathione and trypsin as indicated. Membranes were repelleted, solubilized in 1 % Brij : 35 immunoprecipitation buffer and lysates were loaded onto 10–40 % continuous sucrose gradients and centrifuged for 20 h. Fractions were collected from the bottom of the tubes, immunoprecipitated with mAb 4H3 and analysed by SDS-PAGE followed by autoradiography. The loss of p73 signal in fractions 1–4 and appearance of proteolytic fragments in fractions 5–8 in (b) indicates destabilization of the capsid in the presence of GSSG.