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Journal of Virology, April 2006, p. 3157-3166, Vol. 80, No. 7
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.7.3157-3166.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

African Swine Fever Virus pB119L Protein Is a Flavin Adenine Dinucleotide-Linked Sulfhydryl Oxidase

Irene Rodríguez, Modesto Redrejo-Rodríguez, Javier M. Rodríguez, Alí Alejo, José Salas, and María L. Salas*

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Received 14 October 2005/ Accepted 5 January 2006


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ABSTRACT
 
Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway.


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INTRODUCTION
 
Proteins of the Erv1p/Alrp family are flavin adenine dinucleotide (FAD)-dependent sulfhydryl oxidases catalyzing the formation of disulfide bonds in protein substrates at different subcellular compartments (18, 21). These enzymes contain a redox-active CXXC motif in the protein sequence and noncovalently bound FAD and use O2 for oxidation. The different members of this family are characterized by the presence of highly divergent amino-terminal regions, which are involved in subcellular distribution, and conserved carboxy termini, containing the enzymatic activity (12). Concerning their biological effects, yeast Erv1p is essential for mitochondrial biogenesis, a function that can be complemented by the carboxy-terminal domain of human Alrp (4, 22). It has also been demonstrated that mammalian Alrp has a regulatory role on mitochondrial gene expression and can also act as immunoregulator via its effect on gamma interferon expression (25, 26). Furthermore, human hepatopoietin (HPO), as the Alrp protein is also designated, has been described as a hepatotrophic growth factor that binds to a specific cell surface receptor in hepatocytes and hepatoma cells and induces the stimulation of the mitogen-activated protein kinase cascade (20). In addition to this, intracellular HPO has been shown to interact with the transcriptional coactivator Jun activation domain-binding protein 1, designated JAB1, to regulate AP-1 transcriptional activity (23). Moreover, the potentiation of AP-1 activation by HPO appears to be dependent on its function as a sulfhydryl oxidase (6).

Homologues of this type of sulfhydryl oxidases in some cytoplasmic double-stranded DNA viruses have also been described. Thus, vaccinia virus, the prototypal member of the poxvirus family, encodes a protein, E10R, that belongs to the Erv1p/Alrp family and participates in a viral cytoplasmic pathway of disulfide bond formation. E10R is the upstream component of this pathway, also composed of protein A2.5L, which forms a disulfide-bonded heterodimer with E10R, and G4L, a thioredoxin-like protein that directly oxidizes thiols of several structural components of the virion membrane, this process playing an essential role in virus morphogenesis (31).

African swine fever virus (ASFV), an enveloped icosahedral deoxyvirus that shares a number of properties with the poxviruses, including the cytoplasmic site of virus assembly (29), has also been shown to encode an Erv1p/Alrp homologue, the product of gene B119L (19, 35). Gene B119L encodes a 119-amino-acid protein of 14 kDa that conserves the PCXXC active-site motif, as well as a number of residues that participate in FAD binding in Alrp and Erv2p (11, 34). Protein pB119L has been described as a nonstructural protein that is expressed late in ASFV-infected macrophages (19). As in the case of vaccinia virus E10R, the ASFV B119L gene also appears to be involved in virus morphogenesis, since its deletion from the virus genome strongly affects virion maturation and virus production in infected macrophages (19).

To obtain more information about the role of gene B119L during the ASFV infective cycle, we have examined the sulfhydryl oxidase activity of a purified recombinant protein, pB119L, and have analyzed the redox state of the protein in infected cells, as well as its interaction with other viral proteins, in an attempt to identify possible substrates or intermediates in a putative pathway for disulfide bond formation. Our results show that protein pB119L is a FAD-linked sulfhydryl oxidase, which exists in infected cells mainly in an oxidized state containing a disulfide bond, and forms noncovalent dimers. We also show that pB119L interacts with the viral protein pA151R, which contains a CXXC redox motif and binds, in turn, to protein pE248R, a late structural component of ASFV, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus.


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MATERIALS AND METHODS
 
Cloning of ASFV genes B119L, E248R, and A151R. The B119L open reading frame (ORF) of ASFV strain BA71V was amplified by PCR using oligonucleotides 5'-CGCGGGATCCATGTTGCATTGGGGACCTAA-3', containing a CGCG tail and a BamHI restriction site, and 5'-CGCGGAATTCTTATAGAGATGACCAGGCTC-3', which contains a CGCG tail and an EcoRI site, and cloned in the expression vector pGEX2T (Amersham Biosciences). The A151R ORF of BA71V was amplified by PCR using oligonucleotides 5'-CGCGGATCCATGATGGCGTTGTTACACAA-3', with a CGCG tail and a BamHI site, and 5'-CGCGGTACCTTATTGGAATATATTGGGC-3', with a CGC tail and a KpnI site, and cloned in pRSETA (Invitrogen). BA71V ORF E248R was obtained from plasmid pRSETA-E248R by digestion with EcoRI and BamHI and cloned in plasmid pGEX2T digested with the same enzymes.

Expression and affinity purification of the recombinant proteins pB119L, pA151R, and pE248R. The pB119L protein fused to glutathione S-transferase (GST) was induced in Escherichia coli XL-1 cells at 25°C in the presence of 0.4 mM IPTG (isopropyl-ß-D-thiogaloactopyranoside). After 3 h, the cells were collected by centrifugation, resuspended in phosphate-buffered saline (PBS), and sonicated on ice. Triton X-100 was then added to a final concentration of 1%, and the suspension was incubated for 20 min at 4°C. This was followed by the addition of 10 µg of DNase I/ml and incubation for 20 min at room temperature. An aliquot of this suspension was withdrawn as a total extract fraction. The suspension was cleared by centrifugation for 20 min at 10,000 x g at 4°C, and the supernatant was used for affinity purification of the protein on glutathione-Sepharose 4B columns (Amersham Biosciences) previously equilibrated in PBS containing Triton X-100. The supernatant was passed twice through the column; after being washed extensively with PBS, the recombinant protein was eluted with 10 mM reduced glutathione. Control GST protein was expressed and purified as before with plasmid pGEX2T. Plasmid pGEX2T-E248R was expressed in E. coli XL-1 at 30°C in the presence of 0.4 mM IPTG. The cells were collected by centrifugation after 3 h, cell extracts were prepared, and the recombinant protein was purified as described before. The purified proteins were dialyzed against PBS and concentrated with Centriprep-30 (Millipore).

Plasmid pRSETA-A151R was expressed in E. coli strain BL21(DE3)pLysS at 37°C in the presence of 0.4 mM IPTG for 5 h. The cells were collected by centrifugation; resuspended in 50 mM Na2HPO4, 500 mM NaCl, 20 mM imidazole, and 10 mM ß-mercaptoethanol; and sonicated on ice. After centrifugation for 10 min at 2,500 rpm at 4°C, the supernatant was collected and centrifuged for 20 min at 12,000 x g at 4°C. The sediment of this centrifugation was resuspended in buffer A (6 M HCl guanidine, 0.1 M Na2HPO4, 10 mM Tris-HCl [pH 8.0], 500 mM NaCl, 20 mM imidazole, and 10 mM ß-mercaptoethanol) and incubated for 1 h with shaking. For protein purification, Ni-nitriloacetic acid agarose beads (Qiagen), previously equilibrated in buffer A, were added to the above solution containing the recombinant protein, and the suspension was stirred for 1 h at room temperature and then loaded into a column. The resin was first washed with buffer A and then with buffer C (8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl [pH 7], 500 mM NaCl, 20 mM imidazole, and 10 mM ß-mercaptoethanol), and the recombinant protein was eluted in buffer E (8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl [pH 7], 500 mM NaCl, 20 mM imidazole, and 10 mM ß-mercaptoethanol) adjusted to pH 4.5 with HCl.

Antibodies. Antibodies against purified GST-B119L and GST-E248R proteins were raised in rabbits. For the preparation of antibodies against the purified His-tagged pA151R protein, the protein was run in a polyacrylamide gel, and the corresponding band was extracted and used to raise antibodies in rabbits. The mouse monoclonal anti-p72 antibody 17LD3 has been described previously (30). Proteins P2 and P3 from phage {Phi}29 and antibodies against them were kindly donated by Margarita Salas.

Spectroscopy of purified GST-B119L protein. Solutions (each, 7 µM) of GST-B119L, GST, Erv1p (73 to 179 fragment) (18), obtained from X-Zyme, and FAD (Sigma) in 100 mM potassium phosphate buffer, pH 7.5, were used for spectroscopy. The visible spectra of these solutions were recorded with a U-2000 Double-Beam spectrophotometer (Hitachi) in the range from 350 to 550 nm.

Assay of sulfhydryl oxidase activity. Sulfhydryl oxidase activity was determined with reduced lysozyme as a substrate, prepared as indicated by Lee et al. (18) or with dithiothreitol (DTT), following the procedure described by Hofhaus and Lisowsky (13). Purified recombinant proteins GST-B119L, GST, or Erv1p (73 to 179 fragment) were used in these assays. For assays with reduced lysozyme, the proteins corresponding to 50 pmol of bound FAD per time point were diluted in measurement buffer (2 M Urea in 100 mM potassium phosphate buffer, pH 7.5, containing 1 mM EDTA), together with reduced lysozyme corresponding to 70 nmol reduced thiol groups. The initial content of thiol groups was determined before the addition of the proteins to be assayed. The assay mixture was incubated at 30°C; at different times, 200-µl samples were diluted with 790 µl of measurement buffer, and then DTNB (Ellman's reagent; Sigma) (8) was added to a final concentration of 100 µM. After 30 s, the extinction at 412 nm was measured, and the thiol content was calculated using an extinction coefficient of 13.6 mM–1 cm–1. When DTT was used as a substrate, the proteins corresponding to 50 pmol bound FAD per time point were incubated with 10 mM DTT in PBS at 30°C. At different times, the thiol content was determined with 100-µl samples as before.

Analysis of protein expression in ASFV-infected Vero cells. Vero cells in Dulbecco's modified Eagle's medium containing 2% fetal calf serum were infected with the Vero-adapted ASFV strain BA71V (9) at a multiplicity of infection (MOI) of 10 PFU per cell; at different times postinfection, the cells were lysed in electrophoresis sample buffer. Equivalent amounts of the cell lysates were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and analyzed by Western blotting.

Western blot analysis. Samples were electrophoresed on 12% SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were incubated with antibodies against protein GST-B119L (1:1,000 dilution), GST-E248R (1:1,000 dilution), or pA151R (1:500 dilution) and then with a 1:10,000 dilution of peroxidase-labeled anti-rabbit serum (Amersham Biosciences). The proteins were then detected with the ECL system (Amersham Biosciences) according to the manufacturer's recommendations.

Immunofluorescence microscopy. Vero cells, grown on coverslips, were mock infected or infected with BA71V at an MOI of 1 PFU/cell and fixed at 14 h postinfection (hpi) with methanol at –20°C for 5 min. The cells were then incubated with anti-GST-B119L antibody (1:100 dilution) and the monoclonal antibody against p72 (1:20 dilution) for 1 h at room temperature and then with the secondary antibodies (1:500 dilution) for 45 min at room temperature. The secondary antibodies used were goat anti-mouse immunoglobulin G (IgG) coupled to Alexa 488 and goat anti-rabbit IgG coupled to Alexa 594, both from Molecular Probes. DAPI (4',6-diamidino-2-phenylindole) was used along with the secondary antibodies to stain DNA in nuclei and viral factories. Coverslips were mounted with Mowiol/Dabco on glass slides, and the preparations were examined with a Zeiss Axioskop 2 Plus microscope coupled to a charge-coupled device color camera. Images were processed using Adobe Photoshop software.

Glycerol gradient centrifugation. Vero cells were infected with BA71V at an MOI of 5 PFU/cell and collected at 20 hpi in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% NP-40 and supplemented with protease inhibitor cocktail tablets (Roche). After 15 min on ice, the lysate was centrifuged for 10 min at 10,000 x g. The extract was loaded onto a 5-ml glycerol gradient (15 to 30%) containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% NP-40, and protease inhibitor cocktail tablets. After centrifugation in a Beckman SW-65 rotor at 62,000 rpm for 24 h at 4°C, 25 fractions of 200 µl each were collected from the bottom of the tube and analyzed by SDS-PAGE and Western blotting, using the anti-GST-B119L antibody.

Analysis of disulfide bonds. Vero cells were infected with BA71V at an MOI of 5 PFU/cell for 14 h. The cells were then washed with PBS containing 20 mM N-ethylmaleimide (NEM; Sigma) and collected in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, and protease inhibitor cocktail tablets) with 20 mM NEM. Samples were analyzed by Western blotting after SDS-PAGE under reducing and nonreducing conditions. For trichloroacetic acid (TCA) treatment, the cells were washed with PBS, treated with 10% TCA, collected with a rubber policeman, and washed three times with cold acetone. After resuspension in nonreducing electrophoresis sample buffer containing 20 mM NEM, the samples were analyzed as before. For alkylation, the cells were precipitated with TCA as before and suspended in 50 mM Tris-HCl (pH 7.5) and 1% SDS with or without 20 mM 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS; Molecular Probes) or 20 mM NEM. To reduce the proteins before alkylation, DTT at different concentrations was added to the culture medium at 14 hpi, and incubation was continued for 30 min at 37°C. Cells were collected by centrifugation, precipitated with TCA, and suspended as before in 50 mM Tris-HCl (pH 7.5) and 1% SDS with or without 20 mM AMS or NEM. Before electrophoresis, samples were boiled in nonreducing electrophoresis sample buffer, and the proteins were resolved by SDS-PAGE in 16% Tricine gels and analyzed by Western blotting with the different antibodies.

Coimmunoprecipitation analysis. Vero cells grown on 24-well plates were mock infected or infected with BA71V at an MOI of 5 PFU/cell and labeled with 1-mCi/ml [35S]methionine-[35S]cysteine (Promix in vitro cell labeling mixture; Amersham Biosciences) from 14 to 18 hpi. Unlabeled cell cultures in P60 dishes were mock infected or infected as before. The cultures were harvested at 18 hpi. For each immunoprecipitation, labeled cells from two wells of the 24-well plates or unlabeled cells from one P60 dish were lysed by incubation for 20 min on ice in 2 ml of a buffer containing 25 mM Tris-HCl (pH 8.1), 50 mM NaCl, 0.5% deoxycholate, and 1% NP-40. Lysates were clarified by centrifugation at 14,000 rpm for 15 min at 4°C and incubated with 100 µl of 50% protein A-Sepharose beads for 1 h at 4°C. After centrifugation at 14,000 rpm for 10 min, the supernatant was incubated overnight at 4°C with a 1:40 dilution of either anti-GST-B119L or anti-GST-E248R antibody or with a 1:20 dilution of the anti-A151R antibody. The antigen-antibody complexes were then rotated with 100 µl of protein A-Sepharose beads for 1 h at 4°C. The immune complexes were washed three times with a buffer containing 50 mM Tris-HCl (pH 8.1) and 100 mM NaCl and finally resuspended in electrophoresis sample buffer. Samples were subjected to reducing SDS-PAGE and transferred to nitrocellulose membranes. The proteins were visualized by autoradiography or by immunoblot analysis with anti-GST-B119L, anti-GST-E248R, or anti-A151R antibodies.


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RESULTS
 
Expression and purification of the recombinant protein pB119L. To assess whether the ASFV pB119L protein possessed sulfhydryl oxidase activity, like yeast Erv1p and human Alrp, a recombinant protein was expressed and purified. To this end, the B119L ORF was cloned in pGEX2T vector and expressed in E. coli as a fusion protein to GST. As shown in Fig. 1, a protein of about 40 kDa, the size expected for the fusion protein, was detected in total lysates from cells induced with IPTG. A soluble fraction of these lysates was used for purification by affinity chromatography on glutathione-Sepharose columns, as described in Materials and Methods. After purification, a major band of about 40 kDa, corresponding to the fusion GST-B119L protein, and an additional minor band of 26 kDa, which corresponds to GST, were detected.


Figure 1
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  		FIG. 1. Expression and purification of ASFV recombinant pB119L protein. Coomassie blue staining after reducing SDS-PAGE separation of total extracts (T) from uninduced (–) or IPTG-induced (+) E. coli XL-1 cells transformed with the recombinant plasmid pGEX2T-B119L, the soluble fraction (S) obtained from the total extract, and the purified GST-B119L protein after glutathione-Sepharose chromatography. Molecular mass markers are indicated on the left, and the GST-B119L and GST proteins are indicated by arrows.

Protein pB119L is a flavoprotein with sulfhydryl oxidase activity. We first tested whether the pB119L protein contains FAD, as has been described for other members of the Erv1p/Alrp family (18, 21). For this purpose, solutions of the purified GST-B119L protein and of Erv1p (carried as a control), as well as of FAD, were analyzed by spectroscopy. As shown in Fig. 2, the spectrum of GST-B119L was essentially identical to that of Erv1p, exhibiting the small but typical differences with that of free FAD, thus indicating that ASFV pB119L is a flavoprotein.


Figure 2
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  		FIG. 2. Spectroscopy of purified pB119L. The absorbance in the range from 350 nm to 550 nm was measured for 7 µM solutions of FAD, Erv1p (positions 73 to 179), GST-B119L, and GST. Arrows mark the position of the absorbance maximum for protein-bound and free FAD.

We next investigated whether the viral protein showed sulfhydryl oxidase activity in assays carried out as described in Materials and Methods, using either DTT or reduced lysozyme as a substrate. As can be seen in Fig. 3, GST-B119L was able to oxidize both substrates in a time-dependent manner and with a similar efficiency. The turnover numbers with DTT and lysozyme were 13.4/min and 16/min, respectively, somewhat higher than those obtained with Erv1p in parallel assays (10/min with both substrates). As shown, neither FAD alone nor GST was able to oxidize those substrates. We therefore conclude that the viral protein pB119L exhibits FAD-linked sulfhydryl oxidase activity.


Figure 3
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  		FIG. 3. Sulfhydryl oxidase activity of GST-B119L and Erv1p (positions 73 to 179). (A) The assays were carried out using lysozyme (70 nmol reduced thiol groups) as substrate and with an amount of each protein corresponding to 50 pmol bound FAD per time point, as described in Materials and Methods. Oxidation of thiol groups was measured spectroscopically at 412 nm after the addition of 100 µM DTNB by determining the decrease of extinction at the indicated times. (B) Sulfhydryl oxidase activity of GST-B119L and Erv1p with DTT (10 mM) as a substrate. Other assay conditions were as described in the legend to panel A. Controls without enzyme, with FAD (50 pmol), and with GST are also shown.

Protein pB119L is a late protein that localizes at the viral factory. As described by Lewis et al. (19), protein pB119L is expressed at late times in macrophages infected with the virulent Malawi Lil-20/1 strain of ASFV. To confirm this in our system, we have examined the pattern of pB119L expression in Vero cells infected with the ASFV BA71V strain. As can be seen in Fig. 4A, the anti-GST-B119L antibody, but not the preimmune serum, recognized a band migrating at the expected position for protein pB119L (14 kDa) in ASFV-infected cells specifically by Western blotting. An immunoblot analysis of extracts from infected cells collected at different times postinfection showed that this band was first observed at 14 hpi and accumulated up to 20 hpi (Fig. 4B). Furthermore, the protein band was not detected in the presence of cytosine arabinoside, an inhibitor of viral DNA replication and late transcription, indicating that protein pB119L is a late protein. Also in agreement with Lewis et al. (19), we did not detect the pB119L protein in ASFV particles purified from the extracellular medium by Western blot analysis or by immunoelectron microscopy (data not shown).


Figure 4
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  		FIG. 4. Expression and localization of protein pB119L in ASFV-infected Vero cells. (A) Specificity of the anti-GST-B119L antibody in Western blot analysis after reducing SDS-PAGE. Immunoblotting was carried out for mock-infected cells (M) or ASFV-infected cells (I) harvested at 16 hpi. The results with anti-GST-B119L ({alpha}B119L) and preimmune serum (PI-B119L) are shown. Molecular mass markers are indicated on the left. (B) Expression of protein pB119L during the ASFV infection. Extracts from mock-infected (Mock) or ASFV-infected cells collected at different hours postinfection were analyzed by Western blotting with the anti-GST-B119L antibody after reducing SDS-PAGE. Results obtained with cells infected for 16 h in the presence of 40 µg of cytosine arabinoside (AraC)/ml are also shown. The band corresponding to protein pB119L is indicated by an arrow. (C) Immunofluorescence microscopy analysis of ASFV-infected Vero cells. Mock-infected (Mock) or ASFV-infected Vero cells (14 hpi) were fixed at 14 hpi and double labeled with anti-GST-B119L antibody detected with Alexa 594 goat anti-rabbit IgG (B119L) and with anti-p72 antibody detected with Alexa 488 goat anti-mouse IgG (p72). Cells were counterstained with DAPI to visualize cellular and viral DNA. Arrows indicate the position of the viral factories.

To study the intracellular localization of protein pB119L, mock-infected and ASFV-infected Vero cells were fixed at 14 hpi and analyzed by immunofluorescence with anti-GST-B119L serum and with an antibody against the capsid protein p72. As shown in Fig. 4C, a very low background signal was observed in mock-infected cells. In contrast, the antibody against protein pB119L strongly stained discrete cytoplasmic areas of the virus-infected cells, which were identified as virus factories by immunolabeling with a monoclonal antibody that recognizes the capsid protein p72 and by staining the viral DNA with DAPI. In addition, a diffuse staining of the cytoplasm was detected with the antibody against protein pB119L.

Protein pB119L contains intramolecular disulfide bonds. Sulfhydryl oxidases of the Erv1p/Alrp family form homodimers stabilized by disulfide bonds (18, 21, 34). On the other hand, vaccinia virus E10R protein is capable of forming disulfide-bonded heterodimers with protein A2.5L, the second component of the disulfide bond formation pathway (31). The ASFV pB119L protein contains two unique cysteine residues at the conserved redox-active CXXC motif. To determine if these cysteines were able to form intermolecular or intramolecular disulfide bonds, extracts from Vero cells infected with ASFV were prepared in the presence of the alkylating agent NEM (125 Da) to avoid oxidation during the extraction procedure and analyzed by Western blotting under reducing and nonreducing conditions. As can be seen in Fig. 5A, neither pB119L homodimers nor mixed disulfides between pB119L and other proteins were detected in the absence of DTT. Furthermore, the monomeric pB119L band showed the same electrophoretic mobility under the two conditions. To rule out the possible existence of transient disulfide bonds in pB119L, we treated the cell cultures directly with TCA to denature and precipitate whole-cell proteins, thus preventing thiol-disulfide interchange, as has been previously described (16, 17, 32). Again, no homodimers or mixed disulfides were detected under nonreducing conditions, and the electrophoretic mobility of the monomeric protein was identical when analyzed in the presence or in the absence of DTT (Fig. 5B). This latter result could indicate that the cysteines of pB119L are not disulfide bonded or that reduction of disulfide bonds in the protein does not change its electrophoretic mobility. To resolve this issue, we used the alkylating agent AMS, which increases the molecular mass of a protein in 536 Da per thiol group. ASFV-infected Vero cells were treated or not with DTT at 14 hpi, and the cell pellets were collected, precipitated with TCA, and resuspended in denaturing buffer with or without NEM or AMS, as indicated in Fig. 5C. The proteins were then resolved by SDS-PAGE, and protein pB119L was detected by Western blotting. A single pB119L band was observed in extracts from cells treated with NEM and reduced or not with DTT, whereas after treatment with AMS two bands were detected (Fig. 5C). The upper, very weak band, migrating more slowly, should correspond to the reduced, AMS-alkylated form, while the faster-migrating and more-abundant band should be the oxidized form of the protein containing an intramolecular disulfide bond. In confirmation of this, the sample previously reduced with DTT and alkylated with AMS showed a single band with the mobility of the upper form of the pB119L protein (Fig. 5C).


Figure 5
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  		FIG. 5. Analysis of disulfide bonds in protein pB119L. (A) ASFV-infected Vero cell extracts, prepared in the presence of 20 mM NEM were separated in a 12% SDS-PAGE gel with (+) or without (–) 10 mM DTT in the sample buffer. Protein pB119L was detected by Western blotting with anti-GST-B119L antibody. (B) ASFV-infected cells were treated with 10% TCA as described in Materials and Methods and resuspended in electrophoresis sample buffer with 20 mM NEM and containing (+) or not (–) 10 mM DTT. The samples were analyzed as described in the legend to panel A. Arrowheads (A and B) indicate the position of the monomeric pB119L protein. Molecular mass markers are indicated on the left. (C) ASFV-infected cells were treated as indicated in Materials and Methods with the following reagents in 50 mM Tris-HCl (pH 7.5)-1% SDS, from left to right: NEM (20 mM) and DTT (10 mM), NEM (20 mM), AMS (20 mM), DTT (10 mM) and AMS (20 mM), and DTT (100 mM) and AMS (20 mM). The proteins were separated by SDS-PAGE in 16% Tricine gels and analyzed by Western blotting with the antibody against protein GST-B119L.

Taken together, these results indicate that in the infected cells the pB119L protein does not form disulfide-bonded dimers and that it is present mainly in an oxidized state.

Protein pB119L sediments as a dimer after centrifugation of ASFV-infected cell extracts in glycerol gradients. To investigate whether the pB119L protein could form non-disulfide-bonded dimers, we subjected extracts from infected cells to glycerol gradient centrifugation, analyzing the gradient fractions by Western blotting with the anti-GST-B119L antibody. As shown in Fig. 6, essentially all the pB119L protein sedimented at a position near the 30-kDa size marker. Taking into account the size of the pB119L monomer (14 kDa), this result suggests that the protein is present as a dimer in the infected cell. However, the possibility that protein pB119L forms an heterodimer of this size with another cellular or viral protein cannot be ruled out at present.


Figure 6
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  		FIG. 6. Analysis of protein pB119L by glycerol gradient centrifugation. Extracts from ASFV-infected Vero cells were mixed with phage {Phi}29 monomeric and heterodimeric P2 (30-kDa) and P3 (60-kDa) proteins used as internal molecular mass markers and were loaded on top of a 15 to 30% glycerol gradient. After ultracentrifugation, 24 fractions were collected, and an aliquot of each fraction was analyzed by Western blotting using antibodies against protein GST-B119L or against the phage {Phi}29 proteins. Other molecular mass markers (trypsinogen [30 kDa] and lysozyme [14 kDa]) were run in a parallel gradient, and the gradient fractions were analyzed by Coomassie blue staining after reducing SDS-PAGE. The pB119L band was quantified by densitometric analysis, and the values obtained are expressed as percentages of intensity with respect to the intensity of the band of fraction 15. The positions of the markers are indicated by arrows.

Protein pB119L interacts with other viral proteins. To identify possible substrates of the ASFV sulfhydryl oxidase and/or intermediates of a redox pathway, mock-infected and ASFV-infected Vero cells were metabolically labeled, and the cell extracts were immunoprecipitated with the anti-GST-B119L antibody or preimmune serum as described in Materials and Methods. Upon analysis of the immunocomplexes by SDS-PAGE and autoradiography, we detected two specific bands of approximately 16 and 14 kDa (Fig. 7A). Other labeled bands are unspecific, since they were also detected in mock-infected cells or in infected cells immunoprecipitated with preimmune serum (Fig. 7A). The 14-kDa band corresponded to the pB119L protein, as demonstrated by Western blot analysis of the immunoprecipitate (Fig. 7B). Regarding the 16-kDa band, it should be mentioned that ASFV encodes a protein, pA151R, of 151 amino acids (35) that contains at its C terminus (between amino acids 131 to 135) a WCTKC sequence resembling the active-site motifs of members of the thioredoxin superfamily (7, 15) and which might thus form part of a putative ASFV redox pathway. It should be noted, however, that the pA151R protein does not contain the characteristic fold of thioredoxins (3). To investigate the possible participation of protein pA151R in a redox pathway, we prepared an antibody against the purified recombinant pA151R protein, as described in Materials and Methods. This antibody recognized a band with the size expected for protein pA151R in ASFV-infected cells but not in mock-infected cells in Western blots (Fig. 8A). Furthermore, this band was not detected with the preimmune serum, demonstrating the specificity of the anti-pA151R antibody. As can be seen in Fig. 8B, the 16-kDa pA151R protein was expressed at both early and late times of infection and was not detected in purified virus. In experiments of alkylation with AMS, protein pA151R, treated or not with DTT, was detected as an alkylated form, which migrated more slowly than the untreated control protein, indicating that the majority of the protein was reduced at 14 hpi (Fig. 9A). On the other hand, no intermolecular disulfide-bonded complexes containing protein pA151R were found in the absence of DTT (not shown). We considered the possibility that the 16-kDa band coimmunoprecipitating with pB119L might be the ASFV pA151R protein. To test this, we immunoprecipitated labeled cell extracts with the anti-A151R antibody. As shown in Fig. 7A, we detected a band of 16 kDa in the immunoprecipitate, which corresponded to the pA151R protein as confirmed by the Western blot analysis presented in Fig. 7B. In addition, a specific labeled band of 14 kDa coimmunoprecipitating with the pA151R protein was shown to be the pB119L protein by immunoblotting this immunoprecipitate with the antibody specific for pB119L (Fig. 7B). These results indicated that the ASFV sulfhydryl oxidase interacts with the viral protein pA151R, suggesting that these two proteins could be components of a redox cascade for the formation of disulfide bonds in ASFV proteins. Regarding the question of possible substrates of this pathway, an ASFV protein, designated pE248R, has been described as belonging to a superfamily of myristoylated proteins related to vaccinia virus L1R, a structural, disulfide bridge-containing protein, which is one of the substrates of the virus disulfide bond formation pathway (24, 31). As shown in Fig. 8A, an antibody prepared against the purified recombinant protein pE248R recognized a single protein of 34 kDa in Western blots of extracts from infected cells. This protein was not detected in mock-infected cells or when preimmune serum was used, thus indicating that the 34-kDa band corresponded to protein pE248R. The results presented in Fig. 8C show that this protein was expressed at late times of infection and is a structural component of the virus particle. Furthermore, the protein, which contains four Cys residues, migrates in SDS-PAGE gels as a single band with the same mobility under reducing or nonreducing conditions. However, after treatment with AMS in the absence of DTT, two bands were detected. The faster one, which was more abundant, corresponds to the oxidized state of the protein, whereas the slower one represented protein pE248R with free thiols that were alkylated with AMS (Fig. 9B). As expected, the protein reduced with DTT and treated with AMS migrated as a single band with a mobility corresponding to its reduced, alkylated state (Fig. 9B). As in the case of proteins pB119L and pA151R, no higher-order complexes of protein pE248R were detected under nonreducing conditions (not shown). These results indicated that protein pE248R is mainly present in the infected cells forming intramolecular disulfide bonds; these results are consistent with the possibility that pE248R might indeed be a substrate of the ASFV redox pathway. To investigate this, we immunoprecipitated labeled extracts from ASFV-infected cells with the anti-pE248R antibody. Analysis of the immune complexes by SDS-PAGE and autoradiography revealed a labeled band of 34 kDa, as well as an intensely labeled 16-kDa protein (Fig. 7A). Western blot analysis of the immunoprecipitate showed that, as expected, the 34-kDa band corresponded to the pE248R protein (Fig. 7B). The size of the other band and its comigration in SDS-PAGE gels with pA151R (Fig. 7A) suggested that it could be the viral pA151R protein. To corroborate this, the immunoprecipitate was analyzed by immunoblotting with the anti-A151R antibody. As shown in Fig. 7B, the 16-kDa band was recognized by the pA151R-specific antibody, thus identifying this protein as pA151R. These results provide evidence of an interaction between pA151R and pE248R. This interaction, together with the presence of disulfide bonds in pE248R and the relationship of this protein with vaccinia virus L1R, suggests that the ASFV protein is a final substrate of the redox pathway. It should be noted that a weak band with the mobility of protein pE248R is also observed in the immunoprecipitates with the antibodies against proteins pB119L and pA151R (Fig. 7A), although this band could not be confirmed to be protein pE248R by immunoblotting (not shown).


Figure 7
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  		FIG. 7. Interaction of proteins from ASFV-infected cells with pB119L protein. (A) 35S-labeled extracts from mock-infected (M) and ASFV-infected (I) cells were prepared and coimmunoprecipitated with anti-GST-B119L ({alpha}-B119L), anti-A151R ({alpha}-A151R), or anti-E248R ({alpha}-E248R) antibodies or with control preimmune serum (PI), as described in Materials and Methods. The immune complexes were analyzed by reducing SDS-PAGE, followed by autoradiography. The positions of molecular mass markers are indicated on the left. Specific immunoprecipitated bands are indicated by asterisks. (B) Western blot analysis of the immunoprecipitates. The immunoprecipitates with the antibodies indicated in panel A (IP) were analyzed by Western blotting with anti-B119L, anti-A151R, and anti-E248R antibodies (WB), as indicated. The 14.8- and 37-kDa markers are shown on the left. The asterisks indicate the protein bands recognized specifically by each antibody.


Figure 8
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  		FIG. 8. Expression of pA151R and pE248R proteins in ASFV-infected Vero cells. (A) Specificity of the antibodies used. The specificity of the anti-pA151R ({alpha}A151R) and anti-pE248R ({alpha}E248R) antibodies was tested in Western blots of extracts from mock-infected (M) or ASFV-infected cells (I) harvested at 16 hpi. The results with the corresponding preimmune sera (PI-A151R and PI-E248R) are also shown. Molecular mass markers are indicated on the left. (B) Expression of protein pA151R during ASFV infection. Mock-infected cells (Mock) or ASFV-infected cells harvested at different times postinfection were analyzed by Western blotting with the anti-pA151R antibody. (C) Expression of protein pE248R during ASFV infection. Western blot analysis with the anti-E248R antibody was carried out for mock-infected cells (Mock) or ASFV-infected cells harvested at different times postinfection. In panels B and C, results obtained with cells infected for 16 h in the presence of 40-µg/ml AraC and with 2 µg of purified ASFV are also shown. The arrowheads indicate the position of the 16-kDa pA151R protein (B) and the 34-kDa pE248R protein (C). Reducing SDS-PAGE was used for the analysis of the proteins in all cases.


Figure 9
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  		FIG. 9. Analysis of disulfide bonds in proteins pA151R and pE248R. ASFV-infected cells were treated as indicated in Materials and Methods with the following reagents in 50 mM Tris-HCl (pH 7.5)-1% SDS, from left to right: NEM (20 mM) and DTT (10 mM), NEM (20 mM), AMS (20 mM), DTT (10 mM) and AMS (20 mM), and DTT (100 mM) and AMS (20 mM). The proteins were separated by SDS-PAGE in 16% Tricine gels and analyzed by Western blotting with antibodies against protein pA151R (A) or GST-E248R (B).


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DISCUSSION
 
ASFV assembles, like the poxviruses, in the reducing environment of the cell cytosol. This raised the possibility that the formation of disulfide bonds in some proteins at the viral factory might require a protein-oxidation pathway similar to that described for vaccinia virus. In keeping with this, ASFV encodes a protein, pB119L, which belongs to the Erv1p/Alrp family of sulfhydryl oxidases. To begin exploring this putative pathway, we have performed a biochemical characterization of purified recombinant protein pB119L in the present work, showing that pB119L is a FAD-containing sulfhydryl oxidase capable of efficiently catalyzing in vitro the formation of disulfide bonds in both a protein substrate, like lysozyme, and the small molecule DTT. To our knowledge, this is the first viral Erv1p homologue shown to have sulfhydryl oxidase activity in vitro and to contain bound FAD.

Consistent with a role for protein pB119L as an oxidase in vivo, the protein contains the active-site cysteines mainly in an oxidized state in ASFV-infected cells. However, disulfide-bonded homo- or heterodimers were not detected, even in extracts prepared after the cells were treated with TCA to stabilize the disulfide bonds, although the protein forms noncovalent dimers, as indicated by sedimentation on glycerol gradients. This is in contrast with results obtained with other members of the Erv1p/Alrp family. Thus, in Erv1p and Erv2p, intermolecular disulfide bridges are formed by the interaction of N-terminal CXXC or C-terminal CXC motifs, respectively, with the CXXC active-site motif of the opposite monomer (11, 14), while in Alrp the terminal cysteine residues C15 and C124 form two head-to-tail intermolecular disulfide bonds in the homodimer (34). On the other hand, vaccinia virus E10R forms a mixed disulfide in infected cells with protein A2.5L, the second component of the viral redox pathway, as well as homodimers linked by a disulfide bridge when overexpressed under transfection conditions (31). It may be that, in the case of ASFV, the detection of similar complexes would require a further stabilization of transient intermediates by mutation of the C-terminal cysteine of the pB119L active site and overproduction of the mutated form in infected cells, which might facilitate trapping the mixed disulfides, as described by Frand and Kaiser (10) for the interaction of Ero1p with PDI or PDI homologues.

Studies of the interaction of pB119L with other viral proteins in immunoprecipitation experiments have revealed that the ASFV sulfhydryl oxidase interacts with a viral protein encoded by gene A151R and that this interacts in turn with a late structural protein, pE248R, which contains intramolecular disulfide bonds and belongs to a family of myristoylated proteins related to vaccinia virus L1R, one of the substrates of this virus redox pathway. These results suggest that the oxidation of pE248R is performed by pA151R and that this protein is an intermediate of the ASFV protein oxidation pathway, the FAD-linked sulfhydryl oxidase encoded by gene B119L being the first component of this system.

A proposed mechanism for thiol-disulfide transfer involves the formation of disulfide-linked complexes between the components of the redox pathway. An example of this mechanism is the vaccinia virus protein oxidation system, where the intermediates of the chain form transient heterodimers bound through disulfide bridges (31). However, the experiments presented here indicate that the complexes containing pB119L, pA151R, or pE248R are not held together by disulfide bonds. Although as discussed above, the detection of transient covalent interactions between these proteins through disulfide bridges may require overexpression of the proteins or mutation of one of the active-site cysteines, a mechanism involving the participation of a small thiol-containing molecule such as glutathione could also operate. Further studies using recombinant virus inducibly expressing the pB119L or pA151R proteins will be needed to demonstrate the existence of the putative ASFV redox pathway and to investigate the mechanisms involved in thiol-disulfide transfer in this system.

Although both vaccinia virus E10R and ASFV pB119L are late proteins required for correct virus assembly, significant differences are found between the two proteins. Thus, while E10R is a membrane-associated structural protein, pB119L lacks a transmembrane region and is not detected in purified extracellular virus. Similarly, the ASFV pA151R protein is not structural, in contrast with the presence of the vaccinia virus glutaredoxin, an intermediate in the redox pathway, in the vaccinia virus particles (33). However, at least one of the possible protein substrates of the ASFV pathway, protein pE248R, is a structural component of the virus and is likely to associate with the inner viral membrane, taking into account the presence of a putative myristoylation site and predicted transmembrane regions (24). Furthermore, the N-terminal region of the protein containing the four cysteine residues is predicted to be facing the cytoplasm (28), thus probably requiring a cytoplasmic redox system for the generation of disulfide bridges in the protein. It is therefore possible that the ASFV sulfhydryl oxidase and protein pA151R exert their catalytic function on protein pE248R and other possible structural substrates during the process of virus assembly, being finally excluded from the mature virions. This possibility is sustained by the fact that the deletion of gene B119L dramatically affects virus morphogenesis, decreasing the number of virus particles by 90% and leading to the formation of aberrant virions, which are probably noninfectious and account for 90% of the total particles produced (19). The formation of disulfide bridges in two other ASFV structural proteins has been described. Thus, the inner envelope protein p54 forms disulfide-bonded dimers in the virus particle (27), but since this dimerization must occur through the unique cysteine residue located at the N terminus of the protein that resides in the relatively oxidant endoplasmic reticulum lumen, it is unlikely that either protein pB119L or pA151R is involved in the formation of the disulfide bond in p54. The virus attachment protein p12 is also a membrane protein that forms disulfide-bound dimers with the participation of cysteine residues present at the C terminus (2, 5), which according to the topological model proposed for the polypeptide, would be found in the lumen of the endoplasmic reticulum, again suggesting that proteins pB119L and pA151R are not involved in the dimerization of p12. Preliminary experiments suggest the presence of disulfide bridges in other ASFV structural proteins, including the polyprotein-processing proteinase. In connection with this, it is worthwhile mentioning that infection with the ASFV Malawi strain lacking gene B119L (9GL in this strain) leads to the formation of particles with a phenotype essentially identical to that obtained when the expression of the virus proteinase is repressed in cells infected with the recombinant BA71V vS273Ri, inducibly expressing the proteinase gene (1, 19). This raises the interesting question of whether protein pB119L might control proteinase activity through the formation of disulfide bonds, although we have not so far been able to detect an interaction of the proteinase with protein pB119L or pA151R. An examination of the redox state of the viral enzyme and of polyprotein processing in infections with deletion mutants or inducible virus in gene B119L would help to answer this question.

It is interesting to note that protein pA151R is expressed at both early and late times of infection. While the protein expressed at late times could participate together with the sulfhydryl oxidase in the formation of disulfide bonds in structural virus proteins, additional roles could be proposed for the protein synthesized at early times postinfection. These early functions could be related to DNA precursor synthesis or regulation of signal transduction pathways, in keeping with alternative roles described for thioredoxins (for a review, see reference 3). On the other hand, the fact that the A151R gene cannot be deleted from the virus genome (unpublished results) is consistent with the proposed role of the encoded protein in disulfide bond formation in structural proteins during the late stages of infection.

In summary, the studies presented here advance our understanding of disulfide bond formation in ASFV proteins, with the characterization of the viral pB119L protein as a FAD-containing sulfhydryl oxidase and the identification of the viral pA151R protein and protein pE248R, a new structural component of ASFV particles, as likely intermediate and substrate, respectively, of a virus-encoded redox pathway. Further studies based on these new observations will be needed to confirm and extend the redox system proposed in this work.


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ACKNOWLEDGMENTS
 
We thank J. F. Santarén for helpful comments.

This study was supported by grants from the Spanish Ministerio de Educación y Ciencia (BFU2004-00298) and the European Commission (QLK2-CT-2001-02216) and by an institutional grant from Fundación Ramón Areces. J.M.R. was supported by the "Ramón y Cajal" program of the Ministerio de Educación y Ciencia. M.R.-R. was a predoctoral fellow of the Ministerio de Educación y Ciencia.


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FOOTNOTES
 
* Corresponding author. Mailing address: Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Phone: 34 91 497 84 78. Fax: 34 91 497 47 99. E-mail: mlsalas{at}cbm.uam.es. Back


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Journal of Virology, April 2006, p. 3157-3166, Vol. 80, No. 7
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.7.3157-3166.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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