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Previous Article | Next Article 
Journal of Virology, March 2002, p. 2654-2666, Vol. 76, No. 6
0022-538X/02/$04.00+0 DOI: 10.1128/JVI.76.6.2654-2666.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
Received 11 October 2001/ Accepted 13 December 2001
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ABSTRACT |
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INTRODUCTION |
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The virus particle contains about 50 proteins (22) and consists of several concentric domains with an overall icosahedral shape and an average diameter of 200 nm (3, 4, 14). The viral core is composed of two domains: a central DNA-containing nucleoid and a surrounding thick protein coat referred to as the core shell. The core is enwrapped by an inner lipid envelope and an icosahedral protein capsid. Extracellular particles possess an additional envelope derived from the plasma membrane (9). ASFV particles are formed within cytoplasmic viral factories (3, 9, 10, 33) from precursor membranous structures (3), which are thought to be cisternal structures derived from the endoplasmic reticulum (ER) (4, 37). Morphological evidence indicates that viral membranes become icosahedral particles by the progressive construction of the outer capsid layer, which is composed mainly of viral protein p72 (3, 24). Biochemical data also suggest that protein p72 is assembled in a time-dependent fashion into large membrane-bound complexes that may correspond to capsid-like structures (17). Concomitantly, the developing particles engulf the core material through a complex and poorly understood process that probably begins with the construction of the core shell underneath the viral envelope (3, 37). Subsequently, the viral DNA and nucleoproteins would be encapsulated and finally condensed into the characteristic electron-dense nucleoid (3, 11). The resulting intracellular mature virions are infectious (6), although their relevance for in vivo infection has not yet been established. A fraction of the intracellular particles reach the plasma membrane, likely by a microtubule-mediated transport (2, 15), and are released by budding (9) to give rise to the infectious extracellular enveloped virions.
One of the most striking features of ASFV gene expression is the synthesis of polyprotein precursors to produce several major structural components. The largest ASFV gene, CP2475L, encodes polyprotein pp220, an N-myristoylated polypeptide that after proteolytic processing gives rise to proteins p150, p37, p34, and p14 (40). These proteins are present in equimolecular amounts within the core shell of the mature virions and account for about one-fourth of their total protein mass (3). On the other hand, the major proteins p35 and p15 are processing products of the higher-molecular-weight precursor pp62, encoded by gene CP530R (42). The two polyproteins are expressed late after infection and processed through an ordered cascade of proteolytic events that take place after the second glycine of the consensus motif Gly-Gly-X (31, 40, 42). We have recently shown that the viral protein pS273R is a cysteine proteinase responsible for polyprotein processing (5). Interestingly, ASFV proteinase localizes within the core region of the assembling virions and within the core shell of the mature particles. Together, these data indicate that polyprotein expression and processing must be relevant for core formation.
This report analyzes the role of polyprotein pp220 in ASFV morphogenesis. We describe the construction and properties of a conditional lethal mutant of ASFV with an inducible copy of gene CP2475L. The results indicate that polyprotein pp220 is crucial for the assembly and envelopment of the core shell and for the subsequent steps of core formation, including DNA encapsidation and nucleoid maturation. We also show that precursor pp220, when expressed alone, assembles into large membrane-bound coats reminiscent of the core shell and that N myristoylation is required for its anchoring to membranes.
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MATERIALS AND METHODS |
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Antibodies. The monospecific rabbit polyclonal sera against the structural proteins p150, p37/p14, p34, p35, p15, pS273R, pE120R, p72, and p12 and the mouse monoclonal antibody 17K.G12 against the structural protein p17 have been described previously (5, 6, 7, 32, 39, 40, 42). The monoclonal antibody specific for single- and double-stranded DNAs (MAB3032) was purchased from Chemicon International. To prepare antibodies against proteins p10 (34) and pA104R (8), the complete open reading frames (ORFs) K78R and A104R, respectively, were expressed in E. coli and purified using the His-Patch ThioFusion expression system according to the procedure of the manufacturer (Invitrogen BV). Antibodies against the purified soluble recombinant proteins were raised in rabbits.
Plasmid construction. The intermediate transfer vectors pIND1 and pIND2, designed to allow inducible expression of a target gene after homologous recombination with virus vGUSREP, have been reported previously (6). They contain a cassette formed by the viral inducible promoter p72.I, the lacZ gene under control of the strong late promoter p72 (24), and two multiple cloning sites to allow the cloning of the target gene and the corresponding upstream and downstream flanking sequences.
A synthetic DNA fragment of 1,045 bp, which contains the nucleotide sequence from position -1079 to -55 relative to the translation initiation codon of the CP2745L gene, was obtained by PCR using BA71V genomic DNA as a template and the oligonucleotides 5'-TATGGTACCAATCATATAAGAATAAC and 5'-CGGCGGCCGCGACCAGGACTGCTTCTC, which contain KpnI and NotI restriction sites (underlined) at their respective 5' ends. Plasmids pIND1.pp220.Fl and pIND2.pp220.Fl were generated by inserting the KpnI- and NotI-digested PCR fragment into the KpnI- and NotI-linearized plasmids pIND1 and pIND2, respectively. An 8-kb XbaI DNA fragment containing the complete CP2745L ORF was excised from pGEM-CP2475L (5) and inserted into the XbaI sites of plasmids pIND1.pp220.Fl and pIND2.pp220.Fl to obtain the final transfer vectors pIND1.pp220 and pIND2.pp220, respectively. These vectors differ in the direction of transcription of the lacZ gene with respect to the inducible promoter.
Generation of recombinant virus v220i. Recombinant viruses were generated essentially as previously described (36) with minor modifications. Briefly, Vero cells were infected with virus vGUSREP (24) and transfected with plasmid pIND1.pp220 or pIND2.pp220 in the presence of different concentrations of IPTG (isopropyl-ß-D-thiogalactopyranoside). At 48 h postinfection (hpi), the cells were harvested and the recombinant viruses were isolated by sequential rounds of plaque purification in the presence of IPTG. The inducer concentration at which virus production was maximal, 0.15 mM, was chosen for the growth and purification of the recombinant virus. Similar results were obtained with the two plasmids used, and one virus clone from the pIND2.pp220-transfected cells was selected for further characterization. The genomic structure of this recombinant virus, named v220i, was confirmed by DNA hybridization analysis.
Plaque assays. Vero cell monolayers, in six-well plates, were infected with 600 PFU of recombinant v220i or parental BA71V. After 1 h, the inoculum was removed and the cells were overlaid with DMEM containing 0.6% Noble agar and 2% FCS in the presence or absence of 0.15 mM IPTG. Five days later, the medium was removed and the monolayers were stained with 1% crystal violet.
One-step virus growth curves. Vero cell monolayers, in 24-well plates, were infected with 5 PFU of recombinant v220i or parental BA71V per cell. After a 1-h adsorption, the cells were incubated in DMEM supplemented with 2% FCS. IPTG (0.15 mM) was added immediately after the adsorption period or at 12, 18, or 24 hpi. Infected cells with their culture supernatants were harvested at different times postinfection and sonicated, and titers were determined by plaque assay in the presence of 0.15 mM IPTG.
Immunoprecipitation of polyprotein pp220 from extracts of v220i-infected cells. Vero cells were mock infected or infected with 10 PFU of BA71V or v220i virus per cell in the absence or in the presence of 0.15 mM IPTG. The cells were pulse-labeled from 12 to 18 hpi with 500 µCi of [35S]methionine-[35S]cysteine (Promix in vitro cell labeling mix; Amersham Pharmacia Biotech) per ml. After labeling, the cells were lysed and immunoprecipitated with anti-pp220/p150 antibodies immobilized on protein A-Sepharose as previously described (6). Total extracts and immunoprecipitated proteins were resolved by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-12% PAGE) and detected by autoradiography. Quantification of protein bands was performed with a GS710 densitometer and Quantity One software (both from Bio-Rad Laboratories).
Purification and analysis of mutant v220i particles. A simplified version of the procedure described by Carrascosa et al. (12) was used to purify extracellular v220i particles from culture supernatants. Vero cells, cultured in roller bottles (500 cm2), were infected with 5 PFU of recombinant v220i per cell in the presence or absence of 0.15 mM IPTG. At 36 hpi, the culture media were centrifuged at 2,000 rpm for 10 min in a GS3 Sorvall rotor to remove cell debris, and the extracellular virions were concentrated by centrifugation in a GS3 Sorvall rotor at 8,500 rpm for 6 h at 4°C. Virus pellets were washed in phosphate-buffered saline, mixed with Percoll to a final concentration of 45% in phosphate-buffered saline, and subjected to equilibrium gradient sedimentation in a T865 Sorvall rotor at 20,000 rpm for 30 min at 4°C (12). Aliquots of the fractions were analyzed for virus content by negative staining with 2% uranyl acetate (4), for protein content by Western immunoblotting with anti-p150 and anti-p72 antibodies, and for DNA content by dot blot hybridization with an ASFV-specific probe. Fractions 2 to 8 of both gradients, containing virus particles essentially free of contaminant membrane vesicles, were subsequently pooled and gel filtered through a Sephacryl S-1000 (Amersham Pharmacia Biotech) column to remove Percoll particles (12).
For immunoblotting of v220i particles, samples were electrophoresed in SDS-12% polyacrylamide gels, transferred to nitrocellulose, and probed with the indicated antibodies against ASFV structural proteins. Protein detection was performed using peroxidase-conjugated antibodies and the ECL system (Amersham Pharmacia Biotech).
For dot blot hybridization analysis, equivalent amounts of the gradient fractions were diluted in 100 µl of H2O containing 100 ng of sheared salmon sperm DNA per ml and applied to nitrocellulose membrane (Protran [Schleicher & Schuell]; pore size, 0.2 µm) in a vacuum manifold. The DNA was denatured, neutralized, and bound to the membrane according to the manufacturer's protocol. The probe was the BA71V EcoRI fragment C' labeled with [32P]dCTP by random priming with a NEBlot kit (New England Biolabs). Hybridization was carried out at 65°C as described previously (35). The radioactive DNA signal was visualized by autoradiography.
For electron microscopy of purified v220i particles, virus pellets were fixed with 2% glutaraldehyde in 200 mM HEPES (pH 7.4) for 1 h at room temperature and processed for conventional Epon embedding as described below.
Electron microscopy. For conventional Epon section analysis, Vero cells were infected with 10 PFU of BA71V or recombinant v220i per cell in the absence or presence of 0.15 mM IPTG and fixed at the indicated times with 2% glutaraldehyde in 200 mM HEPES (pH 7.4) for 1 h at room temperature. Postfixation was carried out with 1% OsO4 and 1.5% K3Fe(CN)6 in H2O at 4°C for 30 min. Samples were dehydrated with acetone and embedded in Epon according to standard procedures.
For freeze-substitution, the v220i-infected Vero cells were fixed at the indicated times after infection for 1 h with 4% formaldehyde and 0.1% glutaraldehyde in 200 mM HEPES (pH 7.2) on ice. The specimens were cryoprotected with 30% glycerol for 30 min, rapidly frozen in liquid propane (-180°C), and stored in liquid nitrogen. Freeze-substitution and embedding in Lowicryl K4M were carried out as described previously (3, 4). Ultrathin sections were collected on nickel grids coated with Formvar and carbon and processed for immunogold labeling of ASFV proteins as described previously (3, 4). For DNA detection, sections were preincubated for 30 min at 37°C with 20 µg of proteinase K per ml in a buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 10 mM CaCl2. Labeling was performed as described previously (27) using a monoclonal antibody to DNA and a goat anti-mouse antibody coupled to 10-nm-diameter gold particles. Specimens were examined at 80 kV in a Jeol 1010 electron microscope. For quantification of the immunolabeling experiments, four to six electron micrographs of virus factories were analyzed for each condition at a final magnification of x75,000. Gold particles were counted on cross-sectioned virions.
Site-directed mutagenesis of gene CP2475L. Plasmid pGEM-CP2475L, containing the CP2475L gene under control of the T7 polymerase promoter, has been described elsewhere (5). Plasmid pGEM-CP2475L-G2A, in which the glycine residue at position 2 of ORF CP2475L was mutated to alanine, was obtained as follows. Site-directed mutagenesis was performed by PCR using plasmid pGEM-CP2475L as a template; the oligonucleotide 5'-GAGATgcatgcATGGCTAACCGTGGATCTTCAACC, which contains a point mutation (underlined) and an SphI restriction site (lowercase), as the upstream primer; and the oligonucleotide 5'-GGTGGAACCGCTCGAGGATCGGGCAG as the downstream primer. The resulting amplified fragment was digested with SphI and EcoRV and cloned into the SphI- and EcoRV-digested plasmid pGEM-CP2475L. The presence of the G2A mutation within the resulting plasmid was confirmed by sequencing.
Transient expression in COS cells. COS-7 cells were transfected for 1 h at 37°C with 250 ng of DNA of the plasmids pGEM-CP2475L and pGEM-CP2475L-G2A per 105 cells by using Lipofectamine Plus reagent (Life Technologies) and following the manufacturer's indications. The transfected cells were then infected with 5 PFU of the recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase (22) per cell.
For metabolic labeling experiments, the cells were incubated from 3 to 6 hpi with 500 µCi of [35S]methionine-[35S]cysteine in methionine- and cysteine-free DMEM per ml or with 250 µCi of [3H]myristic acid (Amersham Pharmacia Biotech) in DMEM supplemented with 2% delipidated FCS per ml. After the labeling periods, the cells were lysed and processed for immunoprecipitation with an anti-pp220/p150 antibody.
Cell fractionation was performed at 15 h after infection as previously described (6). The expression of myristoylated and nonmyristoylated polyprotein pp220 was analyzed by Western immunoblotting of equivalent amounts of the different cellular fractions.
For electron microscopy, COS-7 cells were transfected with plasmid pGEM-CP2475L or pGEM-CP2475L-G2A and infected with 5 PFU of vTF7-3 per cell as described above. In some experiments, the cells were incubated in the presence of 40 µg of cytosine arabinoside, an inhibitor of vaccinia virus DNA replication and late protein synthesis, per ml. At 15 hpi, the cells were processed for Epon embedding or freeze-substitution.
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RESULTS |
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In a further approach, one-step growth curves for v220i virus were performed in the presence or absence of 0.15 mM IPTG. As shown in Fig. 1C, under permissive conditions the growth curve of recombinant v220i virus was similar to that obtained with parental BA71V virus. In the absence of inducer, the yields of v220i virus did not increase over time, remaining about 2.6 log units below those observed under permissive conditions from 24 to 48 hpi. In the same experiment, we also tested the ability of v220i grown for different times under nonpermissive conditions to produce infectious particles upon IPTG addition. As shown in Fig. 1C, the later the time of IPTG addition, the lower was the final virus titer obtained. When IPTG was added at 12 hpi, an early time for virus assembly (3, 10), the maximal virus yield, reached at 48 hpi, was about 60% of that observed for v220i grown under permissive conditions throughout the infection. When the inducer was added at 18 or 24 hpi, the maximal yields were 20 and 4% of those obtained with the control, respectively. Together, the results of the plaque assays and one-step growth curves indicate that recombinant v220i is an IPTG-dependent conditional lethal mutant.
Inducible expression of polyprotein pp220. To confirm that the expression of polyprotein pp220 can be regulated by inducer during v220i infections, cells infected with the recombinant under permissive or nonpermissive conditions were labeled with [35S]methionine-[35S]cysteine from 12 to 18 hpi, a period in which late gene expression is in progress. Mock- and BA71V-infected cells labeled under the same conditions were used as negative and positive controls, respectively. As shown in Fig. 2A, similar overall protein profiles were observed when parental BA71V and recombinant v220i infections were compared, with the exception that extra bands corresponding to the reporter ß-glucuronidase (GUS) and ß-galactosidase gene products were expressed in the cells infected with the inducible recombinant. As expected, during BA71V infections, synthesis and processing of polyprotein pp220 were equivalent in the presence and absence of IPTG. In contrast, during v220i infections, the pp220 expression level was drastically reduced when the inducer was not added. This inhibition became more evident when the extracts were immunoprecipitated with a serum against both the polyprotein pp220 and the mature structural protein p150 (Fig. 2B). Densitometric quantification revealed that under permissive conditions, polyprotein expression was about 150% of that determined for control BA71V infections, whereas under nonpermissive conditions, it was reduced to about 5%. In summary, these results show that the conditional lethal phenotype of recombinant v220i is related to the inducer-dependent expression of the viral polyprotein.
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Immunoelectron microscopy of intracellular defective v220i particles. To explore in more detail the consequences of pp220 repression for virus formation, we compared the composition of the defective v220i particles with that of the mature virions by immunoelectron microscopy. Cells infected with recombinant v220i under permissive or restrictive conditions were processed at 24 hpi by freeze-substitution followed by Lowicryl embedding. Ultrathin sections from both samples were then incubated with a repertoire of antibodies against major structural proteins followed by protein A-gold. Table 1 shows a quantification of the labeling density obtained for each antibody on mature icosahedral particles generated in the presence of IPTG and on defective virions (without a proper core) produced in the absence of inducer. Examples of the labeling patterns for some of these proteins are illustrated in Fig. 4. Antibodies against peripheral proteins, like the capsid proteins p72 (24) (Fig. 4A and B) and pE120R (6) or the transmembrane polypeptide p17 (41), labeled mature and defective core-less particles equally. In contrast, the labeling of proteins localized at the core shell, like the pp220-derived protein p150 (3) (Fig. 4C and D) or the pp62-derived protein p35 (42; our unpublished results), was practically absent in the defective v220i particles. Similarly, the detection of nucleoid markers, such as the DNA-binding proteins pA104R (8) (Fig. 4E and F) and p10 (34; our unpublished results), within defective core-less particles was considerably reduced. The exception was a very minor population of nucleoid-containing particles that probably result from the residual expression of the CP2475L gene under restrictive conditions.
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Purification and analysis of extracellular v220i defective particles. To further characterize the mutant v220i particles, we attempted to purify them from the supernatants of infected cells. Extracellular virions collected from the culture media of permissive and restrictive v220i infections were sedimented into an isopycnic Percoll gradient as previously established (12). Samples were previously adjusted to the same content of capsid protein p72 in order to analyze equivalent amounts of virus particles. Under permissive conditions, a cloudy band corresponding to the sedimentation peak described for infectious ASFV particles (12) was visible close to the bottom of the gradient. No distinctive band was detected in the virus samples obtained from nonpermissive infections. The gradients were then fractionated from the bottom, and each fraction was evaluated for virus content by negative staining (not shown) and by Western immunoblotting with antibodies against protein p72 and the pp220-derived protein p150 (Fig. 5A). In both samples, virus particles were found mainly in two broad zones: one close to the top of the gradient, which contained significant amounts of contaminant membrane vesicles, and the other close to the bottom, which was essentially free of membrane vesicles. A similar virus distribution has been described for parental BA71V virus (12). As shown in Fig. 5A, proteins p72 and p150 were also detected predominantly in the broad peaks of both gradients, whereas unprocessed pp220 precursor was detected only in the top peak containing contaminant membranes. As expected, proteins pp220 and p150 were only weakly detected in the sample obtained under restrictive conditions. Next, we analyzed the gradient fractions for viral DNA content by dot blot hybridization with a specific ASFV DNA probe. As shown in Fig. 5B, under permissive conditions, the viral DNA was detected essentially in fractions 3 to 5, which correspond to the band of infectious virus particles. Under restrictive conditions, DNA hybridization was also maximal around fractions 3 to 5, but the intensity of the signal, as judged by densitometric quantitation, was significantly (5 to 6 times) lower than that of the positive control. This finding supports the conclusion that recombinant v220i is a DNA-deficient mutant.
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Next, we analyzed the protein composition of the purified defective v220i particles by Western immunoblotting with a spectrum of antibodies against major ASFV structural proteins. Figure 5D shows the results of a comparison between the mutant and control particles pooled from fractions 2 to 8 of the Percoll gradients. Some ASFV proteins in samples of control mature particles obtained under permissive conditions and collected only from the position where the infectious virus particles band (fractions 3 to 5 of the Percoll gradient) were also analyzed. When the virus samples were adjusted to the same p72 content, other peripheral major proteins, such as the capsid protein pE120R and the transmembrane polypeptides p17 and p12 (1, 6, 13, 24, 41), were detected equally in control and mutant particles. In contrast, the analysis of major core components revealed marked differences between the preparations. Thus, the presence of the mature products derived from polyproteins pp220 (p150, p37, and p34) and pp62 (p35 and p15), as well as the proteinase pS273R, all located at the core shell (3, 5; unpublished data), drastically decreased in the defective v220i particles. Similarly, the levels of the DNA-binding proteins p10 and pA104R (8, 34) were significantly reduced. The differences in these two proteins were even more pronounced when the defective v220i particles were compared with the control particles recovered from the band of infectious particles. This observation probably reflects the fact that the infectious virus band contains essentially mature virions (12), whereas the control sample obtained from fractions 2 to 8 of the Percoll gradients contains a higher proportion of immature virions and therefore a lower content of nucleoid proteins.
In conclusion, the analysis of purified extracellular mutant v220i particles, together with the data obtained from immunoelectron microscopy of intracellular defective virions, indicates that pp220 repression blocks the incorporation of other major components of the core shell and the nucleoid, including the viral genome, into the virus particles.
Effect of pp220 postinduction on virus assembly. Our next goal was to study the effect of IPTG addition on virus assembly in v220i-infected cells previously maintained under restrictive conditions. Figure 6 shows an electron microscopy analysis of v220i-infected cells maintained during 12 h in the absence of IPTG or incubated with the inducer at 12 hpi for an additional 12-h period. In the first case (Fig. 6A), the assembly areas were relatively small and contained mainly precursor membranous structures as well as icosahedral empty particles, with a significant proportion of them being open. After the induction with IPTG (Fig. 6B), the viral factories increased notably in size and, concomitantly, large amounts of both mature and immature icosahedral particles were observed. The proportion of mature virions was, however, clearly lower than that observed in v220i infections maintained during 24 h under permissive conditions (Fig. 3A), which is consistent with the strong reduction (fivefold) observed in the infectious virus yield (Fig. 1C). Concerning the immature virions, a close inspection revealed that they often appeared to contain a distinctive matrix-like domain underneath the viral envelope (Fig. 6C). Immunoelectron microscopy with an antibody against the polyprotein pp220 confirmed that both mature and developing particles were strongly labeled at the core shell (Fig. 6D). Moreover, specific pp220 labeling was clearly detected in open icosahedral virions in which the core shell appeared to be partially assembled (Fig. 6D, inset). In contrast, no gold particles were found within apparently closed, core-less virions, which were likely formed during the initial 12-h period under restrictive conditions (Fig. 6D). When IPTG was added at later times of infection (from 18 or 24 hpi onwards), the proportion of mature virions decreased and, concomitantly, the number of core-less particles increased (not shown). These findings suggest that core formation occurs when the developing particles are still open. We therefore interpret the partial restoration of infectivity observed after IPTG addition (Fig. 1C) to be a consequence of the de novo assembly of mature virions from precursor membranes, although we cannot completely rule out that it might be due to a further maturation of the preformed icosahedral core-less particles.
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Transient expression of pp220 in COS cells. It has previously been reported that polyprotein pp220 is N myristoylated (40). Since myristoylation may serve as a membrane anchoring signal, we studied whether precursor pp220 associates with membranes through its myristic moiety. Transient-expression experiments were performed with COS-7 cells transfected with a plasmid containing the CP2475L gene under control of the T7 bacteriophage promoter and subsequently infected with a recombinant vaccinia virus expressing the T7 RNA polymerase (23). A mutant nonmyristoylable version of polyprotein pp220, in which the N-terminal Gly residue was replaced by Ala, was also expressed. As a negative control, cells were transfected with the empty plasmid and infected with the recombinant vaccinia virus. The transfected cells were labeled with [35S]methionine-[35S]cysteine or with [3H]myristic acid from 3 to 6 hpi, and the extracts were analyzed by SDS-PAGE or immunoprecipitated with an antibody to pp220. Both forms of the ASFV polyprotein were expressed in similar amounts (Fig. 7A) and were efficiently immunoprecipitated from the 35S-labeled extracts (Fig. 7B), but only the wild-type polyprotein was detected after immunoprecipitation of cell extracts labeled with [3H]myristic acid (Fig. 7C). In a second experiment, the transfected cells were homogenized and fractionated into a low-speed (500 x g) sediment and a postnuclear supernatant, which was, in turn, fractionated into a high-speed (100,000 x g) sediment and a supernatant cytoplasmic fraction. Surprisingly, both the myristoylated and the nonmyristoylated forms of pp220 were detected almost exclusively in the low-speed sediment. Since the fractionation experiment may not distinguish between sedimentation due to membrane binding and that due to protein aggregation, we performed electron microscopy of the transfected cells (Fig. 8). Inspection of cells expressing the wild type polyprotein revealed a great accumulation of electron-dense structures associated with the cytosolic sides of a variety of cellular membranes. These pp220-induced membrane-bound coats covered large extensions of the plasma membrane (Fig. 8A, B, and D) or virtually enwrapped intracellular cisternae (Fig. 8A and C), preferentially from the endosomal-lysosomal system. Interestingly, many images (Fig. 8B) of the cross-sectioned coats exhibited a regular thickness (about 30 nm) similar to that of the aberrant zipper-like viral structures (Fig. 6B, E, and F). Equivalent pp220-induced structures were observed in the presence of cytosine arabinoside, a drug that inhibits DNA replication and consequently the morphogenesis of vaccinia virus (not shown). To confirm that these structures were composed of polyprotein pp220, Lowicryl sections of freeze-substituted cells were incubated with an anti-pp220 antibody followed by protein A-gold complexes. As shown in Fig. 8C and D, the electron-dense membrane coats were intensely labeled. On the other hand, the transfected cells expressing the nonmyristoylable polyprotein showed an accumulation of large electron-dense aggregates at the cytoplasm that did not apparently associate with membranes (Fig. 8E). In fact, an extensive examination of the membrane profiles of the cell surfaces did not reveal any structure comparable to those described above. As shown in Fig. 8F, immunoelectron microscopy with an anti-pp220 antibody showed strong labeling on the cytoplasmic aggregates. Thus, the results of the fractionation experiment can be explained by the formation of dense membrane-bound coats by the wild-type polyprotein and of dense cytoplasmic aggregates by the mutant pp220.
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DISCUSSION |
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Analysis of recombinant v220i led to two important findings: (i) the polyprotein pp220 is essential for the core assembly, and (ii) the formation of icosahedral particles is not dependent on core maturation. Under nonpermissive conditions, v220i was essentially unable to form lysis plaques, and its infectious virus yield was about 2.6 log units lower than that obtained in control infections. At the electron microscopic level, repression of pp220 synthesis caused a dramatic effect on virus morphogenesis. Intracellular mature virions were rarely found within the virus factories, whereas large quantities of icosahedral particles essentially devoid of the characteristic electron-dense nucleoid and the surrounding core shell were observed. Interestingly, the assembly of the outermost viral domains, i.e., the inner envelope, the capsid, and the outer envelope, was not apparently affected under restrictive conditions. Indeed, the defective icosahedral particles were efficiently released from the plasma membrane, as occurs with the mature virions in normal infections. This mutant phenotype is consistent with the localization of the pp220-derived proteins p150, p37, p34, and p14 at the core shell, a matrix-like domain placed between the internal DNA-containing nucleoid and the inner viral envelope (3). According to our working model for ASFV morphogenesis, based mainly on the ultrastructural analysis of viral intermediates from parental BA71V infections, the core assembly takes place, at least in its initial stages, within the developing icosahedral particles while they are still open (3). The first stage of this sequential maturational process would be the progressive construction of the core shell underneath the inner lipid envelope. The subsequent uptake of the DNA together with the viral nucleoproteins would lead to the formation of the nucleoid, whose final condensation might take place once the virus particles are already closed (3, 11). From this model, it seems conceivable that a blockage in the assembly of the core shell will have major effects on the subsequent steps of core maturation, as occurs with recombinant v220i. In support of this view is the fact that the defective v220i particles lack not only the pp220-derived proteins but also other examined major core proteins from the core shell (p35 and p15) and the nucleoid (p10 and pA104R). Moreover, the mutant v220i virions were shown to be DNA-deficient particles. This finding, together with the absence of preformed nucleoids or highly dense DNA-containing structures within both normal (3) and mutant v220i (this report) viral factories, is also consistent with the idea that DNA condensation and nucleoid maturation occur within the developing particles and depend on the previous assembly of the core shell. It therefore seems likely that the core-less v220i particles produced under nonpermissive conditions are defective virions irreversibly arrested in their assembly rather than maturation intermediates.
Collectively, our findings show that polyprotein pp220 is directly or indirectly involved in the incorporation of other major core components, including the viral genome, into the virus particles. An interesting question that remains to be addressed is the specific role of polyprotein processing in core assembly. It is tempting to speculate that proteolytic processing might be an essential mechanism for the patio-temporal control of the interactions between the core proteins. Thus, only properly processed pp220 precursors would enter the assembly pathway and lead to the subsequent incorporation of other core components like the nucleoid proteins and the viral genome. In relation to this, it should be noted that other complex DNA-containing viruses, such as the adenoviruses and the poxviruses, use proteolytic processing at cleavage sequences similar or identical to those of ASFV for the maturation of their core precursors. In the case of vaccinia virus, conditional lethal mutations of several genes were shown to affect both the cleavage of the precursor proteins and the assembly of the core (16, 21, 27, 28, 29). A remarkable example is the temperature-sensitive mutant ts16, which exhibits a blockage in the maturation of the core structure and in the proteolytic processing of the core precursors P4a, P4b, and L4 but not in DNA uptake (21, 28). Interestingly, the ts16 mutation maps to the I7 gene (21), which encodes a core protein having some similarity to the recently identified SUMO-1-specific proteases as well as to the proteinases of adenovirus and ASFV (30). It will be of interest to know whether the repression of ASFV proteinase has consequences for core assembly similar to those reported for vaccinia virus mutant ts16 or to those described here for ASFV recombinant v220i.
Besides proteolytic processing, the other major modification of polyprotein pp220 is the covalent addition of myristic acid to its N-terminal glycine residue (40). Using a COS cell expression system, we found that polyprotein pp220 assembles into large membrane-bound coats somewhat reminiscent of the core shell of the mature virus particles and to the aberrant zipper-like viral structures (3, 4, 24). In contrast, a mutant nonmyristoylated version of the polyprotein did not associate with membranes but formed large cytoplasmic aggregates. These data strongly suggest that N myristoylation functions as a membrane-anchoring signal and that polyprotein pp220 possesses an intrinsic capability of self-assembly into a higher-order structure. Both properties could be exploited during ASFV morphogenesis for the assembly of the viral core. Polyprotein pp220 could bind to the inner viral envelope via the myristoyl moiety and multimerize underneath the lipid layer to construct the protein scaffold of the developing core shell. As described above, the proteolytic processing of precursor pp220 might trigger the interactions required for the subsequent incorporation of additional core components. In relation to this, the possible role of polyprotein pp220 as a matrix-like linker protein is similar to that described for the matrix proteins of other enveloped viruses (25). For example, some retroviral Gag polyproteins use N myristoylation for targeting to the plasma membrane and are able to multimerize to produce virus-like particles (26). Interestingly, while polyprotein pp220 preferentially associates with ER-derived viral membranes in ASFV infections (3, 4), it is bound predominantly to the plasma membrane and endosome-lysosome membranes when expressed in COS cells. This observation suggests that, as occurs for other myristoylated proteins, proper membrane targeting of polyprotein pp220 involves other factors besides its acylation.
In summary, this report shows that polyprotein pp220 is essential for the assembly and envelopment of the core shell and for the subsequent steps of core maturation, including DNA encapsidation and nucleoid condensation, but that it is not required for the construction of the external viral domains. The generation of icosahedral virus-like particles by using core-deficient mutants like the one described here may facilitate the identification and characterization of the proteins that compose the viral envelopes and the capsid and may also provide a useful tool for the investigation of effective vaccines against ASFV infection.
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ACKNOWLEDGMENTS |
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This study was supported by grants from the Dirección General de Investigación Científica y Técnica (BMC 2000-1485), the European Community (FAIR-CT97-3441), and the Ministerio de Educación y Cultura (AGF98-1352-CE) and by an institutional grant from Fundación Ramón Areces. G. Andrés was supported by a fellowship from Comunidad Autónoma de Madrid.
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