VIROLOGY 181, 251-257 (1991)

Protein p22 of African Swine Fever Virus: An Early Structural Protein That Is Incorporated into the Membrane of Infected Cells

ANA CAMACHO AND ELADIO VI&JELA’

Centro de Biologia Molecular (CSIC-UAM), Facultad de Ciencias, Universidad Autdnoma. Canto Blanco. 28049 Madrid, Spain

Received April 16. 1990; accepted November 13, 1990

The open reading frame K’177, located at the left end of the African swine fever virus genome, codes for an early induced structural protein of 22,000 Da (~22). which is released from the viral particle by the nonionic detergent n-octyl-g-o-glucopyranoside under conditions that solubilize external viral structural proteins. The predicted amino acid sequence of the protein contains a hydrophobic region at ifs N-terminus with characteristics of a signal peptide and, at early times after virus infection of Vero cells, the protein can be detected in the cell membrane by immunolabeling. 0 1991 Academic Press, Inc.

INTRODUCTION

African swine fever (ASF) virus is responsible for sig- nificant morbidity and mortality of domestic swine in countries where ASF is an endemic disease (Becker, 1987). A striking aspect of the ASF virus infection is that no neutralizing antibodies against the virus have been detected so far in recovered or chronically in- fected pigs, although serum from these animals is able to fix complement (De Boer 1967). If an effective im- mune response against ASF virus infection exists, it should be directed against external virion proteins or membrane polypeptides induced early after infection.

To identify viral proteins that may be involved in pro- tective immunological reactions, we have searched those DNA regions coding for early transcripts (Salas et a/., 1986). One of these regions, a segment of 10 kb, located at the left end of the virus genome, was se- quenced and the open reading frames (ORFs) were de- fined (Gonztilez era/., 1989) (see Fig. 1 a). In the present study, we report the identification and characterization of the protein encoded in ORF K’177, which is an early, structural protein of apparent molecular weight 22 kDa, located externally in the viral particle. The protein contains a hydrophobic region at the N-terminus with the characteristics of a signal peptide and seems to appear transiently in the plasma membrane early after ASF virus infection.

The K’177 DNA sequence and corresponding amino acid se- quence have been deposited in GenBank and under Accession No. M57546.

’ To whom reprint requests should be addressed.

MATERIALS AND METHODS

Cells and virus

Vero cells (CCL8 1) were obtained from the American Type Culture Collection and grown in Dulbecco’s modi- fied Eagle’s (DME) medium supplemented with 5%

newborn calf serum. The origin and properties of Vero-adapted ASF virus

BA71V strain, as well as virus growth and titration in Vero cells, have been described (Enjuanes et a/., 1976). The inoculum used in this study was extracellu- lar virus concentrated by high-speed centrifugation. Cultures of 5 X 1 O4 Vero cells/cm2 were infected with ASF virus at a multiplicity of infection of 20 PFU per cell. The infection was performed in DME medium supple- mented with 2% calf serum with an adsorption period of 2 hr at 37’. Virus purification was as described (Carrascosa et a/., 1985).

Plasmids

Plasmid pUC8HH 10, obtained from A. Talavera, is a pUC8 recombinant with the HindIll H restriction frag- ment of ASF virus DNA (containing the DNA sequence within nucleotides 2900-6800), where the ORF K’177 is included. pUC8HHI 0 was restricted with Accl, which cuts at position -54 of gene K’177 (Gonztilez et al., 1989) and at the multiple cloning site of pUC8. At position -38 there is a TGA termination codon that was removed by treatment with 429 DNA polymerase for 3 min at 25”, due to its 3’+ 5’exonuclease activity (Blanc0 and Salas, 1985) and with mung bean nu- clease for 30 min at 30”. After restriction with Xbal,

251 0042-6822/91 $3.00 Copyright B 1991 by Academic Press. Inc All rights of reproduction in any form reserved.

252 CAMACHO AND VIhJELA

which cuts 56 nucleotides from the termination triplet of gene K’177, the latter, also containing 32 bp up- stream the initiation triplet, was cloned, in phase, at the C-terminus of the /acZ gene of fscherichia co/i plasmid pEX-2 (Stanley and Luzio, 1984). The recombinant was named ~~117-14.

Immunologic reagents

Sera directed against the structural proteins ~17, ~37, and p35 separated by gel electrophoresis and serum directed against purified ASF virus particles were obtained from A. L. Carrascosa and are de- scribed elsewhere (A. L. Carrascosa and E. Viiluela, in preparation). ‘251-labeled goat anti-rat immunoglobu- lins were purchased from Amersham, International.

Polyvalent serum directed against the product of ex- pression of gene K’177 was prepared by immunizing rats with the 9 M urea-treated protein pellet of Esche- n’chia co/i harboring the recombinant plasmid ppl77- 14 and control serum with the corresponding treated protein pellet off. co/i harboring pEX-2 plasmid. lmmu- nization was achieved with four doses of 20 pg of pro- tein in 150 ~1 of PBS (phosphate saline solution) per rat, with an equal volume of complete Freund’s adjuvant in the first injection, incomplete adjuvant in the second, and two more injections with antigen alone. Each serum was titrated by enzyme-linked immunoabsor- bent assay (ELISA) (Voller et al., 1980).

Western blotting assays

Approximately 100 pg of purified virus or 2 mg of protein from infected or uninfected cells was loaded in a 5.5-cm well of a polyacrylamide gel in the presence of SDS (SDS-PAGE) according to Laemmli (1970). After separation of the proteins, the gels were blotted to a Millipore polyvinylidene difluoride (PVDF) membrane (0.45~pm pore size) as described elsewhere (Souter and Wate, 1989). Strips of about 0.3 cm were cut, satu- rated with 0.1% skim milk in 20 mM Tris-HCI, pH 7.5: 0.5 M NaCl (TBS), and incubated for at least 4 hr with the first antibody diluted 1: 10 in TBS. Strips were washed with TBS containing 0.05Ob Tween 20 and in- cubated for 1 hr with the appropriate anti-rat peroxi- dase or alkaline phosphatase-conjugated secondary antibody, diluted 1:500 or 1 :lO,OOO in TBS: 0.25% BSA. After washing with TBS: 0.05% Tween, strips were developed as described by Garfin and Bers (1989).

lmmunolabeling of ASF virus-infected Vero cells

Cultures of 1 O5 Vero cells were mock-infected or in- fected with virus. At 2.5, 4, 5, 6, 7, and 8 hr postinfec- tion the cells were fixed with 1% formaldehyde in PBS,

permeabilized or not with cooled acetone, and incu- bated with antifusion protein or anti-p37 sera at a 1: 10 final dilution in PBS: 0.25% BSA for 3 hr at 37”. The cells were then rinsed with 0.259/o BSA in PBS and incubated with 0.1 &i/l O5 cells of lz51-rabbit anti-rat immunoglobulins for 45 min at room temperature. After extensive rinsing with 0.259/o BSA: 0.05% Tween 20 in PBS, 2% SDS was added to the wells and the radioac- tivity was counted.

Computer analysis

Hydropathy profiles were obtained following the pro- cedure of Kyte and Doolittle (1982). Routine analysis of DNA and protein sequences was carried out with the software package of the University of Wisconsin Genet- ics Computer Group (UWGCG, Devereux et al., 1984). Protein data base searches were done using the FASTA package (Pearson and Lipman, 1988) or the FIND program of the UWGCG package.

RESULTS

Predicted amino acid sequence, hydropatic plots, and secondary structure of the protein coded by ORF K’177

The ORF K’177 is located between nucleotides 4877 and 5410 at the left end of the ASF virus genome (Fig. la). The 534 nucleotides of the coding strand are shown in Fig. 1 b, beneath the predicted amino acid sequence of the translation product. Assuming that the translation starts at the first ATG in the ORF (nu- cleotides 4877-g), the corresponding methionine is followed by 176 amino acids, yielding a core protein of 20,143 Da.

The presence of 10 cysteines in the protein should be noted, 5 of them within the motif CX,CX,CX,CX,C (amino acid positions 39-61). A search at the NBRF sequence data bank of this motif did not render any significant similarity to known proteins,

Another distinctive feature, confirmed by hydropathy analysis (Fig. 2), is the hydrophobic N-terminal se- quence with the characteristics of a signal peptide (von Heijne, 1985) (Fig. 1 b): (i) five amino acids (positions 1 to 5) at the N-terminus with a net positive charge, which correspond fairly well with the n-region of a sig- nal peptide; (ii) a hydrophobic stretch of 22 amino acids (positions 6-27) similar in size and composition to the h-region of a signal peptide; and (iii) a positively charged region (positions 28 to 37) which differs from the cleavage c-region of a signal peptide because the rule -l ,-3 (von Heijne and Gavel, 1988) is not fol- lowed. No other significant hydrophobic region was detected in the protein sequence (Fig. 2).

ASF VIRUS PROTEIN p22 253

3

0 2 Ir 6 8 10 I 1 I I I I I I I I ' kb

RK' 1 RL t I

TIR K'360 KY62 K'177 L356 L270

' n h &:i:i---x: n T I s T L L I A L I I L " I A~MTA~~~M~ACA~A~A~ATA~A~A~ 60

V . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 1 1 L v v F L~.~..Y..I(..LQ..Q..P..P..LK:v c K

ATTAl-lTl’-AmATAmAT AAAAMCMCMCCAO3AAAAA~T?~ 120

VDKDIG~CEHFVRGTZSTLS GTAGATMAGA'FIGDXT-mA- GTGGAACATCCAGCACA'Tl,XGC 180

:LDAVKIDKRNIKIDSKISS ~~CCCG~TA~-ATAGA~-A~~A 260

:EFTPNFYRFTDTAADEQQE XTGAAl.lTA~CCAA~AC CGTFITACGGATA- CGA'IGAWACCMOM 300

FGKTRHPIKITPSPSESHSP TlWGAMM-lCC~CA~CAA~TCCCATAGCCCC 360

Q E V C'E K Y: S W G TddAAEAG W E Y CMmTAmAm -TAT 620

VCDEKECT:YVYNNPHHPVL C~ACA~ACCC~ 680

KYGKDHIIALPRNHKHA AAATAI-XTMGGAWACA~T AGCClTACCTAGAAA’EATAAACATGCATM

FIG. 1. Location of the gene in ASF virus genome and amino acid sequence of protein ~22. (a) ORFs on the left-end region of ASF virus DNA. Arrows represent the proteins, with arrowheads at the C-ter- minus. Proteins are named by the letter of the EcoRl restriction frag- ment where their initiation codons are included, followed bythe num- ber of amino acids (Gonzalez et al., 1990). TIR, terminal inverted repeats; RK’ and RL, EcoRl K’ and L restriction fragments, respec- tively. (b) Nucleotide sequence of the coding strand of the ORF K'177

and predicted amino acid sequence. Cysteines are indicated by a closed circle. The signal peptide is boxed, the n-region is marked by a dashed box, the h-region with a continuous line box, and the hydro- philic domain with a dotted-line box.

Expression in E. co/i of the K’177 encoded polypeptide

To define the expression product of ORF K’177, the corresponding DNA sequence was cloned in plasmid pEx-2 as described under Materials and Methods. The viral sequence was placed in phase at the C-terminal region of the cro-lac-Z gene giving the recombinant ~~177-14. The expression of P-galactosidase was in- duced by heat shift as described by Stanley and Luzio (1984). As can be seen in Fig. 3, E. co/i harboring the recombinant ~~177-14 produced a 140-kDa protein in contrast to the 1 16-kDa ,&galactosidase expressed by

the control pEx-2 plasmid. This 140-kDa protein corre- sponds to the 1 16-kDa bacterial gene product plus 214 amino acids expressed from the coding viral se- quence and upstream region.

In an attempt to purify the fusion protein, pellets con- taining the 140-kDa protein, obtained after sonication of E. co/i cells harboring ~~177-14, were treated with different concentrations of urea in 100 mM Tris-HCI, pH 9.0. After 9 M urea treatment, about 40% of the total 140-kDa protein was found in a purified form in the pellet fraction (Fig. 3). These preparations were used to raise serum directed against the product of gene K’177.

Characterization and location in infected cells and viral particles of the protein encoded by gene K’177

As shown in Fig. 4, serum directed against the fu- sion protein recognizes a specific protein band of about 22 kDa in virus-infected cells, while no corre- sponding band was detected by the serum in mock-in- fected cells or in infected cells when control serum against P-galactosidase (&gal) was used. The protein is a structural component of the virus since it is also detected with the antibody in purified virions (Fig. 5 lane d). By reaction of the virion proteins with a mix of sera against structural proteins of 37 kDa (~37) and 17 kDa (~17) and the anti-fusion protein serum (Fig. 5 lane e) a protein of 22 kDa was detected, in addition to the 37- and 17-kDa proteins. Thus, the product of ORF K’177, with a molecular weight of 22 kDa relative to the 37- and 17-kDa proteins, was named ~22.

Protein p22 is detected in the absence and in the presence of cytosine arabinoside (AraC) indicating that it is an early induced protein (Fig. 4). Although the band is less intense in the presence of the inhibitor, no virus was produced in the AraC-treated cultures and the late structural proteins ~150, ~72, and ~37, recognized by serum against ASF virus structural proteins (aASFV), were not detected. Only a specific band of about 14 kDa is present, which corresponds to an early induced, but otherwise uncharacterized, protein.

In an attempt to determine the location of p22 in the viral particles, purified virus was treated with octylglu- coside and the soluble and insoluble protein fractions were analyzed by Western blots. In Fig. 5, lane g shows that p22 was solubilized to an extent similar to that of the external viral protein ~35, while the internal struc- tural protein p37 was present only in the pellet fraction, as previously shown by A. L. Carrascosa and E. ViAuela (manuscript in preparation). These results sug- gest that p22 is externally located in the virus particle. The amount of p22 in virions, calculated by densitome- try of an autoradiogram of virus labeled with a mixture of 14C-amino acids, is less than 0.3% (data not shown).

254 CAMACHO AND VIkJELA

Amino acid HOOC

(1 Hydrophiliclty >=I.3

6 Hydrophobicityk1.3

FIG. 2. Predicted secondary structure and hydropathy of protein ~22. (a) The secondary structure was predicted by the method of Chou and Fassman (1978). a-helices are Indicated by a sinusoidal wave, P-sheets by a saw-tooth wave, random cotls by a dull saw-tooth wave, and p-turn regions by line turns of 180”. Hydrophilic regions according to Kyte and Doolitle (1982) are indicated by octagons and hydrophobic regions by diamonds. (b) Hydropathic plot with a 1:7 window.

The external location of p22 in the viral particle led us to study a possible relation of the protein to the cell membrane. During the first 6 hr of the virus infection, most p22 could be immunolabeled without permeabil- ization of the cells, suggesting that it is associated ex- ternally to the plasma membrane (Fig. 6a). Afterward, although the synthesis of p22 continues in a linear way, the label bound to unpermeabilized cells clearly decreases, suggesting that p22 is somehow masked, blocked, or internalized (Fig. 6a). No plasma mem- brane label was detected in control-infected cultures incubated with serum directed against the late struc- tural protein p37 (Fig. 6b). Although the background of mock-infected cells is quite high, the experiment has been repeated three times with the same results.

In agreement with this transient detection of p22 in the cell surface, when intact infected cells were briefly treated with trypsin, the protein seemed to be suscepti- ble to the protease at 6 hr postinfection, while after 7.5 hr most of the protein remained undigested (Fig. 7).

DISCUSSION

The ORF K’177, located between nucleotides 4877 and 5410 at the left end of the ASF virus genomic map, has been expressed in E. co/i as a protein fused to the C-terminus of P-galactosidase. With the partially puri-

fied fusion protein (Fig. 3) we have raised a serum able to recognize the product of the ASF virus gene K’177.

The gene product, with an apparent molecular weight of 22 kDa in SDS-PAGE, is induced early after infection and has been characterized as one of the structural components of the purified virus particle re- leased from Vero cells. The protein can be solubilized from these particles by octylglucoside treatment, under conditions that release only external proteins, indicating that p22 is externally located in the viral par- ticle, or otherwise associated with the external poly- peptides released by the detergent.

Virion external proteins are frequently glycoproteins, and their corresponding precursors contain a signal sequence at its N-terminus to initiate the export of the nascent chain across the rough endoplasmic reticu- lum, and a strong hydrophobic domain near its C-ter- minus to anchor the protein in the plasma membrane (Sabatini eta/., 1982). ASFvirus, however, has an atypi- cal envelope where no glycoproteins have been de- tected so far (Val et al., 1986). When a hydropathic plot of the p22 protein was carried out, a single hybropho- bit sequence of 22 amino acids was apparent at the N-terminus (Figs. 1 and 2). However, the C-terminus of the protein not only does not contain a hybrophobic domain, but is one of the most hydrophilic segments of p22.

ASF VIRUS PROTEIN p22 255

P cu rl ’ 7 1:

UREA a : OM 5M 9M -- P s P s P SP SP

KDa

140-

116-

FIG. 3. Expression and purification of the @-gal-p22 fusion protein. The pEx vectors were used in the f. co/i strain pop 2136 containing the clts857 repressor. The plasmids were amplified at 30” and then transient expression was induced shifting the cultures at 42” for 90 min. Cells suspended in phosphate buffered saline were sonicated and centrifuged in a Hettich microfuge. Supernatants (s) and pellets (p) corresponding to 1 ml of the initial culture were run in SDS-PAGE to characterize the fusion product. To purify the protein, after sonica- tion and centrifugation, three pellets with the proteins corresponding to 1 ml of initial culture were treated with 0, 5, and 9 M urea in 0.1 M Tris-HCI, pH 9, for 2 hr at room temperature. After this treatment, the samples were centrifuged and supernatants (s) and pellets (p) were run in 7% SDS-PAGE. Gels were stained with Coomassie blue R-250.

The N-terminal hydrophobic region of p22 has char- acteristics of a signal peptide (von Heijne, 1985): The positively charged amino terminus n-region of five resi- dues, a central hydrophobic h-region of 22 residues, large enough to be a membrane-spanning segment, and a polar carboxy-terminal c-region.

A signal sequence can be cleaved upon transloca- tion or can remain in the polypeptide chain (von Heijne, 1988). When the Folz and Gordon (1987) computer pro- gram was applied to the amino acid sequence of p22 it gave only one positive value for probability of cleavage, between residues 27 and 28, but this value was low.

Although the N-terminal sequence of the p22 protein present in virions has not been directly determined so far, due to the low virus production in infected cells which makes it difficult to obtain sufficient amounts of this minor structural component, the similarity be- tween the theoretical molecular weight of 20,143 D

deduced from the amino acid sequence and that of 22,000 D obtained by SDS-PAGE for the protein in the viral particle (not the 16,000 D protein expected if the signal peptide were cleaved) suggests that the N-ter- minus might remain in the mature protein. Moreover, the findings that (i) p22 is probably a viral external pro- tein, (ii) it seems to appear transiently at the cell surface early after virus infection, and (iii) the only significant hydrophobic sequence is the N-terminal one lead to the idea that the signal peptide of p22 may be an an- chored transmembrane domain. This may be a situa- tion analogous to that found in a small group of trans- membrane proteins (Clement, 1983; von Heijne and Gavel, 1988) of which the Influenza virus neuramini- dase (NA) is one of the best studied examples. NA protein has a signal peptide at its N-terminus that is not cleaved from the mature protein. This peptide provides the signal function in translocation across membranes

fusidn Serum ASF prot. Rgal --- ASFV ++ ++- ++ Ara C --f -f- -+

pl50-

p 72 -

p 37 -

p12-

FIG. 4. Western blotting of proteins from Vero cells infected in the presence or in the absence of AraC and from mock-infected cells. About 40 pg of proteins per lane from cells infected in the absence or in the presence of 40 pg/ml of AraC, collected at 24 hr postinfection, or from mock-infected Vero cells, was separated by electrophoresis in a 15% polyacrylamide gel, transferred to immobilon membrane, and incubated with serum against virus structural proteins (aASFV), anti-Sgalactosidase (&gal) sera or anti-fusion protein serum. The background present in the strip incubated with the serum directed against viral particles is due to an overexposition with the developer, in order to better detect all proteins.

256 CAMACHO AND VINUELA

-0G treatement +OG treatement

obcde LA!- h PS PSPS

FIG. 5. Western blots of virions and octyl-P-D-glucopyranoside (OG)-treated virions. One hundred micrograms of virion proteins was treated or not with 0.5% OG in PBS for 1 hr at 4”. Solubilized (s) and insoluble (p) proteins separated by a 150,000 g centrifugation were resolved in 7 to 20% polyacrylamide gradient gels. Polypeptides were transferred to an immobilon membrane and the strips were incubated with control serum anti-p gal (a); serum antl-p37 (b); serum anti-p17 (c); serum anti-fusion protein (d, g); a mixture of anti-p37, anti-p17. and anti-fusion protein sera (e, f); or serum anti-p35 (h). Protem concentration per lane is about 10 pg.

(Bos et a/., 1984) and remains embedded in the mem- brane serving as an anchor (Block et a/., 1982). If p22 is anchored in the plasma membrane by the N-terminal hybrophobic region, according to the von Heijne theory for proteins with uncleaved signal peptide (von Heijne, 1988), the amino terminus should be found in the cyto- plasm and the carboxyl one externally located.

0

/”

r t

J’ I I I

I I

P- -0’ b

/ /

I I I I / I- ArA*A 2 4 6 6 2 4 6 8

Time, h

FIG. 6. lmmunolabehng of ASF virus Vero-infected cells. ASF virus- Infected cells were fixed and permeabilized (0) or not (0) at the times postinfection indicated. Cultures were incubated with anti-fusion protein (a) or anti-p37 (b) sera, as described under Materials and Methods. Background radioacttvlty. corresponding to permeabilized (3250 cpm) and nonpermeabilized (21 15 cpm) mock-infected cells carried In parallel, has been subtracted.

ab c d Time, h 12 6 7.5 12

Trwxin - - + - + - +

FIG. 7. Treatment of intact infected cells with trypsin: 1 O6 infected Vero cells were collected at 6, 7.5, and 12 hr postinfection by treat- ment with 0.05% trypsin-0.016% EDTA for 10 min at 37” (+) or after scraping the cells (-). The proteins were resolved by SDS-PAGE, transferred to an immobilon membrane, and incubated with the anti- fusion protein (b, c, d) or control @-gal (a) sera.

Since p22 is an early induced and possibly mem- brane-related protein as well as an external structural protein, it might function as a critical antigen, able to elicit an efficient immune response against virus infec- tion.

ACKNOWLEDGMENTS

We are grateful to R. Blasco for his help in the secondary structure prediction, to F. Jimenez for the comments on the manuscript, and to J. Palacin for his technical assistance. This research has been aided by grants from Comislbn Asesora para el Desarrollo de la Investiga- ci6n Cientifica y TBcnica, the Consejeria de Agricultura de la Junta de Extremadura, the European Economic Community, and the Fun- daci6n Ram& Areces. A.C. was a recipient of a fellowship from Fundaci6n Juan March.

REFERENCES

BECKER, Y. (1987). African swine fever. “Developments in Veterinary Virology.” Nijhoff, Boston.

BLANCO, L., and SALAS, M. (1985). Charactenzatlon of 3’+ 5’exonu- clease activity in the 429.encoded DNA polymerase. Nucleic Acds Res. 13. 1239-1249.

BLOCK, J., AIR, G. M., LEVER, W. G., WARD, C. W., LILLEY, G. S., WOODS, E. F., ROXBURGH, C. M., and INGLIS, A. S. (1982). Studies on the size, chemical compositlon and partial sequence of the neuraminidase (NA) from type A Influenza vtruses show that the N-terminal region of the NA is not processed and serves to anchor the NA rn the viral membrane. Virology 119, 109-l 2 1,

Bos, T. J., DAVIS, A. R., and DEBI, P. N. (1984). NH,-terminal hydro- phobic region of influenza virus neuramlnldase provides the signal function in translocation. Proc. Nat/. Aced. Sci. USA 81, 2321- 2331.

CARRASCOSA, A. L., DEL VAL, M., SANTA&N, J. F., and VIKJUELA, E. (1985). Purification and propertles of African swine fever virus. /. Virol. 54, 334-344.

CHOW, P. Y., and FASSMAN, G. D. (1978). Prediction of the secondary structure of proteins from their amino acid sequence. A& fnzy- mol. 47, 45-148.

CLEMENT, J. M. (1983). On the tnsertlon of proteins into membranes. Biochemie 65, 325-338.

DE BOER, C. 1. (1967). Studies to determine neutralizing antibody in sera from animals recovered from African swine fever and labora-

ASF VIRUS PROTEIN p22 257

tory animals inoculated with African swine fever virus with adju- vants. Arch. Gesamte Virusforsch. 20, 164-l 79.

DEVEREUX, J., HAEBERLI, P., and SMITHIES, 0. (1984). A comprehen- sive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387-395.

ENJUANES, L., CARRASCOSA, A. L., MORENO, M. A., and VIAUELA, E. (1976). Tritation of ASFV virus. /. Gen. Viral. 32, 47 l-477.

Folz, R. J., and GORDON, J. I. (1987). Computer-assisted predictions of signal peptidase processing sites. Biochem. Biophys. Res. Commun. 146,870-877.

GARFIN, D. E., and BERS, G. (1989). Basic aspect of protein blotting. In “Protein Blotting” (B. A. Baldo, E. R. Tovey. S. T. Leonards, Eds.), pp. 5-42. Karger, Basel.

GONZ&LEZ, A., CALVO, V., ALMAN, F., ALMENDRAL, J. M., RAM/REZ, J. C., DE LA VEGA, I., BLASCO, R., and VI~UELA, E. (1990). Multigene families in African swine fever virus DNA. Family 360.1. Viral. 64, 2073-2081.

KYTE, J., and DOOLIITLE, R. F. (1982). A simple method for displaying the hydropathic character of a protein. 1. Mol. Biol. 157, 105-l 32.

LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277, 680-685.

PEARSON, W. R., and LIPMAN, D. J. (1988). Improved tools for biologi- cal sequence comparison. Proc. Nat/. Acad. Sci. USA 85, 2444- 2448.

SABATINI, P. D., KREIBICH, G., MORIMOTO, T., and ADESNIK, M. (1982).

Mechanism for the incorporation of proteins in membranes and organelles. J. Cell Biol. 92, l-22.

SALAS, M. L., REY-CAMPOS, J., ALMENDRAL, J. M., TALAVERA, A., and VI&ELA, E. (1986). Transcription and translation maps of African swine fever virus. Virology 152, 228-240.

SOUTER, A. K., and WATE, D. P. (1989). Ligand blotting. In “Protein Function” fl. C. Creighton, Ed.), p. 55. IRL Press, Oxford/New York/Tokyo.

STANLEY, K. K., and LUZIO, J. P. (1984). Construction of a new family of high efficiency bacterial expression vectors: Identification of cDNA clones coding for human liver proteins. EMBO J. 3, 1429- 1434.

VAL, M., CARRASCOSA, J. L., and VII;IUEL% E. (1986). Glycosylated components of African swine fever virus particles. Virology 152, 39-49.

VOLLER, A., BIDWELL, D., and BARTLE~, A. (1980). Enzyme linked immunoadsorbent assay. ln “Manual Clinical Immunology” (N. Rose and H. Friedmon, Eds.), 2nd ed. p. 359. American Society for Microbiology, Washington DC.

VON HEIJNE, G. (1985). Signal sequences: The limits of variation. /. Mol. Biol. 184, 99-105.

VON HEIJNE, G. (1988). Transcending the impenetrable: How proteins come to terms with membranes. Biochim. Biophys. Acta 947, 307-333.

VON HEIJNE, G.. and GAVEL (1988). Topogenic signals in integral mem- brane proteins. Eur. J. Biochem. 174, 671-678.