African swine fever virus trans-prenyltransferase

African Swine Fever Virus trans-Prenyltransferase* (Received for publication, October 7, 1996, and in revised form, January 22, 1997) Alı´ Alejo‡, Rafael J. Ya´n˜ ez‡§, Javier M. Rodrı´guez¶, Eladio Vin˜ uela, and Marı´a L. Salasi From the Centro de Biologı´a Molecular “Severo Ochoa” (Consejo Superior de Investigaciones Cientı´ficas-Universidad Auto´noma de Madrid), Universidad Auto´noma, Cantoblanco, 28049 Madrid, Spain The present study describes the characterization of an African swine fever virus gene homologous to prenyltransferases. The gene, designated B318L, is located within the EcoRI B fragment in the central region of the virus genome, and encodes a polypeptide with a predicted molecular weight of 35,904. The protein is characterized by the presence of a putative hydrophobic transmembrane domain at the amino end. The gene is expressed at the late stage of virus infection, and transcription is initiated at positions 2118, 2119, 2120, and 2122 relative to the first nucleotide of the translation start codon. Protein B318L presents a colinear arrangement of the four highly conserved regions and the two aspartate-rich motifs characteristic of geranylgeranyl diphosphate synthases, farnesyl diphosphate synthases, and other prenyltransferases. Throughout these regions, the percentages of identity between protein B318L and various prenyltransferases range from 28.6 to 48.7%. The gene was cloned in vector pTrxFus without the amino-terminal hydrophobic region and expressed in Escherichia coli. The recombinant protein, purified essentially to homogeneity by affinity chromatography, catalyzes the sequential condensation of isopentenyl diphosphate with different allylic diphosphates, farnesyl diphosphate being the best allylic substrate of the reaction. All-trans-polyprenyl diphosphates containing 3–13 isoprene units are synthesized, which identifies the B318L protein as a trans-prenyltransferase. Prenyltransferase is the generic name for a family of enzymes catalyzing the sequential condensation of isopentenyl diphosphate (IPP) 1 with allylic diphosphates to form prenyl diphosphates of different chain length in the biosynthetic pathway of isoprenoid compounds (1–3). Farnesyl diphosphate (FPP) synthase and geranylgeranyl diphosphate (GGPP) synthase, the best characterized members of the prenyltransferase family, catalyze reactions at central positions in the isoprenoid pathway, giving rise to the formation of FPP and GGPP, respectively. These compounds are, in turn, precursors for the synthesis of a variety of products including cholesterol, carotenoids, and prenyl proteins, which are involved in basic cellular functions, such as membrane structure, synthesis of steroid hormones, and intracellular trafficking (3, 4). On the other hand, cisand trans-prenyltransferases catalyze the cis or trans additions of IPP to trans,trans-FPP producing polyprenyl diphosphates, which are precursors of dolychols and ubiquinones, involved, respectively, in synthesis of glycoproteins and electron transport (5, 6). Increasing evidence indicates that isoprenoid compounds also play essential roles during the life cycle of viruses. Thus, it has been recently shown that prenylation of hepatitis d virus large antigen is required for the formation and release of virus particles (7, 8). In addition, other studies with murine leukemia virus have indicated the existence of a relationship between isoprenoid biosynthesis and intracellular transport and processing of the viral envelope protein (9). These authors have suggested that prenylation of cellular Rab proteins is required for the incorporation of viral envelope precursors into the virions. Despite this role of isoprenoids in the multiplication of certain viruses, no virus-encoded prenyltransferase has been described so far. We report here the characterization of an African swine fever virus (ASFV) gene, designated B318L (10), encoding a protein with homology to prenyltransferases. ASFV is a large, enveloped DNA virus, that causes a severe disease in domestic pigs (11, 12). Its genome is formed by a double-stranded, linear DNA molecule of 170–190 kilobase pairs with hairpin loops and terminal inverted repeats (13, 14), this structure being remarkably similar to that of the poxvirus DNA (15, 16). Also in common with the poxviruses (17), ASFV particles contain all of the enzymatic machinery required for early RNA synthesis (18–20). The virion has about 50 structural proteins (21) and consists of a nucleoprotein core surrounded by a lipoprotein envelope, the capsid, and an outer membrane where the virus attachment protein p12 is located (22, 23). The assembly of virions is known to occur in the cytoplasm of the infected cell, but the steps involved in the virus morphogenesis are still poorly understood. We present a comparison of the amino acid sequences of the ASFV and cellular prenyltransferases, along with experiments describing the transcriptional expression of the viral gene during infection of cultured cells. We also show that the protein expressed in Escherichia coli and purified catalyzes the synthesis of all-trans-polyprenyl diphosphates, which identifies it asatrans-prenyltransferase. EXPERIMENTAL PROCEDURES Materials—[114 C]IPP (specific activity 56 Ci/mol) was obtained from Amersham Corp. Nonlabeled IPP, dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), all-trans-FPP, all-trans-GGPP, geraniol, farnesol (mixed isomers), all-trans-geranylgeraniol, solanesol, ficapre- * This work was supported by Direccio´n General de Investigacio´n Cientı´fica y Te´cnica Grant PB93-0160-C02-01, European Community Grant AIR-CT93-1332, and an institutional grant from Fundacio´n Ramo´n Areces to the Centro de Biologı´a Molecular “Severo Ochoa.” The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ The first two authors contributed equally to this work. § Present address: Gene Targeting Group, MRC Clinical Sciences Centre, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK. ¶Present address: Institute for Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, UK. iTo whom correspondence should be addressed. Centro de Biologı´a Molecular “Severo Ochoa” (CSIC-UAM), Universidad Auto´noma, Cantoblanco, 28049 Madrid, Spain. Tel: 34-1-3978478; Fax: 34-1-3974799; E-mail: [email protected]. 1 The abbreviations used are: IPP, isopentenyl diphosphate; ASFV, African swine fever virus; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GPP, geranyl diphosphate; GGPP, geranylgeranyl diphosphate; ORF, open reading frame; kb, kilobase(s). THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 14, Issue of April 4, pp. 9417–9423, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www-jbc.stanford.edu/jbc/ 9417 This is an Open Access article under the CC BY license. nols C 45 –C 65 , and prenols C 40 –C 60 were purchased from Sigma. 13 HLabeled trans,trans,cis-geranylgeraniol was from American Radiolabeled Chemicals, Inc., and acid phosphatase was from Boehringer. Silica gel 60 and RP-18 thin layer plates were obtained from Merck. Prenols C 35 –C 125 were kindly provided by Dr. T. Chojnacki (Polish Academy of Sciences, Warsaw). Cells and Virus—Vero cells (CCL81) were obtained from the American Type Culture Collection and grown in Dulbecco’s modified Eagle’s medium containing 10% newborn calf serum. The Vero-adapted ASFV strain BA71V was propagated and titrated as described previously (24). Computer Analysis—General analyses of DNA and protein sequences were performed with the software package of the University of Wisconsin Genetics Computer Group (25). Data base searches were done with programs FASTA and TFASTA (26). Computation was also performed at the National Center for Biotechnology Information using the BLAST (27) network service. Protein patterns were searched using the MacPattern program (28) and the PROSITE (29) and BLOCKS (30) data bases. Preparation and Analysis of RNA—Vero cells were mock-infected or infected with ASFV (BA71V strain) at a multiplicity of 20 plaqueforming units/cell. To obtain early RNA, the cells were infected in the presence of 100 m g/ml cycloheximide or 40 m g/ml cytosine arabinoside for 7 h. Late RNA was isolated from cells infected for 18 h in the absence of inhibitors. Whole cell RNA was prepared by the guanidinium isothiocyanate/cesium chloride extraction procedure (31). Northern blot hybridization was carried out as reported elsewhere (32), using as probe the 32 P-end-labeled oligonucleotides 59-GTATTCCATGAAAAAGCGCGACACTACGCG-39(RB-8), 59-CTGTGATTCTAAAATACTTAGGTTTGCGCG-39(RB-3), or 59-ATAATTCGGTATAGGGCTTGAGTAGTTGGC-39(RB-10), complementary to nucleotides 1124 to 195, 191 to 162, and 2200 to 2229, respectively, of the B318L gene noncoding strand. Oligonucleotides RB-8 and RB-3 were used for primer extension analysis, performed essentially as described by Sambrook et al. (33). After 59-end labeling with 32 P, the primer was annealed, at 43 °C, to the different classes of RNA and extended with avian myeloblastosis virus reverse transcriptase for 15 min at 42 °C. The primer extension products were then electrophoresed in a 6% polyacrylamide gel. Cloning and Expression of Gene B318L—The B318L gene lacking the first 62 nucleotides was amplified by polymerase chain reaction using oligonucleotides 59-GCGCTCTAGAGCGCAAACCTAAGTATTTTA-39 and 59-CGCGCTGCAGTTAGGTCCCCAATGCAACAT-39, and cloned in the expression vector pTrxFus (34). The first primer was designed with a GCGC tail and a XbaI restriction site, and the second primer contains a CGCG tail and a PstI restriction site. Both restriction sites are absent in the viral sequence. The polymerase chain reaction product was digested with XbaI and PstI and cloned into XbaI/PstI-cut pTrxFus vector under the control of the P L promoter from bacteriophage l . The viral gene was thus expressed as a fusion to the E. coli protein thioredoxin. Sequencing of the cloned product revealed a silent mutation in Ala-117 (an A to T change in nucleotide 351 of the B318L open reading frame (ORF); third base on codon 117). E. coli strain GI724 cells (34) were transformed with either the wild-type pTrxFus vector or the recombinant plasmid pTrxFus-B318L, and protein expression was analyzed as indicated by the supplier (Invitrogen, Thiofusion TM Expression System Instruction Manual). Briefly, the transformed cells were grown overnight at 30 °C in RM medium containing 100 m g/ml ampicillin. The cultures were then diluted 1:20 in induction medium and grown at 30 °C to an A 550 of 0.5. The cells were induced with 100 m g/ml tryptophan at 34 °C for 2 h. Samples were analyzed in SDS-polyacrylamide gels, and the polypeptides were visualized by Coomassie Blue staining. Affinity Purification of the Recombinant B318L Protein—A 250-ml culture of E. coli cells harboring the pTrxFus-B318L plasmid was induced with tryptophan at 34 °C for 2 h. The cells were harvested by centrifugation, suspended in 5 ml of running buffer (100 mMTris-HCl, pH 7, 150 mMNaCl, 1 mMEDTA, 1 mM b -mercaptoethanol), and sonicated with five 15-s bursts. After centrifugation for 15 min at 10,000 3g, the supernatant was collected and incubated with 2 ml of ThioBond TM resin (Invitrogen), activated as indicated by the supplier, for 60 min at 4 °C with rotation. The resin was allowed to settle, and, after collecting the flow-through, the column was washed with 30 bed volumes of running buffer containing 20 mM b -mercaptoethanol and eluted with 3 bed volumes of running buffer containing increasing concentrations of b -mercaptoethanol. The B318L protein eluted over a range of concentrations of b -mercaptoethanol (50, 100, 200, and 500 mM). The fractions containing the protein were pooled and dialyzed against 50 mMTris-HCl, pH 7.5, 50 mMNaCl, 1 mMEDTA. This pool was then incubated with 1 ml of activated ThioBond TM resin for1hat 4 °C with rotation, the resin was allowed to settle, and the column was washed with 10 bed volumes of running buffer containing 2 mM b -mercaptoethanol. The protein was “knocked” off the column with 3 volumes of a high b -mercaptoethanol concentration (1 M). The eluate was dialyzed as described before and concentrated using a Centricon-10 device. The concentrated sample was analyzed for purity by SDS-polyacrylamide gel electrophoresis with Coomassie Blue staining. Assay of Prenyltransferase Activity—The assay mixture contained, in a final volume of 0.1 ml, 50 m M[114 C]IPP (specific activity 24 cpm/ pmol), 100 m Mall-trans-FPP or GPP, 2 mMMgCl 2 ,1mMdithiothreitol, 20 mMTris-HCl, pH 7.5, 50 m g/ml of bovine serum albumin, and enzyme protein as indicated. After 15 min at 37 °C, the reactions were stopped by chilling in ice, and the products were extracted as follows: 0.15 ml of H 2 O saturated with n-butyl alcohol were added to the reaction mixtures, and the products were extracted with 0.5 ml of n-butyl alcohol saturated with H 2 O. The aqueous phase was reextracted with 0.5 ml of n-butyl alcohol, and the pooled butanol extracts were back-washed with H 2 O. The radioactivity in the butanol phase was determined by scintillation counting. Product Analysis—The butanol extracts were treated with acid phosphatase by the method of Fujii et al. (35). The hydrolysates were extracted with hexane, and the hexane-soluble products were analyzed by reversed-phase RP-18 thin layer chromatography in acetone:H 2 O (195:5) or by normal silica gel thin layer chromatography in benzene: ethyl acetate (9:1). The position of authentic standards was visualized with iodine vapor. For autoradiography, the thin layer plates were exposed on a Fujifilm BAS-MP 20405 imaging plate at room temperature. The exposed imaging plate was analyzed with a Fuji BAS 1500 analyzer. RESULTS Properties and Transcriptional Analysis of ORF B318L— ASFV ORF B318L, located within the EcoRI B fragment of the virus genome (Fig. 1A), encodes a protein of 318 amino acids, with a predicted molecular weight of 35,904, homologous to prenyltransferases (10). The hydropathy profile shown in Fig. 1Bpredicts that the B318L protein contains a transmembrane region of 21 amino acids at the amino end of the protein. This region is followed by four positively charged amino acids (Arg or Lys residues at positions 22, 23, 25, and 28), thus classifying the B318L protein as a putative class III membrane protein, FIG.1.Localization of ORF B318L within the ASFV genome and hydropathy profile of the protein encoded. A,EcoRI map of ASFV BA71V DNA and localization of ORF B318L within the EcoRIB(RB) fragment. B, hydropathy profile of protein B318L. The hydropathy profile was obtained following the method of Kyte and Doolittle (65), using a window of 7 residues. The line at 11.6 is a preset cut-off value for predicting transmembrane helices (66). The amino-terminal hydrophobic domain of protein B318L is shown below. The statistical significance of this putative transmembrane domain was assessed by using the maximal segment score method of Karlin and Altschul (67) with the MacPattern program (28). ASFV Prenyltransferase9418 with the carboxyl terminus facing the cytoplasm (36). To study the expression of the B318L gene during the viral infection and to determine the transcription initiation site, Northern blot and primer extension analyses were carried out, using as a hybridization probe or as a primer the 32 P-labeled oligonucleotide RB-3 (see “Experimental Procedures”) specific for ORF B318L. Hybridization of this oligonucleotide to Northern blots containing RNA from mock-infected cells and early (cycloheximide and cytosine arabinoside) and late RNA from cells infected with ASFV revealed eight RNA species with sizes of 1.1, 1.7, 2.4, 3, 4.4, 6.6, 8, and 9.4 kb in the sample of late RNA (Fig. 2). Hybridization with another oligonucleotide (RB8), also specific for ORF B318L, as indicated under “Experimental Procedures,” detected the same RNA bands (data not shown). To map the 59-ends of the transcription products of ORF B318L, the 32 P-labeled oligonucleotide RB-3 was annealed to the classes of RNA described before and extended with avian myeloblastosis virus reverse transcriptase, as described under “Experimental Procedures.” In agreement with the Northern blot results, the oligonucleotide primer hybridized exclusively with late RNA. After extension with reverse transcriptase, four specific bands of 209, 210, 211, and 213 nucleotides were detected (Fig. 2), corresponding to initiation of transcription at positions 2118, 2119, 2120, and 2122, relative to the first nucleotide of the translation start codon, respectively, as indicated by circles in the sequence shown in Fig. 2. These transcription initiation sites were confirmed using oligonucleotide RB-8 as primer (data not shown). A motif composed of seven consecutive thymidylate residues (the 7T motif), recently identified as a signal for 39-end formation of ASFV RNAs (37, 38), is found 32 nucleotides downstream of the translation stop codon of ORF B318L. Transcription termination at this site would produce an RNA of approximately 1.1 kb, which is the size of the smaller RNA band detected by Northern hybridization. Termination of transcription at alternative 7T motifs located further downstream might account for the 6.6-, 8-, and 9.4-kb RNAs, while the remaining species might correspond to transcripts of upstream ORFs, running through the B318L gene (10). In agreement with this possibility, it has been found that oligonucleotide RB-10, corresponding to a region upstream of the transcription initiation sites determined above, hybridizes in Northern blots to the 1.7-, 2.4-, 3-, and 4.4-kb species but not to the 1.1-, 6.6-, 8-, and 9.4-kb RNAs (not shown). Comparison of the Amino Acid Sequences of ASFV Protein B318L and Cellular Prenyltransferases—Searches in the data bases with the derived amino acid sequence of ORF B318L showed significant similarity to proteins of the prenyltransferase family. The highest optimized FASTA scores, with values ranging from 142 to 170, were obtained with the sequences of GGPP synthases from Capsicum annuum,Erwinia uredovora, and Erwinia herbicola and with the FPP synthase sequence from Bacillus stearothermophilus. To compare the amino acid sequence of protein B318L with the sequences of prenyltransferases, the ASFV protein was FIG.3. Multiple alignment of conserved regions in protein B318L and prenyltransferases. The multiple alignment was generated using the PILEUP program (25). The amino acid sequences of prenyltransferases were obtained from the SWISSPROT data base. GGPPS-CAPAN, GGPP synthase from C. annuum (39); FPPS-ECOLI, FPP synthase (ispA gene) from E. coli (68); GGPPS-RHOCA, GGPP synthase from Rhodobacter capsulatus (69); GGPPS-ERWHE, GGPP synthase (crtE gene) from E. herbicola (70); HXPPS-SCHCE, hexaprenyl diphosphate synthase (coq1 gene) from S. cerevisiae (40); B318LASFV, protein B318L from ASFV; GGPPS-NEUCR, GGPP synthase from N. crassa (71); FPPS-SCHCE, FPP synthase from S. cerevisiae (72); HPPPS-BSTEA, heptaprenyl diphosphate synthase from B. stearothermophilus (73); OPPS-ECOLI, octaprenyl diphosphate synthase (ispB gene) from E. coli (74). Numbers at the beginnings and ends of the amino acid sequences indicate the positions in the protein sequence, while numbers above the alignment refer to the position in the multiple alignment. The number of residues between the different regions are shown in parentheses.Boxes enclose identical amino acids in at least 6 of the 10 sequences compared. FIG.2.Transcriptional analysis of ORF B318L. Autoradiograms of RNA hybridization (left) and primer extension analysis (right)ofRNA from mock-infected cells (M), early RNA from cells infected with ASFV in the presence of cycloheximide (C) or cytosine arabinoside (A), and late RNA from cells infected in the absence of inhibitors (L). In the left panel, the sizes (in kilobases) of RNA molecular weight markers are shown. The samples in the right panel were electrophoresed beside a DNA sequencing reaction (DNA ladder) of the corresponding region of the gene performed with the same oligonucleotide used for primer extension. The sizes (in nucleotides) of the four DNA fragments obtained in the primer extension analysis are indicated. The transcription initiation sites of gene B318L are shown at the bottom and are indicated by open circles. The box encloses a consensus sequence for 39-end formation of RNA. ASFV Prenyltransferase 9419 first aligned with 16 demonstrated or putative prenyltransferases by using the PILEUP program, and a dendrogram displaying clustering relationships was constructed. Representative members of the different clusters obtained were then selected for the comparison of conserved regions, as shown in Fig. 3. All of these regions, designated I to IV according to Kuntz et al. (39), are also present in the B318L protein and have nearly the same spatial arrangement in the ASFV polypeptide and other prenyltransferases. Regions II and IV contain the PROSITE library signatures 1 and 2, used to identify proteins belonging to the prenyltransferase family. The consensus sequences of signatures 1 and 2 are L(L/I/V/ M)XDDX 2,4 DX 4 RRG and (L/I/V/M/F/Y)GX 2 FQ(L/I/V/M)XDD(L/ I/V/M/F/Y)X(D/N), respectively, in which the amino acids enclosed in parentheses signify alternatives for that position found in different prenyltransferases, each Xrepresents a nonconserved amino acid, and each subscript indicates the number of intervening residues. As can be seen in Fig. 3, these sequences are also conserved in the ASFV B318L protein, with only a substitution of the Gly residue for an Asn in signature 1. It has been suggested that the motif DDX 2,4 D in signatures 1 and 2 serves as binding site for the substrates and that this binding is facilitated by the formation of salt bridges between the aspartic residues and magnesium (39, 40). In keeping with this notion, mutational studies of the aspartate and arginine residues within signature 1 and of the two first aspartates within signature 2 of FPP synthase indicate that these amino acids are involved in enzyme catalysis (41–43). The proposed roles of these domains have been confirmed following crystallization of FPP synthase (44). On the other hand, sequence analysis of the FPP synthase gene of a yeast mutant defective in FPP synthase shows that the conserved lysine in region III is critical for catalytic activity (45). Conservation of these motifs and residues in the ASFV B318L protein strongly supports the proposal that the B318L gene encodes a prenyltransferase. The percentages of identity and similarity between protein B318L and prenyltransferases throughout the regions aligned in Fig. 3 show that the ASFV protein is more similar to the GGPP synthases from C. annuum,R. capsulatus, and E. herbicola and to the FPP synthase and octaprenyl diphosphate synthase from E. coli than to the hexaprenyl diphosphate synthase and FPP synthase from Saccharomyces cerevisiae, the GGPP synthase from Neurospora crassa and the heptaprenyl diphosphate synthase from B. stearothermophilus (Table I). Expression and Purification of the B318L Protein—To confirm that the B318L ORF encodes a prenyltransferase, the gene was cloned in the expression vector pTrxFus without the amino-terminal 21-amino acid hydrophobic region, to facilitate the expression of the protein in a soluble form. The resulting construction encodes a hybrid protein containing the E. coli thioredoxin followed in frame by 297 amino acids of the B318L protein. As shown in Fig. 4A, the SDS-polyacrylamide gel electrophoresis of total cell lysates of E. coli GI724 cells carrying the expression vector pTrxFus-B318L, harvested after induction with tryptophan, showed a band of 47 kDa, which was not detected in uninduced cells or in cells harboring the control pTrxFus plasmid. The size of the induced protein is consistent with that calculated for the fusion protein (46.9 kDa). To determine whether the protein encoded by ORF B318L has prenyltransferase activity, extracts from E. coli cells transformed with plasmid pTrxFus-B318L and induced with tryptophan were prepared, and the protein was purified by affinity chromatography on ThioBond TM columns, as described under “Experimental Procedures.” Relative to cells transformed with the control pTrxFus plasmid, the pTrxFus-B318L-transformed cells have a 24-fold higher specific prenyltransferase activity (Table II). After affinity chromatography, the purified B318L protein was essentially homogeneous as estimated by SDSpolyacrylamide gel electrophoresis with Coomassie Blue staining (Fig. 4B) and had an specific activity of 167 nmol of IPP incorporated into prenyl diphosphates per 15 min per mg of protein (Table II). Characteristics of the Prenyltransferase Activity of Protein B318L—The B318L protein required a divalent metal for activity. Using FPP as substrate, the optimal Mg 21 concentration was 1–2 mM, while with GPP maximal stimulation was obtained between 2 and 5 mMMg 21 . The activity at the optimal concentrations of Mn 21 of 1–2 mMwas only about 10% of that observed with Mg 21 , using either FPP or GPP as substrate. The purified B318L protein can use DMAPP, GPP, all-transFPP, or all-trans-GGPP as its allylic substrate (Table III), the ratio of activities being DMAPP:GPP:FPP:GGPP 51:2.2:4.1: 1.9, under the standard assay conditions. Thus, the best substrate of the enzyme was FPP. The reaction products derived from [114 C]IPP and the various allylic substrates were analyzed by reversed-phase thin layer chromatography after butanol extraction and hydrolysis with acid phosphatase to the corresponding alcohols, as indicated under “Experimental Procedures.” As shown in Fig. 5, when the allylic substrate was DMAPP, a mixture of C 15 –C 35 prenyl diphosphates was produced, while GPP yielded FPP and GGPP, as well as longer chain polyprenyl diphosphates containing 5–9 isoprene units. In the case of the reaction with FPP, GGPP, and polyprenyl diphosphates with chain lengths of C 25 – C 65 were formed, and when GGPP was used as substrate, polyprenyl diphosphates of 5–12 isoprene residues were synthesized. The product distribution obtained was very similar under a variety of reaction conditions, including different concentrations of Mg 21 or enzyme, and various reaction times. Although bovine serum albumin stimulates the prenyltransferase activity about 2-fold and is therefore present in the standard reaction mixture, it has no effect on the chain length of the products formed. However, the proportion of long chain TABLE I Percentage of identity and similarity (in parentheses) between prenyltransferases according to the multiple alignment Percentage of identity, and similarity were calculated with program DISTANCES (24), using threshold values of 1.5 and 0.6, respectively. Abbreviations for proteins are as indicated in the legend to Fig. 3. Protein FPPSECOLI GGPPS-RHOCA GGPPSERWHE HXPPS-SCHCE B318LASFV GGPPS-NEUCR FPPSSCHCE HPPPS-BSTEA OPPSECOLI GPPS-CAPAN 71.8 (80.8) 61.5 (76.9) 59.0 (78.2) 57.9 (75.0) 44.9 (60.3) 39.5 (64.5) 35.1 (54.6) 52.6 (75.0) 51.3 (68.4) FPPS-ECOLI 56.4 (71.8) 57.7 (71.8) 51.3 (65.8) 47.4 (64.1) 38.2 (59.2) 35.1 (53.3) 54.0 (71.1) 51.3 (69.7) GGPPS-RHOCA 56.4 (75.6) 50.0 (72.4) 46.2 (61.5) 38.2 (60.5) 33.8 (49.4) 52.6 (75.0) 50.0 (65.8) GGPPS-ERWHE 46.1 (68.4) 48.7 (64.1) 42.1 (55.3) 36.4 (50.7) 46.1 (69.7) 47.4 (65.8) HXPPS-SCHCE 36.8 (54.0) 35.5 (59.2) 36.8 (55.3) 55.3 (76.3) 60.5 (76.3) B318L-ASFV 30.3 (48.7) 28.6 (46.8) 39.5 (63.2) 43.4 (59.2) GGPPS-NEUCR 30.3 (50.0) 35.5 (60.5) 39.5 (57.9) FPPS-SCHCE 34.2 (50.0) 36.8 (54.0) HPPPS-BSTEA 50.0 (75.0) ASFV Prenyltransferase9420 polyprenyl diphosphates tended to increase when the concentration of FPP in the reaction mixture was lowered (data not shown). The dephosphorylated products of the reaction using [114 C]IPP and FPP as substrates were also analyzed on silica gel thin layer chromatography to determine the trans/cis configuration of the GGPP synthesized. The main spot observed on the autoradiogram comigrated with all-trans-geranylgeraniol (Fig. 6, lane 2), indicating that the GGPP synthesized contains the trans double bond at the a -isoprene unit. Other spots detected comigrate with the products of the reaction when GGPP is used as substrate (Fig. 6, lane 1). Since one of these additional spots also comigrates with the trans,trans,cis-geranylgeraniol, the radioactivity comigrating with the geranylgeraniol standard in a reversed-phase chromatogram was eluted and analyzed on silica gel thin layer chromatography, to confirm that all of the GGPP synthesized in the reaction with FPP had atrans configuration. The resulting autoradiogram revealed a main spot comigrating with all-trans-geranylgeraniol, while no product comigrating with trans,trans,cis-geranylgeraniol was detected (Fig. 6, lane 3). DISCUSSION It is shown here that the ASFV protein encoded by ORF B318L is homologous to prenyltransferases, containing the four highly conserved amino acid regions characteristic of these enzymes, and, within these regions, several amino acids that are critical for catalytic activity. To determine whether the B318L protein indeed had prenyltransferase activity, we undertook its expression in E. coli. Since attempts to express the complete protein in a soluble form failed, we cloned the gene without the amino-terminal hydrophobic region in the pTrxFus vector. The viral gene was thus expressed as a fusion to the E. coli protein thioredoxin, producing a soluble protein that could be purified essentially to homogeneity employing an affinity column. The purified protein has been shown to catalyze the sequential condensation of IPP with allylic diphosphates to yield prenyl diphosphates containing 3–13 isoprene units. A study of the stereochemistry of the reaction showed that IPP is added to FPP in a trans configuration, suggesting that all-trans-polyprenyl diphosphates are synthesized. Under the in vitro reaction conditions, the enzyme does not synthesize a single product of specific chain length. It is possible that in the in vivo environment the chain length distribution of the products might vary. Since the B318L protein contains a putative transmembrane domain, the chain length of the products might depend on its interaction with membranes, as appears to be the case for some prenyltransferases synthesizing long chain polyprenyl diphosphates (46). It will therefore be of interest in the future to investigate the possible associaFIG.4.Expression and purification of protein B318L. A, induction of protein B318L expressed in E. coli. Whole lysates of E. coli cells carrying pTrxFus or pTrxFus-B318L plasmids were analyzed by SDSpolyacrylamide gel electrophoresis followed by Coomassie Blue staining. U, uninduced cells; I, cells induced with tryptophan. The sizes in kDa of marker proteins (M) are indicated on the left. The arrow indicates the 47-kDa induced protein. B, purification of protein B318L by affinity chromatography. Protein B318L was purified from E. coli cells harboring the pTrxFus-B318L plasmid as indicated under “Experimental Procedures.” Protein samples were analyzed by SDS-polyacrylamide gel electrophoresis with Coomassie Blue staining. SN, supernatant fraction after sonication of the cells and centrifugation; ThioBond Resin, purified B318L protein after ThioBond TM affinity chromatography. The arrow indicates the B318L protein. The sizes in kDa of marker proteins (M) are indicated on the left. FIG.5.Reversed-phase thin layer chromatogram of the products of the reaction catalyzed by the B318L protein. The products of standard reactions with [114 C]IPP and the different allylic substrates indicated were analyzed after treatment with acid phosphatase as described under “Experimental Procedures.” The migration positions of farnesol, geranylgeraniol, solanesol, and C 40 –C 65 polyprenols used as markers are indicated. The deduced sizes of C 25 –C 35 products are also shown. TABLE II Phenyltransferase activity expressed in E. coli cells Sample Specific activity a Relative specific activity nmol/15 min/mg pTrxFus b 0.31 1 pTrxFus-B318L b 7.5 24 Affinity-purified pTrxFus-B318L c 167 539 a Activity was assayed under standard conditions (see “Experimental Procedures”) with FPP as allylic substrate. b The supernatant cell-free extract obtained as described under “Experimental Procedures” was assayed. c The B318L protein purified by affinity chromatography on ThioBond™ columns was assayed. TABLE III Substrate specificity Substrate Enzyme activity a cpm None b 59 DMAPP 811 GPP 1782 All-trans-FPP 3296 All-trans-GGPP 1510 a Enzyme activity was determined under standard conditions (see “Experimental Procedures”) with the allylic substrates indicated at a concentration of 100 m M. b Only [114 C]IPP was present as substrate. ASFV Prenyltransferase 9421 tion of the B318L protein with membranes and to study its activity in a membrane-bound form. It has also been shown that some of these prenyltransferases require protein factors for the synthesis of the natural products (47–49). Purification of the B318L protein from ASFV-infected cells might reveal the existence of a similar factor for the viral enzyme. To date, the ASFV B318L gene is the only prenyltransferase gene of viral origin identified. Its presence in ASFV raises a number of interesting questions about the significance of this enzyme in the virus life cycle. Since FPP and GGPP are formed in the reaction catalyzed by the viral enzyme, it could be argued that these products might be used as prenyl donors for the farnesylation or geranylgeranylation of cellular or viral proteins. These modifications might, in turn, be required for the assembly and release of virus particles, as has been described for other viruses (7–9). The existence of an ASFV ORF (L83L) encoding a protein with a consensus prenylation sequence (10) as well as the expression of the B318L gene at the late stage of infection, when virus morphogenesis occurs, would be consistent with such a role for the ASFV prenyltransferase. However, it should be noted that the properties of the ASFV prenyltransferase clearly differ from those described for the FPP synthase or GGPP synthase involved in the synthesis of the prenyl donors for protein prenylation. Thus, these enzymes produce FPP (C 15 ) or GGPP (C 20 ) as the ultimate products and are unable to utilize GGPP as substrate (50, 51), while the viral enzyme can form longer products and uses GGPP efficiently. Furthermore, the cellular enzymes synthesizing the precursors for protein prenylation are cytosolic proteins (52–55), whereas the ASFV B318L protein is predicted to be membrane-bound. On the other hand, all-trans-prenyltransferases, such as octaprenyl diphosphate synthase (56) and solanesyl diphosphate synthase (49), catalyze the sequential condensation of IPP to FPP to give long chain polyprenyl diphosphates, which are the precursors of quinone side chains. The ASFV B318L protein may have a similar role. If that is the case, the acquisition by ASFV of a prenyltransferase gene for the synthesis of the ubiquinone (coenzyme Q) prenyl chain could be related to an increase in mitochondrial function that might be required to provide extra energy for viral processes, such as macromolecular synthesis and morphogenesis. In this connection, it should be mentioned that mitochondrial DNA synthesis has been found to increase in herpes simplex type 1 virus-infected Vero cells (57) and that adenovirus infection of human cells stimulates mitochondrial activity (58). On the other hand, as has been previously discussed (10), macrophages and monocytes, the target cells in natural ASFV infection, generate reactive oxygen species for their microbicidal functions, which may cause oxidative damage to the virus DNA and membranes. The reactive oxygen species can also trigger and/or mediate apoptotic cell death (59), whose control is important to develop a productive viral infection. In addition to its role as electron carrier in mitochondria, it has been suggested that coenzyme Q, which is also present in the nucleus, plasma membrane, endoplasmic reticulum-Golgi, and lysosomes (60, 61), can function outside the mitochondria in antioxidation (62–64). 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Commun. 202, 340–345 ASFV Prenyltransferase 9423