(Received for publication, October 7, 1996, and in revised form, January 22, 1997)
From the Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma, 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 118,
119,
120, and
122 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, cis- and 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 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 as a trans-prenyltransferase.
[1-14C]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, ficaprenols C45-C65, and prenols C40-C60 were purchased from Sigma. 1-3H-Labeled 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 C35-C125 were kindly provided by Dr. T. Chojnacki (Polish Academy of Sciences, Warsaw).
Cells and VirusVero 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 AnalysisGeneral 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 RNAVero cells were
mock-infected or infected with ASFV (BA71V strain) at a multiplicity of
20 plaque-forming units/cell. To obtain early RNA, the cells were
infected in the presence of 100 µg/ml cycloheximide or 40 µg/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 32P-end-labeled
oligonucleotides 5-GTATTCCATGAAAAAGCGCGACACTACGCG-3
(RB-8),
5
-CTGTGATTCTAAAATACTTAGGTTTGCGCG-3
(RB-3), or
5
-ATAATTCGGTATAGGGCTTGAGTAGTTGGC-3
(RB-10), complementary to
nucleotides +124 to +95, +91 to +62, and
200 to
229, 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 5
-end labeling
with 32P, 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.
The B318L
gene lacking the first 62 nucleotides was amplified by polymerase chain
reaction using oligonucleotides 5-GCGCTCTAGAGCGCAAACCTAAGTATTTTA-3
and 5
-CGCGCTGCAGTTAGGTCCCCAATGCAACAT-3
, 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 PL promoter from
bacteriophage
. 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, ThiofusionTM Expression System Instruction Manual). Briefly, the transformed cells were grown overnight at 30 °C in RM medium containing 100 µg/ml ampicillin. The cultures were then diluted 1:20 in induction medium and grown at 30 °C to an A550 of 0.5. The cells were induced with 100 µg/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 ProteinA
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 mM Tris-HCl, pH 7, 150 mM NaCl, 1 mM EDTA, 1 mM -mercaptoethanol), and
sonicated with five 15-s bursts. After centrifugation for 15 min at
10,000 × g, the supernatant was collected and
incubated with 2 ml of ThioBondTM 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
-mercaptoethanol and eluted with
3 bed volumes of running buffer containing increasing concentrations of
-mercaptoethanol. The B318L protein eluted over a range of
concentrations of
-mercaptoethanol (50, 100, 200, and 500 mM). The fractions containing the protein were pooled and
dialyzed against 50 mM Tris-HCl, pH 7.5, 50 mM
NaCl, 1 mM EDTA. This pool was then incubated with 1 ml of
activated ThioBondTM resin for 1 h at 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
-mercaptoethanol. The protein was "knocked" off the column with
3 volumes of a high
-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.
The assay mixture contained, in a final volume of 0.1 ml, 50 µM [1-14C]IPP (specific activity 24 cpm/pmol), 100 µM all-trans-FPP or GPP, 2 mM MgCl2, 1 mM dithiothreitol, 20 mM Tris-HCl, pH 7.5, 50 µg/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 H2O 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 H2O. The aqueous phase was reextracted with 0.5 ml of n-butyl alcohol, and the pooled butanol extracts were back-washed with H2O. The radioactivity in the butanol phase was determined by scintillation counting.
Product AnalysisThe 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:H2O (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.
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. 1B predicts 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, 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 32P-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 (RB-8), also specific for
ORF B318L, as indicated under "Experimental Procedures,"
detected the same RNA bands (data not shown).
To map the 5-ends of the transcription products of ORF
B318L, the 32P-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
118,
119,
120, and
122, 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 3-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).
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 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)XDDX2,4DX4RRG
and
(L/I/V/M/F/Y)GX2FQ(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 X represents 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 DDX2,4D 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).
|
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 ThioBondTM 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 SDS-polyacrylamide 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).
|
The B318L protein required a divalent metal for activity. Using FPP as substrate, the optimal Mg2+ concentration was 1-2 mM, while with GPP maximal stimulation was obtained between 2 and 5 mM Mg2+. The activity at the optimal concentrations of Mn2+ of 1-2 mM was only about 10% of that observed with Mg2+, using either FPP or GPP as substrate.
The purified B318L protein can use DMAPP, GPP, all-trans-FPP, or all-trans-GGPP as its allylic substrate (Table III), the ratio of activities being DMAPP:GPP:FPP:GGPP = 1:2.2:4.1:1.9, under the standard assay conditions. Thus, the best substrate of the enzyme was FPP.
|
The reaction products derived from [1-14C]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
C15-C35 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 C25-C65 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
Mg2+ 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 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
[1-14C]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 -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 a trans 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).
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 association 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 (C15) or GGPP (C20) 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). Thus, it can be speculated that the ASFV prenyltransferase might be involved in the synthesis of the prenyl chain of ubiquinone for the production of extra amounts of this compound and that this could be important to protect the virus membranes and/or DNA against oxidation and to prevent cell death by apoptosis of the infected cell.
Studies on the localization of protein B318L and on its prenyltransferase activity in ASFV-infected cells, as well as an examination of protein prenylation induction during the virus life cycle, may help to elucidate the role of the virus-encoded prenyltransferase in the biology of ASFV.
We thank T. Chojnacki for the gift of prenols and J. Salas for critical reading of the manuscript.