From the Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain
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ABSTRACT |
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In a previous study, it was shown that the
protein encoded by the gene B318L of African swine fever
virus (ASFV) is a trans-prenyltransferase that catalyzes
in vitro the condensation of farnesyl diphosphate and
isopentenyl diphosphate to synthesize geranylgeranyl diphosphate and
longer chain prenyl diphosphates (Alejo, A., Yáñez, R. J., Rodríguez, J. M., Viñuela, E., and Salas,
M. L. (1997) J. Biol. Chem. 272, 9417-9423). To
investigate the in vivo function of the viral enzyme, we
have determined, in this work, its subcellular localization and
activity in cell extracts. Two systems were used in these studies:
cells infected with ASFV and cells infected with a recombinant
pseudo-Sindbis virus carrying the complete B318L gene. In
this latter system, the trans-prenyltransferase was found
to colocalize with the endoplasmic reticulum marker protein-disulfide
isomerase, whereas in cells infected with ASFV, the viral enzyme was
present in cytoplasmic viral assembly sites, associated with precursor
viral membranes derived from the endoplasmic reticulum. In addition,
after subcellular fractionation, the viral enzyme partitioned into the
membrane fraction. Extraction of membrane proteins with alkaline
carbonate and Triton X-114 indicated that the ASFV enzyme behaved as an
integral membrane protein. The membrane enzyme synthesized
predominantly all-trans-geranylgeranyl diphosphate from
farnesyl diphosphate and isopentenyl diphosphate. These results indicate that the viral B318L protein is a
trans-geranylgeranyl-diphosphate synthase, being the only
enzyme of this type that is known to have a membrane localization.
In the biosynthetic pathway of isoprenoid compounds, different
enzymes belonging to the family of prenyltransferases catalyze the
condensation of isopentenyl diphosphate
(IPP)1 with allylic
diphosphates to give rise to prenyl diphosphates of different chain
lengths and double-bond stereochemistry (1-3). Thus,
farnesyl-diphosphate synthase and geranylgeranyl-diphosphate synthase
produce the 15-carbon all-trans-FPP and the 20-carbon all-trans-GGPP isoprenoids, respectively, which can serve as
substrates for protein farnesylation or geranylgeranylation (4, 5). FPP
is also the direct precursor for the synthesis of cholesterol and
serves as allylic substrate for cis- and
trans-polyprenyl-diphosphate synthases involved in the
synthesis of the long chain isoprenoids with cis- or
trans-stereochemistry, which are the precursors of dolichols
and the lateral chain of ubiquinones, respectively (6, 7).
Prenyltransferases have been described in a wide variety of organisms,
both prokaryotic and eukaryotic, where their isoprenoid products play
essential roles in cellular processes. More recently, it has been shown
that prenylation of cellular or viral proteins is required for the
multiplication of certain viruses (8-11). In this connection, we have
previously reported (12) the characterization of the first viral
trans-prenyltransferase encoded by African swine fever virus
(ASFV), a large enveloped DNA virus with an icosahedral morphology that
assembles in the cytoplasm of the infected cell and causes a severe
disease in domestic pigs (13, 14). The ASFV gene, designated
B318L, contains an amino-terminal hydrophobic sequence and
conserves all the regions characteristic of prenyltransferases. After
cloning of the gene without the hydrophobic sequence, expression in
Escherichia coli as a fusion to the E. coli
protein thioredoxin, and purification, it was shown that the
recombinant enzyme catalyzed the trans-addition of IPP to allylic diphosphates, with FPP being the best substrate of the reaction
(12). However, under a variety of reaction conditions, the enzyme did
not synthesize a unique product with a specific chain length. Although
the major product of the reaction was GGPP, using IPP and FPP as
substrates, additional longer chain prenyl diphosphates, containing up
to 13 isoprene units, were synthesized in significant amounts. In
addition, the viral enzyme could use GGPP efficiently as substrate.
These properties are clearly distinct from those described for the
cytosolic trans-GGPP synthase, the enzyme involved in the
synthesis of the prenyl donor for protein geranylgeranylation, since
this enzyme synthesizes GGPP as the ultimate product and is unable to
use GGPP as substrate (15-20). On the other hand, specific
all-trans-polyprenyl-diphosphate synthases, such as
octaprenyl-diphosphate synthase (21) and solanesyl-diphosphate synthase
(22), have also been shown to catalyze the synthesis of a defined
length product.
Since the ASFV trans-prenyltransferase contains a putative
transmembrane domain at the amino-terminal end, it was possible that
its interaction with membranes in the cell environment could determine
the chain length of the product synthesized, as in the case of some
long chain polyprenyl-diphosphate synthases (23). Therefore, as a step
toward a better understanding of the in vivo activity and
function of the ASFV trans-prenyltransferase, we have
examined the subcellular localization and activity of the viral enzyme
in cells infected with ASFV or with a recombinant pseudo-Sindbis
virus carrying the ASFV gene. We show that the viral protein is
associated with endoplasmic reticulum (ER) membranes in cells infected
with the recombinant Sindbis virus, whereas in ASFV-infected cells, the
enzyme is localized in the cytoplasmic viral assembly sites, where it
is found associated with membrane structures that have been shown
recently by Rouiller et al. (24) and Andrés et
al. (25) to derive from the ER. We also show that the ASFV enzyme
is an integral membrane protein and that the membrane-bound enzyme
synthesizes preferentially all-trans-GGPP.
Cells, Viruses, and Reagents--
Vero (African green monkey
kidney) cells were obtained from American Type Culture Collection and
grown in Dulbecco's modified Eagle's medium containing 5% newborn
calf serum. Baby hamster kidney (BHK) cells were from Invitrogen and
grown in Antibodies--
The ASFV B318L gene was cloned into a
pRSET vector (Invitrogen), and the recombinant protein was expressed in
E. coli and purified under denaturing conditions using
single-step Ni2+-nitrilotriacetic acid affinity
chromatography. Antibodies against the purified recombinant
trans-prenyltransferase (B318L protein) were raised in
rabbits. The immune serum obtained recognized the recombinant protein
on Western blots. The anti-protein-disulfide isomerase mouse monoclonal
antibody (1D3) was from Stressgen Biotech Corp.
Metabolic Labeling, Immunoprecipitation, and Electrophoretic
Analysis--
Preconfluent monolayers of Vero cells were infected with
ASFV at a multiplicity of infection of 10 plaque-forming units/cell, and at different times post-infection, the cells were pulse-labeled for
2 h with a mixture of [35S]methionine and
[35S]cysteine (150 µCi/1 × 106 cells;
Amersham Pharmacia Biotech). Lysates were prepared by resuspending the
cells in radioimmune precipitation assay buffer (10 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 1% sodium deoxycholate, 1%
Nonidet P-40, and 0.1% SDS), diluted 10-fold in the same buffer, preimmunoprecipitated with preimmune serum and protein A-Sepharose, and
then immunoprecipitated with a 1:100 dilution of preimmune or
anti-B318L protein serum and protein A-Sepharose. Bound proteins were
eluted by boiling in electrophoresis sample buffer and resolved by
SDS-polyacrylamide gel (7-20%) electrophoresis.
Western Blotting--
For Western blots, Vero cells were
infected with ASFV as described above, and at different times
post-infection, the cells were lysed in electrophoresis sample buffer.
Equivalent amounts of the cell lysates were electrophoresed on
SDS-polyacrylamide gels (7-20%) and subsequently transferred to
nitrocellulose membranes. The membranes were blocked in Tris-buffered
saline/Tween buffer (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 0.1% Tween 20) with 5% dry milk powder for
1 h and then incubated overnight at 4 °C with a 1:500 dilution
of the anti-pB318L serum in Tris-buffered saline/Tween with 1% dry
milk powder. The membranes were washed with Tris-buffered saline/Tween
and incubated with a peroxidase-labeled anti-rabbit serum (Amersham
Pharmacia Biotech), and the proteins were detected with an
electrochemiluminescence system (ECL system, Amersham Pharmacia
Biotech) according to the manufacturer's recommendations.
Construction of Recombinant Pseudo-Sindbis Virus--
The ASFV
gene B318L, cloned in the pBluescript plasmid, was subcloned
in the BbrPI restriction site of the pSinRep5 plasmid (Invitrogen Sindbis Expression System, Version C) to obtain the recombinant plasmid pSinRep/B318L, which was linearized with
NotI. The linearized plasmid pSinRep/B318L and the DH-BB
helper template were transcribed in vitro using the
InvitroScript CAP SP6 in vitro transcription kit
(Invitrogen). To obtain recombinant pseudovirus, the in
vitro transcripts were transfected by electroporation into BHK
cells following the instructions provided by Invitrogen. At 24 h
after transfection, the supernatants were collected, and pseudovirions
were concentrated by ultracentrifugation onto sucrose cushions.
Immunofluorescence--
Vero cells, grown on chamber slides,
were mock-infected or infected with ASFV at a multiplicity of infection
of 1 plaque-forming unit/cell and fixed at 14 h post-infection
with methanol at Immunoelectron Microscopy--
Vero cells were mock-infected or
infected with ASFV at a multiplicity of infection of 10 plaque-forming
units/cell and fixed, at 20 h post-infection, with 4%
formaldehyde and 0.1% glutaraldehyde in 200 mM HEPES, pH
7.2, for 1 h at room temperature. After fixation, the cells were
processed for cryosectioning as detailed by Andrés et
al. (25). Ultrathin thawed cryosections were incubated with a 1:40
dilution of the anti-B318L antibody followed by protein A-gold (10 nm).
Subcellular Fractionation--
Subcellular fractionation of Vero
cells was performed as described (28) with slight modifications. Vero
cells were mock-infected or infected with ASFV at a multiplicity of
infection of 5 plaque-forming units/cell, and at 19 h
post-infection, the cells were resuspended in homogenization buffer
containing 20 mM HEPES, pH 7.4, 0.28 M sucrose,
2 mM EDTA, and 1 mM phenylmethanesulfonyl
fluoride and then disrupted by N2 cavitation at 400 p.s.i for 10 min at 4 °C or by passaging through a 25-gauge syringe.
In the case of mock- or Sindbis virus-infected BHK cells, the cells
were resuspended in the same homogenization buffer as described above
and disrupted manually using a Kontes homogenizer. In all cases, the
homogenate was centrifuged at 700 × g for 5 min to
sediment nuclei, and the supernatant fraction was centrifuged at
150,000 × g for 30 min at 4 °C to obtain the
membrane and cytosolic fractions. For prenyltransferase assays, the
membrane fraction was resuspended in homogenization buffer at a protein
concentration of 3-7 mg/ml and stored, as the cytosolic fraction, at
For the analysis of membrane association of the B318L protein, membrane
fractions from mock- or ASFV-infected Vero cells were treated with
sodium carbonate or Triton X-114 according to the methods of Fujiki
et al. (29) and Pryde and Phillips (30). For carbonate
treatment, membrane fractions from 4 × 106 cells were
resuspended in 100 µl of 0.1 M
Na2CO3, pH 11.5; incubated for 30 min at
4 °C; and subsequently centrifuged for 10 min at 100,000 × g. The supernatant and the sediment were dissociated with
electrophoresis sample buffer. For treatment with Triton X-114, an
equivalent amount of the membrane fraction was resuspended in 100 µl
of 2% Triton X-114, 150 mM NaCl, and 10 mM
Tris-HCl, pH 7.5; incubated for 10 min at 4 °C; and then transferred
to 30 °C for 20 min. The samples were briefly spun in a
microcentrifuge to separate the lower detergent-rich phase from the
upper phase, and both phases were dissociated as described above.
Equivalent amounts of each sample were subjected to Western blot analysis.
Assay of Prenyltransferase Activity and Product
Analysis--
The regular assay mixture contained, in a final volume
of 0.1 ml, 50 µM [1-14C]IPP (specific
activity of 24 cpm/pmol), 100 µM
all-trans-FPP, 2 mM MgCl2, 1 mM dithiothreitol, 20 mM Tris-HCl, pH 7.5, 50 µg/ml bovine serum albumin, 20 mM KF, and enzyme protein
as indicated. After 4 h at 37 °C, the products were extracted
with butanol as described before (12), and the butanol extracts were
treated with acid phosphatase by the method of Fujii et al.
(31). The hydrolysates were extracted with hexane, and the
hexane-soluble products were analyzed by reversed-phase RP-18
thin-layer chromatography in acetone/H2O (95:5). The
positions of authentic standards were visualized with iodine vapor. The
thin-layer plates were exposed for autoradiography on a Fuji BAS-MP
20405 imaging plate, and the exposed imaging plate was analyzed with a
Fuji BAS 1500 analyzer. Densitometric analysis of the bands was
performed using TINA Version 2.0 software.
Synthesis and Localization of ASFV trans-Prenyltransferase in
Infected Cells--
We have previously shown that the ASFV
trans-prenyltransferase gene B318L is transcribed
at late times of infection (12). In agreement with this, the results of
immunoprecipitation experiments presented in Fig.
1A indicate that the synthesis
of the enzyme in ASFV-infected Vero cells was initiated at 8-10 h
post-infection, a time at which viral DNA replication is already
underway (32); reached a maximum at 13-15 h post-infection; and
decreased at later times. Furthermore, the protein was not synthesized
in the presence of Ara-C, an inhibitor of viral DNA replication and
late transcription (32), indicating that the ASFV
trans-prenyltransferase is a late protein. Western blot
analysis revealed the presence of a specific band of ~37 kDa in
ASFV-infected cells. This band was first detected at 12 h
post-infection and accumulated up to 24 h post-infection (Fig.
1B). In keeping with the immunoprecipitation results, the
protein band was not seen in the presence of Ara-C (Fig.
1B).
As mentioned in the Introduction, the
trans-prenyltransferase of ASFV contains a putative
transmembrane domain at its amino-terminal end, suggesting an
association of the protein with cellular or viral membranes. To test
this possibility, we first examined the intracellular localization of
the trans-prenyltransferase in BHK cells infected with a
recombinant pseudo-Sindbis virus carrying the ASFV gene to determine
whether the enzyme was targeted to cellular membranes independently of
other ASFV proteins or processes related to the infection with ASFV.
The results of double immunofluorescence experiments using antibodies
against the ASFV trans-prenyltransferase (B318L protein) and
the ER marker protein protein-disulfide isomerase indicated that the
viral enzyme localized to the ER (Fig. 2,
B and C). A very low background
immunofluorescence was observed with the anti-B318L antibody in the
case of mock-infected BHK cells, which served as a control (Fig.
2A). A similar low background signal was detected with this
antibody in mock-infected Vero cells (Fig. 2D). On the other
hand, in Vero cells infected for 14 h with ASFV, the
trans-prenyltransferase was located in discrete cytoplasmic
areas close to the nucleus, which were identified as viral assembly
sites by staining of the viral DNA with bisbenzimide (Fig. 2,
E and G). It should also be noted that at an
earlier time of infection (10 h), when the synthesis of the protein has just been initiated (see Fig. 1), the same immunofluorescent pattern was obtained (data not shown). In contrast, the protein-disulfide isomerase signal was excluded from these areas and was found
surrounding the viral factories, whereas in cells lacking factories,
the signal showed a pattern characteristic of the ER (Fig.
2F).
To examine in detail the localization of the
trans-prenyltransferase within the viral factories,
immunocytochemical studies with electron microscopy were performed. For
this, ultrathin thawed cryosections of Vero cells mock-infected or
infected with ASFV for 20 h were incubated with the anti-B318L
protein antibody followed by protein A-gold. A low immunogold labeling
was observed in the case of mock-infected cells (Fig.
3A). In cells infected with ASFV, the antibody strongly labeled the region of the viral factories, in keeping with the immunofluorescence results (Fig. 3B).
Within the factories, the gold grains were seen decorating mainly
membrane-like structures (Fig. 3C), which are precursors of
the inner envelope of ASFV (33) and are derived from the ER (24, 25).
Gold grains were essentially excluded from mature virus particles (Fig. 3B). In agreement with this, Western blot analysis of highly
purified ASFV using the antibody against the B318L protein has
indicated that the trans-prenyltransferase is not a
structural component of the virus (data not shown).
ASFV trans-Prenyltransferase Is an Integral Membrane
Protein--
The above studies indicated that the ASFV
trans-prenyltransferase was associated in the viral
factories with membranes that are derived from the ER. To further
investigate the association of the viral enzyme with membranes, cell
fractionation experiments were performed, and the distribution of the
viral enzyme into membrane and cytosolic fractions from ASFV-infected
cells was examined using the anti-B318L protein antibody. As a control, a similar fractionation of mock-infected cells was carried out. In the
membrane fraction from ASFV-infected cells, a specific band
corresponding to the ASFV prenyltransferase was detected (Fig.
4, lane 4). This band was not
found in the cytosolic or membrane fractions from mock-infected cells
(Fig. 4, lanes 1 and 2). On the other hand, a
cellular protein that partitioned exclusively into the cytosolic
fraction from mock-infected and infected cells was detected (Fig. 4,
lanes 1 and 3).
To analyze the nature of the association of the viral enzyme with
membranes, washes with alkaline carbonate and extractions with Triton
X-114 were performed as indicated under "Experimental Procedures."
After washing the membranes from infected cells with alkaline carbonate
and centrifugation, the ASFV B318L protein remained associated with the
sediment (Fig. 4, lanes 5 and 6), indicating that
it is not peripherally bound to the membranes. On the other hand, phase
extraction of membrane proteins with Triton-X114 showed that the viral
protein behaved as an integral membrane protein since it partitioned
into the detergent phase (Fig. 4, lanes 7 and
8).
Prenyltransferase Activity in Membrane Fractions of ASFV-infected
Cells--
To determine the activity and products of the ASFV
prenyltransferase in extracts from infected cells, the cytosolic and
membrane fractions were incubated with [1-14C]IPP and FPP
in a standard reaction mixture (see "Experimental Procedures"), and
the reaction products were analyzed by reversed-phase thin-layer
chromatography after butanol extraction and hydrolysis with acid
phosphatase to the corresponding alcohols as described under
"Experimental Procedures." A low GGPP synthase activity was
detected in the cytosolic fraction of mock-infected cells, but this
activity did not increase in the virus-infected cells (data not shown).
The results obtained with the membrane fractions are shown in Fig.
5. In the case of mock-infected cells,
the major products formed were prenyl diphosphates of 30 and 50 carbons, whereas the levels of GGPP synthesized were very low (Fig.
5A, lane 2). In contrast, the membrane fraction
of ASFV-infected cells produced predominantly GGPP, together with
geranylfarnesyl diphosphate, although in considerably lower amounts
(Fig. 5A, lane 4). On the other hand, the
synthesis of the C30 and C50 products was
comparable to that found in mock-infected cells. In the presence of 1%
Triton X-100, the products synthesized remained essentially unchanged (Fig. 5A, lanes 3 and 5). In all
cases, similar levels of 14C-labeled FPP were produced,
probably due to the presence of IPP isomerase in the membrane
preparations. The product distribution obtained with the enzyme from
the membrane fraction of infected cells was very similar using
different amounts of protein (between 50 and 150 µg) in the assay and
various reaction times (1-16 h) (data not shown).
A densitometric quantification of the products synthesized by the
membrane fractions of mock- and ASFV-infected cells showed an increase
of ~25-fold in the amount of GGPP generated by the prenyltransferase
of infected cells with respect to that of mock-infected cells (Table
I). On the contrary, no significant
differences were found in the levels of the C30 and
C50 polyprenyl diphosphates. The C50 isoprenoid
detected in the mock-infected and infected cells could be a precursor
of the ubiquinone (Q) side chain. In keeping with this, an analysis of
the Q species present in Vero cells mock-infected or infected with ASFV
revealed that Q-10, which contains a lateral chain of 50 carbons, was
the predominant form in both
cases.2
As described previously (12) and as shown in Fig. 5A
(lane 1), the purified recombinant ASFV
trans-prenyltransferase produced prenyl diphosphates
containing 4-12 isoprene units. In contrast, chain elongation by the
enzyme present in the membrane fraction from infected cells was more
limited (Fig. 5A, lanes 4 and 5). Thus, the ratio of GGPP to geranylfarnesyl diphosphate
(C25) was 3.6 ± 0.7 (n = 6) in the
case of the recombinant enzyme and 8.5 ± 1.5 (n = 9) for the membrane prenyltransferase. Additional longer chain
isoprenoids were synthesized in very low amounts by the enzyme from
infected cells (Fig. 5A).
To determine the trans/cis-configuration of GGPP
synthesized by the viral enzyme, the radioactivity of the
geranylgeraniol band was eluted from the reversed-phase chromatogram
and analyzed by silica gel thin-layer chromatography. The resulting
autoradiogram revealed a main spot comigrating with
all-trans-geranylgeraniol (Fig. 5B, lane
1), as in the case of the recombinant ASFV
trans-prenyltransferase (lane 2), whereas no
product comigrating with
trans,trans,cis-geranylgeraniol was detected.
Taken together, the above results suggested that the activity detected
in the membrane fraction of infected cells corresponded to the ASFV
trans-prenyltransferase. To confirm this, we first tested
the effect of the anti-B318L antibody on the reaction catalyzed by this
subcellular fraction. Fig. 6A
shows that the specific antibody strongly inhibited the formation of
GGPP and geranylfarnesyl diphosphate by the enzyme from infected cells
(lanes 4 and 5), whereas a preimmune serum had no
effect (lane 6). In contrast, the reaction catalyzed by the
membrane fraction of mock-infected cells was not affected by the
anti-B318L antibody (Fig. 6A, lanes 2 and
3), and this antibody did not decrease the formation of the
C30 and C50 products obtained with the membrane
preparation of infected cells (lanes 4 and 5),
suggesting that these compounds are synthesized by the cellular
polyprenyl-diphosphate synthase.
In a different approach, we assayed prenyltransferase activity in a
membrane fraction prepared from BHK cells infected with the recombinant
Sind/B318L virus expressing the ASFV B318L gene. When
compared with mock-infected BHK cells (Fig. 6B, lane
1), a considerable increase (16-fold as determined by
densitometric scanning) was observed in the amount of GGPP synthesized
by the membrane fraction from cells infected with the recombinant
Sind/B318L virus (lane 2). With this fraction, lower amounts
of geranylfarnesyl diphosphate and C30-40 isoprenoid
compounds were also produced. On the other hand, the synthesis of
C45 and C50 polyprenyl diphosphates increased
only 2.5-fold in the infected cells relative to mock-infected cells.
Thus, the products generated by the enzyme present in the membrane
fraction from cells infected with the recombinant Sindbis virus were
very similar to those synthesized by the corresponding fraction from
ASFV-infected cells.
A previous characterization of the
trans-prenyltransferase encoded by ASFV showed that the
purified recombinant enzyme, lacking an amino-terminal hydrophobic
region and expressed as a fusion with the E. coli protein
thioredoxin, catalyzed the synthesis of GGPP as the major product, as
well as longer prenyl diphosphates containing up to 13 isoprene units,
using FPP and IPP as substrates (12). On the basis of these results,
two possible roles for the viral enzyme during infection were proposed;
one of them was the formation of GGPP, a product that could then be
used for prenylation of proteins during the virus life cycle. If this
were the case, the synthesis of longer isoprenoid compounds could be
due to an inability of the recombinant enzyme to terminate chain
elongation at the GGPP step. As an alternative, the possibility was
also considered that the long chain polyprenyl diphosphates synthesized could serve as precursors of the Q side chains. The synthesis of Q, in
turn, could be related to an increase in mitochondrial function or to a
role of Q as antioxidant during the infection of macrophages, the
target cells in natural ASFV infection, to prevent oxidative damage to
viral components since these cells generate large amounts of reactive
oxygen species for their microbicidal functions.
To obtain further information on the natural products of the ASFV
trans-prenyltransferase and on its function during the
infectious cycle, we have examined, in this study, the subcellular
localization of the viral enzyme and its activity in extracts from
infected cells. Expression of the trans-prenyltransferase in
a recombinant Sindbis virus has allowed us to demonstrate its targeting
to the ER, a result that is consistent with the presence of a
transmembrane domain in the protein. On the other hand, in cells
infected with ASFV, the enzyme is found in the cytoplasmic viral
assembly sites, associated with membrane structures that are precursors
of the viral inner envelope and are derived from the ER (24, 25, 33).
Therefore, it is possible that in ASFV-infected cells, the enzyme is
synthesized within the viral factories, being thus directed to the
precursor viral membranes of the assembly areas or to nearby ER
membranes, which would then be rapidly incorporated into the factories
to form the viral membranes. During the maturation process of the virus
particles from these membranes, the trans-prenyltransferase must be excluded from them since it is not found in the mature virions.
The enzyme present in membrane preparations from cells infected with
ASFV or with the recombinant Sindbis virus expressing the ASFV gene
catalyzes predominantly the synthesis of all-trans-GGPP, with little further elongation to longer chain polyprenyl diphosphates, in contrast to the results obtained in the case of the recombinant enzyme expressed in E. coli. Although this difference could
reflect a better control of polymerization by the membrane-associated enzyme, the fact that the pattern of isoprenoid distribution is not
altered by the presence of detergent suggests that an integral membrane
structure is not required to determine the specificity of products. It
is possible that other factors might influence chain polymerization.
On the other hand, the finding that the formation of GGPP by the
membrane fraction of ASFV-infected cells is greatly increased with
respect to that of mock-infected cells, whereas the synthesis of the
C50 precursor of Q remains essentially unchanged, strongly supports that the natural product of the ASFV enzyme is
all-trans-GGPP, allowing its classification as a
trans-GGPP synthase. However, two features of the viral
enzyme distinguish it from the cellular trans-GGPP
synthases. Thus, the cellular enzymes are cytosolic proteins (17, 20)
and strictly control chain termination at the GGPP step (15, 16). In
contrast, the ASFV trans-GGPP synthase is an integral
membrane protein and elongates to some extent the GGPP product, even
when assayed in a membrane-associated form.
As mentioned above, GGPP synthesized by the ASFV
trans-prenyltransferase could serve as substrate for
prenylation of cellular or viral proteins. The synthesis of the enzyme
at the late stage of infection and its localization in precursor viral
membranes within the viral factories are consistent with the view that
this modification might be required in the morphogenetic processes of
the virus, as has been described for hepatitis Attempts to construct ASFV recombinants depleted of the
trans-prenyltransferase gene have failed,2
suggesting that the gene is essential for virus multiplication. A
system for inducible gene expression from ASFV recombinants, recently
developed in our laboratory (36), might be useful to investigate the
role of the trans-prenyltransferase during virus replication.
The association with membranes of the ASFV enzyme is intriguing in view
of the fact that the cellular trans-GGPP synthases have been
described as cytosolic proteins. Recent studies on the mammalian
protein farnesyltransferase provide clues to the possible physiological
significance of that association. Thus, it has been shown that product
release is the rate-limiting step in catalysis (37) and that
dissociation of the product from the enzyme requires the provision of
additional FPP substrate (38). It has been proposed that the
farnesyltransferase-product complex is directed to a membrane
compartment where FPP would be located, thus triggering the release of
the farnesylated protein (38). In a similar fashion, GGPP generated by
the membrane-bound trans-prenyltransferase of ASFV might
efficiently promote the dissociation of
geranylgeranyltransferase-product complexes targeted to the viral
membranes in the factories. This could facilitate the prenylation of
proteins playing a role in the virus replication cycle.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-modified Eagle's medium supplemented with 2 mM L-glutamine and 5% fetal bovine serum. The
Vero adapted ASFV strain BA71V was propagated and titrated as described
previously (26). [1-14C]IPP (specific activity of 56 Ci/mol) was obtained from Amersham Pharmacia Biotech;
trans,trans,cis-[1-3H]geranylgeraniol
was from American Radiolabeled Chemicals, Inc.; and unlabeled
isoprenoid compounds and cytosine arabinoside (Ara-C) were from Sigma.
Silica Gel 60 and RP-18F254S thin-layer plates were from
Merck, and acid phosphatase was from Roche Molecular Biochemicals.
20 °C for 5 min. The cells were then incubated
with anti-B318L and anti-protein-disulfide isomerase antibodies, as
indicated in the legend to Fig. 2, in phosphate-buffered saline
containing 0.1% bovine serum albumin at 37 °C for 1 h; rinsed
three times for 10 min with the same buffer; and incubated with
fluoresceinated goat anti-rabbit (Tago, Inc.) and Texas Red-linked
sheep anti-mouse (Amersham Pharmacia Biotech) antibodies. Nuclear and
viral DNAs were visualized by staining with 5 µg of bisbenzimide
(Hoechst 33258, Sigma) per ml of phosphate-buffered saline for 5 min.
Cells were examined under a Zeiss Axiovert microscope. BHK cells were grown as described above and infected at a multiplicity of infection of
0.2 infective units/cell (27) with recombinant pseudo-Sindbis virus
carrying the ASFV B318L gene (Sind/B318L), and at 24 h
post-infection, the cells were fixed and processed for
immunofluorescence as detailed above.
20 °C until use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Synthesis of
trans-prenyltransferase in ASFV-infected Vero
cells. A, kinetics of the synthesis of
trans-prenyltransferase in infected cells. Mock-infected
(lane 1) or ASFV-infected Vero cells were pulse-labeled with
a mixture of [35S]methionine and
[35S]cysteine for 2 h at 8 (lane 2), 13 (lane 3), 16 (lane 5), and 20 (lane 6)
h post-infection, and cell extracts were immunoprecipitated with the
anti-B318L antibody and analyzed by SDS-polyacrylamide gel
electrophoresis as described under "Experimental Procedures." The
results obtained with cells infected in the presence of 40 µg/ml
Ara-C and labeled for 2 h at 13 h post-infection are shown
(lane 4). B, accumulation of
trans-prenyltransferase in infected cells. Western blot
analysis with the anti-B318L antibody was carried out for mock-infected
(lane 1) or ASFV-infected Vero cells harvested at 4 (lane 2), 8 (lane 3), 12 (lane 4), 16 (lane 5), 20 (lane 7), and 24 (lane 8)
h post-infection. Results obtained with cells infected in the presence
of Ara-C and harvested at 16 h post-infection are shown
(lane 6). The migration positions of molecular mass markers
are shown on the left. The bands corresponding to the B318L protein are
indicated.
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Fig. 2.
Immunofluorescence microscopy of
trans-prenyltransferase in cells infected with ASFV or
with the recombinant pseudo-Sindbis virus Sind/B318L. BHK cells
were mock-infected (A) or infected with the recombinant
pseudo-Sindbis virus Sind/B318L (B and C) and
fixed at 24 h post-infection, and Vero cells were mock-infected
(D) or infected with ASFV (E-G) and fixed at
14 h post-infection. The cells were double-labeled with the
anti-B318L antibody, detected with the fluoresceinated anti-rabbit
antibody (A, B, D, and E),
and with the anti-protein-disulfide isomerase monoclonal antibody,
detected with the Texas Red-linked anti-mouse antibody (C
and F). The ASFV-infected cells were also labeled with the
fluorescent DNA dye bisbenzimide (G). The arrows
in E-G indicate the viral factory.
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Fig. 3.
Immunoelectron microscopy with anti-B318L
antibodies in ASFV-infected Vero cells. Vero cells were
mock-infected or infected with ASFV, fixed at 20 h post-infection,
and processed for cryosectioning. Ultrathin sections were incubated
with the anti-B318L antibody followed by protein A-gold (10 nm) as
described under "Experimental Procedures." A, section of
a mock-infected cell showing a region of the cytoplasm. M,
mitochondria; G, Golgi. B, section of an infected
cell showing a region of the cytoplasm containing a viral factory
(VF). The upper left area with very few gold particles
corresponds to the cytoplasmic region surrounding the viral factory.
The arrow indicates a mature virion at the limit of the
factory. C, region of a viral factory at a higher
magnification. Arrowheads indicate gold particles decorating
membrane-like structures. Bars = 200 nm in
A-C.
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Fig. 4.
Subcellular distribution and membrane
association of trans-prenyltransferase in
ASFV-infected Vero cells. Mock-infected (lanes 1 and
2) or ASFV-infected (lanes 3 and 4)
cells were disrupted at 20 h post-infection and fractionated into
cytosolic (lanes 1 and 3) and membrane
(lanes 2 and 4) fractions as described under
"Experimental Procedures." Membrane fractions from infected cells
were treated with sodium carbonate or Triton X-114 as indicated under
"Experimental Procedures": the supernatant (lane 5) and
sediment (lane 6) from carbonate-treated membrane fractions
from ASFV-infected cells and the upper (lane 7) and lower
(lane 8) phases from Triton X-114-extracted membrane
fractions from infected cells. Samples were analyzed by Western
blotting with the anti-B318L antibody. The migration positions of
molecular mass markers are shown on the left. The arrow
indicates the band corresponding to the B318L protein.
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Fig. 5.
Analysis of the products formed by the
membrane-associated trans-prenyltransferase of
ASFV. A, reversed-phase thin-layer chromatogram. The
purified recombinant ASFV trans-prenyltransferase (250 ng)
(lane 1) or membrane fractions (100 µg) of mock-infected
cells (lanes 2 and 3) or ASFV-infected cells
(lanes 4 and 5) were incubated with
[1-14C]IPP and FPP in a standard reaction mixture (see
"Experimental Procedures") in the absence (lanes 2 and
4) or presence (lanes 3 and 5) of 1%
Triton X-100. The reaction with the recombinant enzyme was performed in
the absence of detergent. After 4 h at 37 °C, the products were
treated with acid phosphatase and analyzed by reversed-phase
RP-18F254S thin-layer chromatography as described under
"Experimental Procedures." The sizes of some of the most relevant
products synthesized are indicated on the right. B, Silica
Gel 60 thin-layer chromatogram. To determine the stereochemistry of the
double bond, the radioactivity corresponding to the geranylgeraniol
bands was eluted with ether and analyzed on silica gels. Shown is
geranylgeraniol from reactions catalyzed by membrane extracts from
ASFV-infected cells (lane 1) or by the recombinant enzyme
(lane 2). Unlabeled all-trans-geranylgeraniol
(t,t,t-GGOH) and 3H-labeled
trans,trans,cis-geranylgeraniol
(t,t,c-GGOH) were used as markers. The
14C-labeled geranylgeraniol product was detected as
described under "Experimental Procedures." To determine the
migration position of the 3H-labeled
trans,trans,cis-geranylgeraniol
marker, the chromatogram was cut into rectangles, and the radioactivity
was determined by scintillation counting. The unlabeled marker was
revealed with iodine vapors. The positions of the markers are indicated
on the right. ori, origin.
Quantification of isoprenoid compounds produced by membrane-associated
enzyme
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Fig. 6.
Inhibition of the ASFV
trans-prenyltransferase using specific antibodies and
analysis of the products formed by membrane extracts of
Sind/B318L-infected BHK cells. A, the purified
recombinant ASFV trans-prenyltransferase (250 ng)
(lane 1) or membrane fractions (100 µg) of mock-infected
cells (lanes 2 and 3) or ASFV-infected cells
(lanes 4-6) were incubated with [1-14C]IPP
and FPP in a standard reaction mixture with 1% Triton X-100 (see
"Experimental Procedures") containing no antiserum (lanes
1, 2, and 4), an anti-B318L antiserum
(lanes 3 and 5), or a preimmune serum (lane
6). After 4 h at 37 °C, the products were treated with
acid phosphatase and analyzed by reversed-phase RP-18F254S
thin-layer chromatography as described under "Experimental
Procedures." The sizes of some of the most relevant products
synthesized are indicated on the right. B, membrane
fractions (50 µg) of mock-infected cells (lane 1) or
Sind/B318L-infected BHK cells (lane 2) were incubated with
[1-14C]IPP and FPP in a standard reaction mixture
containing 1% Triton X-100, and the products were analyzed as
described for A. The sizes of some of the most relevant
products synthesized are indicated on the right. ori,
origin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
virus and murine
leukemia virus (8-11). In the first case, it has been shown that the
large antigen of the virus is farnesylated (34) and that this
modification facilitates virion assembly (8, 9, 11). On the other hand,
the studies with murine leukemia virus indicate that prenylation of
cellular Rab proteins is required for the processing and incorporation
of viral envelope precursors into the virions (10). We are presently
examining whether viral or cellular proteins are prenylated during the
ASFV infectious cycle. A possible candidate to be modified by
prenylation is the viral protein encoded by the open reading frame
L83L, which contains a carboxyl-terminal CTIL type I
geranylgeranylation motif (35) and is transcribed at a late time of
infection.2 The use of drugs known to interfere with the
prenylation of proteins may also help to understand the role, if any,
of protein prenylation during the infectious cycle.
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ACKNOWLEDGEMENT |
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We thank J. Salas for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by Dirección General de Investigación Científica y Técnica Grant PB96-0902-C02-01, European Community Grant FAIR5-PL97-3441, and an institutional grant from the Fundación Ramón Areces.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Fellow of the Ministerio de Educación y Cultura of Spain.
§ Fellow of the Comunidad Autónoma de Madrid.
¶ To whom correspondence should be addressed. Tel.: 34-91-3978478; Fax: 34-91-3974799; E-mail: mlsalas{at}cbm.uam.es.
2 A. Alejo and M. L. Salas, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: IPP, isopentenyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate; ASFV, African swine fever virus; ER, endoplasmic reticulum; BHK, baby hamster kidney; Ara-C, cytosine arabinoside; Q, ubiquinone.
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