From the Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain
Received for publication, July 31, 2000, and in revised form, October 9, 2000
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ABSTRACT |
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African swine fever virus (ASFV) is a complex DNA
virus that employs polyprotein processing at Gly-Gly-Xaa sites as a
strategy to produce several major core components of the viral
particle. The virus gene S273R encodes a 31-kDa protein that contains a "core domain" with the conserved catalytic residues characteristic of SUMO-1-specific proteases and the adenovirus protease. Using a COS
cell expression system, it was found that protein pS273R is capable of
cleaving the viral polyproteins pp62 and pp220 in a specific way giving
rise to the same intermediates and mature products as those produced in
ASFV-infected cells. Furthermore, protein pS273R, like adenovirus
protease and SUMO-1-specific enzymes, is a cysteine protease, because
its activity is abolished by mutation of the predicted catalytic
histidine and cysteine residues and is inhibited by sulfhydryl-blocking
reagents. Protein pS273R is expressed late after infection and is
localized in the cytoplasmic viral factories, where it is found
associated with virus precursors and mature virions. In the virions,
the protein is present in the core shell, a domain where the products
of the viral polyproteins are also located. The identification of the
ASFV protease will allow a better understanding of the role of
polyprotein processing in virus assembly and may contribute to our
knowledge of the emerging family of SUMO-1-specific proteases.
Positive strand RNA viruses and retroviruses encode polyproteins,
which are proteolytically cleaved by viral proteases to yield the
nonstructural and structural proteins required for replication and
morphogenesis (1-3). On the other hand, DNA viruses, such as
adenoviruses and poxviruses, synthesize precursor proteins whose
maturation by proteolytic removal of terminal peptides plays an
essential role in virion formation (1).
African swine fever virus
(ASFV),1 a large and complex
virus containing a 170-kb double-stranded DNA molecule with 151 potential genes (4, 5), is atypical among DNA viruses in that it
encodes two polyproteins, pp220 and pp62, which are cleaved to produce six major structural components of the virus particle (6, 7). These
proteins, p150, p37, p34, and p14, derived from polyprotein pp220 and
p35 and p15, products of polyprotein pp62, are the major components of
the core shell, a thick protein layer that surrounds the DNA-containing
central nucleoid and that is enwrapped by the inner lipoprotein
envelope and the icosahedral capsid
(8).2 All the proteolytic
cleavages occur after the second Gly of the consensus sequence
Gly-Gly-Xaa, which is also recognized as a cleavage site in the
maturation of adenovirus structural proteins and in some cellular
proteins, including polyubiquitin (9) and ubiquitin-like proteins (10).
A similar cleavage site (Ala-Gly-Xaa) is used for the maturation of
vaccinia virus structural proteins (11, 12). Although the adenovirus
protease that processes at Gly-Gly-Xaa sites is well characterized
(13-15), the enzymes involved in the processing of the ASFV
polyproteins or in the cleavage of vaccinia virus precursor proteins
have not yet been identified.
Recently, Li and Hochstrasser (16) have described a novel cysteine
protease from yeast, designated Ulp-1, that catalyzes the release of
the ubiquitin-like protein SUMO-1/Smt3 from its precursor or from
sumoylated proteins, with the cleavage taking place at Gly-Gly-Xaa
sequences. Since the publication of this report, other SUMO-1-specific
proteases similar to the yeast enzyme have been identified in
vertebrates (17-19). These proteases would regulate the
SUMO-1-modified state of certain proteins whose function in a variety
of cellular processes, such as cell cycle progression and nuclear
import, is dependent on this modification (20). Li and Hochstrasser
(16) have also shown that a "core domain" of ~90 amino acids of
the yeast protease, containing the conserved catalytic cysteine and
histidine residues, presents some similarity to gene products from
several animal viruses, including the adenovirus L3 protease, the I7
products of fowlpox and vaccinia virus, and open reading frame (ORF)
S273R of ASFV. This suggested that the S273R product might be the
protease involved in the processing of the ASFV polyproteins. We show
here, using an expression system in COS cells, the specific processing
of polyproteins pp62 and pp220 in the presence of protein pS273R.
Furthermore, inhibitor profiling and site-directed mutagenesis results
indicate that this protein is a cysteine protease. Studies on the
synthesis and localization of protein pS273R during ASFV infection are
also presented. The results are consistent with a role for the virus protease in the assembly of the virus core.
Cells and Viruses--
Vero and COS-7 cells were obtained from
the American Type Culture Collection and grown in Dulbecco's modified
Eagle's medium containing 5% fetal calf serum. The Vero-adapted ASFV
strain BA71V was propagated and titrated as described previously (21).
Highly purified ASFV was prepared as described (22). The recombinant vaccinia virus vTF7-3 expressing bacteriophage T7 RNA polymerase (23)
was kindly donated by Dr. B. Moss.
Plasmids and Site-directed Mutagenesis--
Plasmid pRSET-S273R
carrying the ASFV S273R gene as an N-terminal hexahistidine fusion was
obtained as follows. The S273R gene was amplified by polymerase chain
reaction using oligonucleotides 273-PCR1
(5'-CGCGCGCTCGAGATGTCTATATTAGAAAAAATTACGT) and 273-PCR2 (5'-GCGCGCGAATTCTTATGCGATGCGAAACAGATGGGT), digested with
XhoI and EcoRI, and cloned into the pRSET-A
vector (Invitrogen).
The S273R gene was subcloned from pRSET-S273R into the T7 promoter
containing mammalian expression vector pcDNA 3.1 (Invitrogen), and
the recombinant plasmid pcDNA-S273R was used for activity assays in
transfection experiments. Site-directed mutagenesis of the S273R gene
was performed by polymerase chain reaction (24) using as a template the
pcDNA-S273R plasmid. The histidine residue at position 168 was
mutated to arginine (S273R,H168R mutant) and cysteine 232 was mutated
to serine (S273R,C232S mutant). Amplified mutant fragments
corresponding to the complete S273R gene were cloned into the pcDNA
3.1 vector, and the presence of the desired mutation and absence of
additional changes were confirmed by sequencing of the resulting
plasmids. The recombinant plasmid KS-CP530R containing ORF CP530R,
which encodes the pp62 polyprotein of 530 amino acids under the control
of the T7 polymerase promoter, was obtained as described (7).
The plasmid KS-p17C'f, used for the generation of antibodies against
both polyprotein pp62 and its mature p35 product, was obtained as
follows. The plasmid KS-CP530R was digested with PstI, blunted with Mung Bean exonuclease, and then digested with
Mlu I and filled in with Klenow fragment. After ligation,
this recombinant plasmid lacks the region spanning amino acid residues
15-175 of pp62 polyprotein. This plasmid was again digested with
XhoI to remove the 3' end of ORF CP530R spanning amino acid
residues 343-530.
Plasmid pGEM-CP2475L was constructed by cloning the 0.7-kb
XbaI/PstI fragment from plasmid pAR-p34 (6) and
the 6.9-kb PstI/XbaI fragment of the RC'
restriction fragment of the ASFV genome (5) into vector pGEM-4
(Promega). The resulting plasmid contained the complete CP2475L ORF,
encoding polyprotein pp220, under the control of the T7 polymerase promoter.
Antibodies--
To prepare antibodies against protein pS273R,
Escherichia coli BL21(DE3) pLysS cells harboring the
pRSET-S273R plasmid were induced for 1.5 h with 0.4 mM
isopropyl-
The mouse monoclonal antibody 18H.H7, which recognizes both the ASFV
polyprotein pp220 and its mature product p150, was used in the
immunofluorescence studies. This antibody, as well as the rabbit
polyclonal anti-p150, anti-p34, and anti-p37/p14 sera, which also
recognize polyprotein pp220, have been previously characterized (6, 8,
25).
The rabbit polyclonal serum against proteins pp62 and p35 was raised
against the 17-kDa recombinant protein encoded by plasmid KS-p17C'f.
For this, KS-p17C'f transformed E. coli BL21(DE3) cells were
induced for 2 h with 1 mM
isopropyl- Coexpression Experiments--
COS-7 cells were transfected with
250 ng of DNA/105 cells of the plasmids indicated in each
case using LipofectAMINE Plus reagent (Life Technologies, Inc.)
following the manufacturer's indications for 1 h at 37 °C. In
cotransfection experiments, the amount of pcDNA-S273R was
Preparation of Polyprotein or Protease-containing Extracts and in
Vitro Assay of Processing Activity--
COS-7 cells were
transfected/infected as described before using the corresponding
plasmids. At 16 h post-infection, the cells were resuspended at
107 cells/ml in homogenization buffer containing 20 mM HEPES, pH 7.4, 0.28 M sucrose, 2 mM EDTA, and passed through a 25-gauge syringe 15 times.
The homogenate was centrifuged at 700 × g for 5 min to
sediment nuclei and unbroken cells, and the supernatant fraction was
subsequently centrifuged at 100,000 × g for 30 min at
4 °C to separate the soluble cytoplasm from the membrane/particulate material. The activity assays in vitro were performed using
the soluble cytoplasmic fraction as source of protein pS273R and
polyprotein pp62 and the membrane/particulate cytoplasmic fraction as a
source of polyprotein pp220. Reactions were performed at 30 °C in a
final volume of 50 µl containing 5 µl of both protease and
polyprotein-containing extracts diluted in 20 mM HEPES, pH
7.4. After the indicated times, the samples were subjected to
SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting.
For inhibitor studies, the reactions were incubated for 6 h as
above in the absence or in the presence of the following protease inhibitors: 10 µM pepstatin, 1 mM
1,10-phenanthroline, 1 mM phenylmethanesulfonyl fluoride,
2.5 mM N-ethylmaleimide, 100 µM
benzyloxycarbonyl-leucyl-valyl-glycyl-diazomethylketone (Z-LVG-CHN2), or 100 µM
L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane (E-64). All the inhibitors were freshly prepared as a 100-fold concentrated stock solution in methanol with the exception of Z-LVG-CHN2 and E-64, prepared in dimethyl sulfoxide and
water, respectively.
Western Blot Analysis--
For Western blots, equivalent amounts
of whole cell extracts or cytosolic fractions were electrophoresed on
SDS-polyacrylamide gels and transferred to nitrocellulose membranes.
The membranes were incubated with a 1:1,000 dilution of the antibodies
and then with a 1:10,000 dilution of peroxidase-labeled anti-rabbit
serum (Amersham Pharmacia Biotech), and the proteins were detected with the ECL system (Amersham Pharmacia Biotech) according to the
manufacturer's recommendations.
Preparation and Analysis of RNA--
Vero 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 (26), and poly (A)+ RNA was selected by oligo
(dT)-cellulose chromatography as described by Sambrook et
al. (27). Northern blot hybridization was carried out as reported
elsewhere (28) with 2 µg of each class of poly (A)+ RNA,
using as probe the 32P-end-labeled oligonucleotide S273R
(5'-TACTTAAACAGCTATCTTTGTTTGTAAGGT), complementary to nucleotides +79
to +50 of gene S273R. Primer extension analysis was performed
essentially as described by Sambrook et al. (27). After 5'
end-labeling with 32P, the oligonucleotide S273R was
annealed at 30 °C to the different classes of RNA and extended with
avian myeloblastosis virus reverse transcriptase for 2 h at
42 °C. The primer extension products were then electrophoresed in a
6% polyacrylamide gel alongside an irrelevant DNA sequence reaction.
Immunofluorescence Microscopy--
Vero cells grown on
coverslips were mock-infected or infected with ASFV at a multiplicity
of infection of 1 plaque-forming unit/cell and fixed at 18 h
post-infection with methanol at Immunoelectron Microscopy--
Vero cells were 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. (29).
Ultrathin thawed cryosections were incubated for 45 min at room
temperature with a 1:10 dilution of the anti-pS273R antibody followed
by an incubation of 30 min at room temperature with a 1:40 dilution of
protein A-gold (diameter, 10 nm; Biocell Research Laboratories).
Specimens were examined with a Jeol 1010 microscope.
Polyprotein Processing Activity of Protein pS273R--
To
investigate the putative protease activity of protein pS273R, we have
used a system of COS-7 cells transiently coexpressing the S273R gene
and the genes coding for polyprotein pp62 and polyprotein pp220, which
served as substrates. During ASFV infection, polyprotein pp62 is first
cleaved at a Gly-Gly-Gly sequence at positions 157-159 to produce the
15-kDa structural protein p15 and an intermediate precursor of 46 kDa
(pp46). A second cleavage at the Gly-Gly-Asn site at positions 462-464
gives rise to the mature structural protein p35 of 35 kDa and to an
8-kDa polypeptide that has not been detected in the infected cells (7)
(Fig. 1A, upper
diagram). An alternative, although minor, processing
pathway, described in the lower diagram of Fig.
1A, has also been found.2 In this case,
processing first takes place at the most C-terminal cleavage site,
producing an intermediate pp52 precursor of 52 kDa. Cleavage of this
intermediate at the Gly-Gly-Gly sequence at position 157-159 then
produces the two structural proteins p15 and p35.
As shown in Fig. 1B (lanes 3 of top
and middle panels), a specific band corresponding to the
unprocessed pp62 polyprotein was detected by Western blot using both
anti-p35 and anti-p15 antibodies in lysates from cells transfected with
the polyprotein gene and infected with vTF7-3. When the polyprotein was
coexpressed with the S273R gene (Fig. 1B, top and
middle panels, lanes 4), the pp62 band strongly
decreased coinciding with the appearance of the proteolytic products
p35 (upper panel) and p15 (middle panel), which
had the same electrophoretic mobility as the structural proteins p35
and p15 detected in the samples of highly purified ASFV (Fig.
1B, top and middle panels, lanes
5). In addition, the pp46 intermediate could be detected with the
anti-p35 serum, whereas the antibody against protein p15 revealed a
specific band of 52 kDa, which probably corresponds to pp52 precursor.
The expression of gene S273R in the transfected cells is shown in the
lower panel of Fig. 1B. The protein had a size of
31 kDa, which is consistent with its predicted molecular weight of
31,550 (30). As shown in Fig. 1B (bottom panel,
lane 5), protein pS273R was also detected in purified virus particles.
To assay the activity of protein pS273R in vitro, cytosolic
extracts from COS-7 cells expressing protein pS273R or polyprotein pp62, prepared as described under "Experimental Procedures," were mixed and incubated for varying time periods, as indicated in Fig.
1C. After incubation, the samples were analyzed by Western blot using antibodies against proteins p35 (upper panel) or
p15 (lower panel). Fig. 1C shows that in the
presence of pS273R polyprotein pp62 is processed in a
time-dependent and cascade fashion, with the formation of
pp52 and pp46 intermediates, which are converted to the mature p35 and
p15 products. As can also be seen in this figure, the p15 product,
detected at the earliest time examined (15 min), increased at 1 h,
and remained constant at later times, whereas the amount of the mature
protein p35 increased considerably from 1 to 3 h of incubation.
These results suggest that, as it occurs during ASFV infection (7), the
main initial event in the in vitro processing of pp62 by
protein pS273R is the recognition of the cleavage site at position
158-159, with the formation of the mature p15 protein and preprotein
pp46. This latter protein would then be cleaved at positions 463-464
to produce the mature protein p35.
A similar in vitro assay was performed to analyze whether
protein pS273R was also able to cleave polyprotein pp220 into its mature products. During ASFV infection, polyprotein pp220 is firstly cleaved at the Gly-Gly-Gly sequence at positions 43-45 and the Gly-Gly-Ala sequence at positions 892-894 to give rise to the mature
product p150 and the intermediate precursor protein pp90 (6) (Fig.
2A). Preprotein pp90 is
subsequently cleaved at a Gly-Gly-Asp sequence at positions 367-369 to
generate the mature protein p34 and the intermediate precursor pp55,
which is finally processed at a Gly-Gly-Ala site at positions 521-523
to produce the mature products p14 and p37. As shown in Fig.
2B (lanes 1), the antibodies against products
p150 (top panel), p34 (middle panel), and p37 and
p14 (bottom panel) detected the unprocessed pp220
polyprotein in pp220 containing extracts incubated for 6 h in the
absence of protein pS273R. Additionally, lower molecular weight bands,
probably corresponding to incomplete forms of the pp220 polyprotein
produced in the transfected cells, were observed. After incubation with
pS273R containing extracts for 1 h (lanes 2), 3 h
(lanes 3), and 6 h (lanes 4), the levels of
polyprotein pp220 decreased in a time-dependent fashion
coinciding with the appearance and accumulation of proteins of 150 kDa
(upper panel), 34 kDa (middle panel), and, more
weakly, of 37 kDa, which was only visualized after a longer exposure
time (lower panel). These proteolytic products comigrated
with the mature proteins p150, p34, and p37 present in samples of
purified ASFV particles (lanes 5). Interestingly, a 90-kDa
protein was also specifically detected by the antibodies against
protein p34 (middle panel, lanes 2-4) and
proteins p37/p14 (lower panel, lanes 2-4),
whereas a 55-kDa protein was recognized only by the anti-p37/p14 serum
(lower panel, lanes 2-4). The detection of these
proteins is consistent with the formation of the intermediate
precursors pp90 and pp55 (Fig. 2A). The other mature product
p14 could not be detected in the in vitro assays because of
the poor recognition of protein p14 by Western blot analysis (see
lane 5 in the lower panel) and, possibly, because
of a low efficiency of processing of precursor pp55. These results
indicate that the two ASFV polyproteins pp220 and pp62 are accurately
cleaved in the presence of protein pS273R to give rise to the same
mature products and intermediate precursors as those produced during
ASFV infection.
Characterization of Protein pS273R as a Cysteine Protease--
As
reported by Li and Hochstrasser (16) and shown in the alignment of Fig.
3A, the ASFV protein pS273R
conserves near its C terminus the catalytic core domain of the
SUMO-1-specific proteases and the adenovirus protease containing the
four key catalytic residues, His, Glu/Asp (Asn in the case of the ASFV
protein), Gln, and Cys, which are characteristic of cysteine proteases. To investigate whether the ASFV protein was also a cysteine protease, a
number of inhibitors specific for the different classes of proteases were tested using the in vitro assay described above. As
shown in Fig. 3B, inhibitors of aspartic proteases
(pepstatin), metallo-proteases (1,10-phenanthroline), or serine
proteases (phenylmethanesulfonyl fluoride) had no effect on pp62
processing. On the other hand, two cysteine protease inhibitors,
N-ethylmaleimide and the peptidyl diazomethane
Z-Leu-Val-Gly-CHN2, completely or partially blocked, respectively, the cleavage of the polyprotein. It is of interest to
note that E-64, which is also a specific inhibitor of cysteine proteases, did not inhibit the processing activity of protein pS273R.
In this connection, it should be mentioned that the adenovirus protease
is also relatively insensitive to this compound (31, 32).
To further characterize the pS273R protein as a cysteine protease, the
conserved cysteine and histidine residues of the putative catalytic
domain were mutated to serine and arginine, respectively, and the
activity of the mutant proteins was tested. As shown in Fig.
3C, no cleavage was detected with the pS273R-C232S or
pS273R-H168R mutants, indicating that the conserved cysteine and
histidine residues are indeed critical for the activity of protein pS273R.
Expression of Gene S273R in ASFV-infected Vero Cells--
To study
the transcription of gene S273R during ASFV infection, RNA
hybridization and primer extension analysis were carried out with the
32P-labeled oligonucleotide S273R (see "Experimental
Procedures") specific for ORF S273R. 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 showed a main band of 4.5 kb, as well as
other species, in the late RNA sample (Fig.
4A). Primer extension analysis
revealed a main band of 93 nucleotides that was detected only in the
case of the late RNA sample (Fig. 4B) and corresponded to
initiation of transcription at position
Western blot analysis revealed the presence of a specific band
migrating at the expected position for protein pS273R (31 kDa) in
ASFV-infected cells (Fig. 4C). This band was first detected at 8 h post-infection, a time at which viral DNA replication in Vero cells is already underway (34), and accumulated up to 20 h
post-infection. Furthermore, the protein band was not seen in the
presence of cytosine arabinoside, an inhibitor of viral DNA replication
and late transcription, indicating that protein pS273R is a late
protein. As can also be seen in this figure, protein pS273R synthesized
in the infected cells comigrated with that present in purified virus
particles (Fig. 4C, lane 9).
Localization of Protein pS273R in Infected Cells--
To study the
intracellular localization of pS273R protein, mock-infected and
ASFV-infected Vero cells were fixed at 18 h after infection and
analyzed by immunofluorescence with the anti-pS273R serum. As shown in
Fig. 5A, a low background
signal was observed in the mock-infected cells used as a control. In
contrast, the antibody against protein pS273R strongly stained discrete
cytoplasmic areas of the virus-infected cells (Fig. 5B),
which were identified as virus factories by immunolabeling with a
monoclonal antibody that recognizes the polyprotein precursor pp220 and
its mature product p150 (Fig. 5C) (8). As previously
reported, the antibody to pp220/p150 proteins also labeled virus
particles scattered throughout the cytoplasm (8). Although more weakly,
the anti-pS273R serum also stained cytoplasmic clusters of virions
outside the assembly areas (Fig. 5B), suggesting that
protein pS273R is incorporated into the virus particles, in agreement
with the immunoblotting experiments performed with purified
virions.
To examine in more detail the localization of ASFV protease within the
virus factories, we carried out an immunoelectron microscopic analysis
of Vero cells infected with ASFV for 20 h. For this, thawed
cryosections were incubated with the anti-pS273R antibody followed by
protein A-gold. As shown in Fig.
6A, the antibody labeled the
region of the viral factories, in keeping with the immunofluorescence
results. Within the assembly sites, labeling was essentially associated
with both intermediate virus structures (Fig. 6B) and mature
virions (Fig. 6C). Interestingly, gold grains were localized
within the core region of immature icosahedral particles that lack the
electron dense DNA-containing nucleoid. Within mature particles, the
signal was associated with the core shell (Fig. 6C), the
protein domain surrounding the electron dense nucleoid and containing
the polyprotein products (8).
A new family of structurally related cysteine proteases that
process SUMO-1-modified proteins at consensus Gly-Gly-Xaa sequences has
been recently described (16). The sequence similarity between the
members of this family is largely confined to a C-terminal region of
about 200 amino acids that contains a 90-amino acid core domain that is
also present in the adenovirus protease, as well as in the poxvirus I7
protein and protein pS273R of ASFV. This core domain contains the
active site triad of the adenovirus protease, composed of residues
His54, Glu71, and Cys122, which in
the crystal structure of this enzyme have a spatial disposition
identical to that of the catalytic triad of the classical cysteine
protease papain (35), although the sequential order of the residues in
the polypeptide chain of this enzyme is Cys25,
His159, and Asn175. In addition, a fourth
residue, Gln115, of the adenovirus protease is in an
equivalent spatial position to the Gln19 of papain involved
in the formation of the oxyanion hole in the active site. The ASFV
protein pS273R conserves the His, Cys, and Gln residues, and, as in the
case of papain, has an Asn at the position of the adenovirus protease
Glu71 residue. This sequence similarity strongly argued
that this protein was the protease implicated in the proteolytic
processing of ASFV polyproteins at Gly-Gly-Xaa sites.
The results presented here show that protein pS273R processes the virus
polyproteins in a specific and accurate way giving rise to the same
intermediates and final mature products as those produced in
ASFV-infected cells, suggesting that this protein is the protease
involved in polyprotein processing during the infection. Furthermore,
and as expected from the sequence comparison, the protein has been
characterized as a cysteine protease, because its activity is abrogated
by mutation of the critical His and Cys residues of the conserved
catalytic domain and is inhibited by sulfhydryl-blocking reagents.
Taking into account the above findings, the ASFV protease can be
included in the same group of cysteine proteases as the SUMO-1-specific proteases and the protease from adenovirus. All of these proteins are
characterized by the presence of unrelated N-terminal extensions of
variable lengths, which most likely account for the specificity of the
substrate. In relation to this, it is worth noting that the ASFV
protease is the only enzyme of this group that processes polyprotein
precursors recognizing several Gly-Gly-Xaa sites in the substrate
sequence. Interestingly, during ASFV infection, this processing occurs
through an ordered cascade of proteolytic cleavages, which may depend
on conformational changes in the intermediate products or on
interaction with other proteins during virus morphogenesis (6, 7). The
identification of the viral protease will help to elucidate the
mechanisms underlying the cascade processing of the viral polyproteins.
It is also possible that the viral enzyme might be able to cleave
cellular or viral ubiquitin-like modified proteins. This is an
interesting possibility that may provide insight into the structural
and functional aspects of this emerging family of cysteine proteases.
The studies on the expression and localization of protein pS273R in
cells infected with ASFV further support a role for this protein in the
processing of the viral polyproteins during the virus morphogenesis.
Thus, protein pS273R, as its polyprotein substrates, is synthesized at
late times of infection and is localized within the cytoplasmic viral
factories where morphogenesis occurs.
We have previously shown that the mature proteins p150, p37, p34, and
p14, products of polyprotein pp220, are major components of the core
shell, representing about 25% of the total protein mass of the virus
particle (8). The same location has been found for the structural
proteins p35 and p15, which are derived from polyprotein
pp62.2 These findings, together with the observed
localization of protein pS273R in the virus morphogenetic
intermediates, suggest a role for the ASFV protease in the assembly of
the viral core. Thus, proteolytic processing might be an essential
mechanism for the spatio-temporal control of the interactions between
core components, in such a way that only preassembled and properly
processed substrates could enter the assembly pathway. In connection
with this, it should be mentioned that other complex DNA-containing
viruses, such as adenoviruses and poxviruses, use proteolytic
processing at similar or identical cleavage sequences for the
maturation of major core proteins. In the case of poxviruses, the
cleavage of core precursors is crucial for the formation of virus
particles, which does not take place if the processing is blocked (36). In contrast, the adenovirus protease acts in newly assembled virions, being needed for their maturation into infectious virions (37). To
ensure that processing of the viral precursor proteins takes place only
after virion assembly, this enzyme employs a unique and complex
regulatory mechanism, involving stimulation of its activity by peptide
and DNA cofactors (13, 14). Further studies will be needed to ascertain
whether similar mechanisms operate to control the temporal and spatial
activity of the ASFV protease.
As has also been described for the adenovirus protease and the I7
protein of poxviruses (38, 39), the ASFV protein pS273R is present in
the core of mature virus particles. This finding could merely reflect a
residual presence of an enzymatic activity needed during the final
steps of ASFV morphogenesis. Another possibility is that protein pS273R
is involved in some early event of viral infection. This is the case
for the adenovirus protease that has been implicated in virus entry
into the host cell and disassembly of the incoming virus particles (40,
41). Whether the ASFV protease, besides a morphogenetic role late in
the virus replicative cycle, might also cleave other viral or even
cellular substrates early in infection is a question that remains to be addressed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside, and the recombinant
protein was purified under denaturing conditions using single-step
Ni2+-nitrilotriacetic acid affinity chromatography.
Antibodies against the purified recombinant pS273R protein were raised
in rabbits.
-D-thiogalactopyranoside and then incubated
for 2 h with rifampicin (0.2 mg/ml). After lysis and
centrifugation, the pellet containing the recombinant protein as the
main component was used to generate antibodies in rabbits. The rabbit
polyclonal serum against ASFV polyprotein pp62 and its mature product
p15 has already been described (7).
of that of the polyprotein gene-containing plasmid.
Subsequently, the cells were infected with vTF7-3 at a multiplicity of
infection of 5 plaque-forming units/cell, allowing the expression of T7
RNA polymerase and thus of the polyprotein and S273R genes. The
infection was carried out in the presence of 40 µg/ml cytosine
arabinoside to inhibit vaccinia virus late protein synthesis. The
expression and processing of the polyprotein was analyzed at 16 h
post-infection by Western blot, as indicated below.
20 °C for 5 min. The cells were
incubated with a 1:250 dilution of rabbit polyclonal anti-pS273R serum
and a 1:10 dilution of mouse monoclonal (18H.H7) anti-pp220 antibody as
indicated in the legend to Fig. 5 and incubated for 45 min at 37 °C
with a 1:500 dilution of Alexa 488 goat anti-rabbit IgG and a 1:500
dilution of Alexa 594 goat anti-mouse IgG antibodies (Molecular Probes,
Inc.). As a control, each antibody was individually tested. Cells were
examined with an Axiovert fluorescent microscope (Carl Zeiss,
Inc.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Proteolytic processing of pp62 polyprotein by
protein pS273R. A, schematic representation of
polyprotein pp62 processing. The two alternative processing pathways
are shown. The precursor proteins are indicated by white
boxes, and mature products are shown by black boxes.
The cleavage sites are indicated by arrowheads. B, COS-7
cells were transfected with KS and pcDNA 3.1 plasmids containing
the pp62 and S273R genes, respectively, and infected with vTF7-3 as
indicated under "Experimental Procedures." 16 h after
infection, expression of the transfected genes was analyzed by Western
blot, using antibodies against proteins p35 (upper panel),
p15 (middle panel), or pS273R (lower panel).
Lanes 1, not infected and not transfected cells; lanes
2, cells infected but not transfected; lanes 3, cells
infected and transfected with pp62 gene; lanes 4, cells
infected and cotransfected with genes pp62 and S273R; lanes
5, 0.5 µg of highly purified ASFV. The bands corresponding to
polyprotein pp62 and its proteolytic products and to protein pS273R are
indicated on the right. The migration position of molecular
mass markers is indicated on the left. C, time course
of polyprotein pp62 processing by protein pS273R in vitro.
Cytosolic extracts containing pp62 or pS273R proteins, prepared as
described under "Experimental Procedures," were mixed and incubated
for 15 min (lanes 2), 1 h (lanes 3), or
3 h (lanes 4). As a control, the extract containing
polyprotein pp62 alone was incubated for 6 h (lanes 1).
Polyprotein processing was analyzed by Western blot using anti-p35
(upper panel) or anti-p15 (lower panel)
antibodies. The bands corresponding to polyprotein pp62 and its
products are indicated on the right.
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Fig. 2.
Proteolytic processing of pp220 polyprotein
by protein pS273R. A, schematic representation of
polyprotein pp220 processing during ASFV infection. The precursor
proteins are indicated by white boxes, and mature products
are indicated by black boxes. The cleavage sites are
indicated by arrowheads. B, time course of polyprotein pp220
processing by protein pS273R in vitro. COS-7 cells were
transfected with pGEM and pcDNA 3.1 plasmids containing the pp220
and S273R genes, respectively, and infected with vTF7-3 as indicated
under "Experimental Procedures." Membrane/particulate extracts,
containing pp220 protein, and cytosolic extracts, containing pS273R
protein, prepared as indicated under "Experimental Procedures,"
were mixed and incubated for 1 h (lanes 2), 3 h
(lanes 3), and 6 h (lanes 4). As a control,
the extract containing polyprotein pp220 alone was incubated for 6 h (lanes 1). Polyprotein processing was analyzed by Western
blot using anti-p150 (top panel), anti-p34 (middle
panel), and anti-p37/p14 (bottom panel) antibodies. A
longer exposure of the region corresponding to protein p37 is shown in
a separate box in the bottom panel. Lanes 5 show the
migration of structural proteins p150, p34, and p37 from 0.5 µg of
highly purified ASFV. The bands corresponding to polyprotein pp220 and
its products are indicated on the right. Note that the
anti-p37/p14 serum (bottom panel) did not detect protein p14
neither in the in vitro assay (lanes 2-4) nor in
the virus sample (lane 5). Molecular masses are indicated on
the left.
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Fig. 3.
Characterization of protein pS273R as a
cysteine protease. A, sequence alignment of the
conserved catalytic core domain of protein pS273R and members of the
SUMO-1-specific protease family. Multiple alignment of 15 sequences was
performed online by using the CLUSTALW program (42) at the European
Bioinformatics Institute. The alignment of five selected sequences is
presented in the figure. Numbers at left and
right indicate the amino acid position relative to the N
terminus of each protein. Residues conserved or identical in at least
80% of the 15 sequences aligned are in bold type over a
gray background. The invariant residues in the 15 sequences
are indicated with an asterisk. Full triangles mark the
amino acid residues of the catalytic triad (His, Asn/Asp/Glu, and Cys)
and the Gln residue involved in the formation of the oxyanion hole in
the active site. Sequences: ASFV S273R, protein pS273R of
ASFV (Q00946); VACCV I7, vaccinia virus I7 protein (P12926);
ADE2 VPRT, human adenovirus 2 protease (P03252); SENP1
Hs, human sentrin-specific protease (AF149770); Ulp1
Sc, Saccharomyces cerevisiae Ulp1 protease (S63462).
B, effect of protease inhibitors on the processing of pp62.
Cytosolic extracts containing polyprotein pp62 or protein pS273R were
mixed and incubated for 6 h at 30 °C in the absence ( lanes) or presence of 10 µM pepstatin
(Peps), 1 mM 1,10-phenanthroline
(Phen), 1 mM phenylmethanesulfonyl fluoride
(PMSF), 2.5 mM N-ethylmaleimide
(NEM), 100 µM Z-LVG-CHN2
(LVG), or 100 µM E-64. A control reaction
containing the proteolytic substrate pp62 alone is also presented
(
). The samples were then analyzed by Western blot using
an anti-p35 antibody. The pp62 and p35 bands are indicated.
C, assay of processing activity of S273R-C232S and
S273R-H168R mutants. Processing activity was determined as in
B after incubation of polyprotein pp62 alone
(
), or with wild type S273R (wt), S273R-C232S
(C232S), or S273R-H168R (H168R). Western blot
analysis of reaction products and protein pS273R was performed using
anti-p35 and anti-pS273R antibodies, respectively. The bands
corresponding to pp62, p35, and pS273R are indicated.
15 relative to the first
nucleotide of the translation start codon of ORF S273R. A motif
composed of seven or more consecutive thymidylate residues (the 7T
motif) has been identified as a signal for 3'-end formation of ASFV
mRNAs (33). The first 7T motif after ORF S273R is found 52 nucleotides downstream of the contiguous P1192R ORF coding for the
virus topoisomerase II (30). Termination of transcription at this motif
would produce an RNA of 4497 bases, which is consistent with the
size of the main RNA band detected by Northern blot.
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Fig. 4.
Expression of gene S273R in ASFV-infected
Vero cells. A, Northern blot analysis of RNA 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). The arrow indicates
the 4.5-kb RNA band corresponding to the S273R gene. B,
primer extension analysis of the 5' end of S273R transcripts. The same
classes of RNA were used. After extension, the samples were
electrophoresed beside an irrelevant DNA sequencing reaction (DNA
ladder). The size of the major DNA fragment obtained is indicated.
C, Western blot analysis with the anti-pS273R antibody was
carried out for mock-infected cells (lane 1) or
ASFV-infected cells harvested at 4 (lane 2), 8 (lane
3), 10 (lane 4), 12 (lane 5), 14 (lane
6), and 20 (lane 7) h post-infection. Results obtained
with cells infected for 20 h in the presence of cytosine
arabinoside (lane 8) and with 2 µg of highly purified ASFV
particles (lane 9) are also shown. The migration positions
of molecular mass markers are shown on the left. The band
corresponding to protein pS273R is indicated on the
right.
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Fig. 5.
Immunfluorescence detection of protein pS273R
in Vero cells infected with ASFV. Mock-infected (A) or
ASFV-infected (B and C) Vero cells were fixed at
18 h post-infection and double-labeled with anti-pS273R antibody
detected with Alexa 488 goat anti-rabbit IgG (A and
B) and with anti-pp220 monoclonal antibody detected with
Alexa 594 goat anti-mouse IgG (C). In B and
C, the viral factories and clusters of virions, labeled with
both antibodies, are indicated by arrows and
arrowheads, respectively.
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Fig. 6.
Immunoelectron microscopy with anti-pS273R
serum in ASFV-infected Vero cells. Vero cells were infected with
ASFV, fixed at 20 h post-infection, and processed for
cryosectioning. Ultrathin sections were incubated with the anti-pS273R
antibody followed by protein A-gold (10 nm) as described under
"Experimental Procedures." A, section of an infected
cell showing a region of the cytoplasm containing a viral factory
(VF). B and C, regions of a viral
factory at a higher magnification. In B, the labeling is
associated with the core region of immature virus particles
(arrows). In C, gold particles are localized in
the core shell of mature virions (arrows). The core shell
domain is delimited in the right upper virus. Bars, 100 nm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank J. Salas and C. López-Otín 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-CT97-3441, Ministerio de Educación y Cultura Grant AGF98-1352-CE, 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.
The first two authors contributed equally to this work.
§ Fellow of the Comunidad Autónoma de Madrid.
¶ Present address: Centro Nacional de Biotecnología (CSIC), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
To whom correspondence should be addressed: Centro de
Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad
Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Tel.: 34-913978478; Fax: 34-913974799; E-mail:
mlsalas@cbm.uam.es.
Published, JBC Papers in Press, October 12, 2000, DOI 10.1074/jbc.M006844200
2 G. Andrés, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: ASFV, African swine fever virus; kb, kilobase(s); ORF, open reading frame; Z-LVG-CHN2, benzyloxycarbonyl-leucyl-valyl-glycyl-diazomethylketone; E-64, L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane.
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REFERENCES |
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---|
1. | Kräusslich, H.-G., and Wimmer, E. (1988) Annu. Rev. Biochem. 57, 701-754[CrossRef][Medline] [Order article via Infotrieve] |
2. | Dougherty, W. G., and Semler, B. L. (1993) Microbiol. Rev. 57, 781-822[Abstract] |
3. | Gorbalenya, A. E., and Snijder, E. J. (1996) Persp. Drug Discov. Design 6, 64-86 |
4. | Viñuela, E. (1985) Curr. Top. Microbiol. Immunol. 116, 151-170[Medline] [Order article via Infotrieve] |
5. | Yáñez, R. J., Rodríguez, J. M., Nogal, M. L., Yuste, L., Enríquez, C., Rodriguez, J. F., and Viñuela, E. (1995) Virology 208, 249-278[CrossRef][Medline] [Order article via Infotrieve] |
6. | Simón-Mateo, C., Andrés, G., and Viñuela, E. (1993) EMBO J. 12, 2977-2987[Abstract] |
7. | Simón-Mateo, C., Andrés, G., Almazán, F., and Viñuela, E. (1997) J. Virol. 71, 5799-5804[Abstract] |
8. | Andrés, G., Simón-Mateo, C., and Viñuela, E. (1997) J. Virol. 71, 2331-2341[Abstract] |
9. |
López-Otín, C.,
Simón-Mateo, C.,
Martínez, L.,
and Viñuela, E.
(1989)
J. Biol. Chem.
264,
9107-9110 |
10. |
Kamitani, T.,
Nguyen, H. P.,
and Yeh, E. T. H.
(1997)
J. Biol. Chem.
272,
14001-14004 |
11. | VanSlyke, J. K., Whitehead, S. S., Wilson, E. M., and Hruby, D. E. (1991) Virology 183, 467-478[Medline] [Order article via Infotrieve] |
12. |
Lee, P.,
and Hruby, D. E.
(1994)
J. Biol. Chem.
269,
8616-8622 |
13. | Webster, A., Hay, R. T., and Kemp, G. (1993) Cell 72, 97-104[Medline] [Order article via Infotrieve] |
14. |
Mangel, W. F.,
Toledo, D. L.,
Brown, M. T.,
Martin, J. H.,
and McGrath, W. J.
(1996)
J. Biol. Chem.
271,
536-543 |
15. | Ding, J., McGrath, W. J., Sweet, R. M., and Mangel, W. F. (1996) EMBO J. 15, 1778-1783[Abstract] |
16. | Li, S.-J., and Hochstrasser, M. (1999) Nature 398, 246-251[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Suzuki, T.,
Ichiyama, A.,
Saitoh, H.,
Kawakami, T.,
Omata, M.,
Chung, C. H.,
Kimura, M.,
Shimbara, N.,
and Tanaka, K.
(1999)
J. Biol. Chem.
274,
31131-31134 |
18. |
Gong, L.,
Millas, S.,
Maul, G. G.,
and Yeh, E. T. H.
(2000)
J. Biol. Chem.
275,
3355-3359 |
19. |
Kim, K. I.,
Baek, S. H.,
Jeon, Y-J.,
Nishimori, S.,
Suzuki, T.,
Uchida, S.,
Shimbara, N.,
Saitoh, H.,
Tanaka, K.,
and Chung, C. H.
(2000)
J. Biol. Chem.
275,
14102-14106 |
20. | Yeh, E. T. H., Gong, L., and Kamitani, T. (2000) Gene (Amst.) 248, 1-14[CrossRef][Medline] [Order article via Infotrieve] |
21. | Enjuanes, L., Carrascosa, A. L., Moreno, M. A., and Viñuela, E. (1976) J. Gen. Virol. 32, 471-477[Abstract] |
22. | Carrascosa, A. L., del Val, M., Santarén, J. F., and Viñuela, E. (1985) J. Virol. 54, 337-344[Medline] [Order article via Infotrieve] |
23. | Fuerst, T. R., Niles, E. G., Studier, F. W., and Moss, B. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8122-8126[Abstract] |
24. | Herlitze, S., and Koenen, M. (1990) Gene (Amst.) 91, 143-147[Medline] [Order article via Infotrieve] |
25. | Sanz, A., García-Barreno, B., Nogal, M. L., Viñuela, E., and Enjuanes, L. (1985) J. Virol. 54, 199-206[Medline] [Order article via Infotrieve] |
26. | Chirgwin, J. M., Przbyla, A. E., McDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299[Medline] [Order article via Infotrieve] |
27. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., pp. 7.26-7.29 and 7.79-7.83 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
28. | Yáñez, R. J., Rodríguez, J. M, Salas, M. L., and Viñuela, E. (1993) J. Gen. Virol. 74, 1633-1638[Abstract] |
29. |
Andrés, G.,
García-Escudero, R.,
Simón-Mateo, C.,
and Viñuela, E.
(1998)
J. Virol.
72,
8988-9001 |
30. | García-Beato, R., Freije, J. M., López-Otín, C., Blasco, R., Viñuela, E., and Salas, M. L. (1992) Virology 188, 938-947[Medline] [Order article via Infotrieve] |
31. |
Tihanyi, K.,
Bourbonnière, M.,
Houde, A.,
Rancourt, C.,
and Weber, J. M.
(1993)
J. Biol. Chem.
268,
1780-1785 |
32. | Sircar, S., Ruzindana-Umunyana, A., Neugebauer, W., and Weber, J. M. (1998) Antiviral Res. 40, 45-51[CrossRef][Medline] [Order article via Infotrieve] |
33. | Almazán, F., Rodríguez, J. M., Andrés, G., Pérez, R., Viñuela, E., and Rodriguez, J. F. (1992) J. Virol. 66, 6655-6667[Abstract] |
34. | Salas, M. L., Rey-Campos, J., Almendral, J. M., Talavera, A., and Viñuela, E. (1986) Virology 152, 228-240[Medline] [Order article via Infotrieve] |
35. | Drenth, J., Kalk, K. H., and Swen, H. M. (1976) Biochemistry 15, 3731-3738[Medline] [Order article via Infotrieve] |
36. | Moss, B., and Rosenblum, E. N. (1973) J. Mol. Biol. 81, 267-269[Medline] [Order article via Infotrieve] |
37. | Weber, J. (1976) J. Virol. 17, 462-471[Medline] [Order article via Infotrieve] |
38. | Anderson, C. W.,. (1990) Virology 177, 259-272[Medline] [Order article via Infotrieve] |
39. | Kane, E. M., and Shuman, S. (1993) J. Virol. 67, 2689-2698[Abstract] |
40. | Cotten, M., and Weber, J. M. (1995) Virology 213, 494-502[CrossRef][Medline] [Order article via Infotrieve] |
41. | Greber, U. F., Webster, P., Weber, J., and Helenius, A. (1996) EMBO J. 15, 1766-1777[Abstract] |
42. | Higgins, D. G., Thompson, J. D., and Gibson, T. J. (1996) Methods Enzymol. 266, 383-402[Medline] [Order article via Infotrieve] |