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J. Biol. Chem., Vol. 276, Issue 38, 35473-35481, September 21, 2001
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From the Institute of Medical Biochemistry, Division of
Biochemistry, University of Vienna, Vienna BioCenter,
Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria
Received for publication, May 9, 2001, and in revised form, July 9, 2001
The leader proteinase (Lpro) of
foot-and-mouth disease virus frees itself from the nascent polyprotein,
cleaving between its own C terminus and the N terminus of VP4 at the
sequence Lys-Leu-Lys- Specific proteolysis is an essential component of the life cycle
of many viruses. Virally encoded proteinases are required not only to
process viral protein precursors into the mature proteins but also to
cleave host proteins to modulate the physiology of the infected cell
(1, 2). A classic example is the inhibition of host protein synthesis
from capped cellular mRNA that occurs during the replication of
most picornaviruses (3). These viruses mediate this through the
cleavage of the two homologues of eukaryotic initiation factor 4G
(eIF4GI1 and eIF4GII) by
either the Lpro of FMDV or by the 2A proteinase of human
rhinoviruses, Coxsackie virus, and polioviruses (4-8). As a
consequence of the cleavage of the eIF4G homologues, the domain of
eIF4G that binds the cap-binding protein eIF4E is severed from the
domain of eIF4G which binds eIF3, so that the infected cell is unable
to recruit its own capped mRNA to the 40 S ribosome (Fig.
1A (9, 10)). Translation of
viral mRNA is unaffected as it initiates internally via an IRES
(11, 12).
The exact mechanism of the cleavage of eIF4GI and eIF4GII by the
picornaviral proteinases is still unresolved. As this cleavage occurs
before measurable amounts of viral proteins can be detected in
vivo (13), it has been proposed that the viral proteinases induce
cleavage of the eIF4G homologues by activating an as yet unidentified
cellular proteinase (5, 14). This idea was supported by the inability
to imitate in vitro the efficiency of eIF4GI cleavage using
purified recombinant proteinases (15-17). However, we have recently
shown that eIF4GI can be efficiently cleaved in RRLs by nascently
translated proteinases at concentrations close to those calculated
in vivo (18).
The Lpro of FMDV, which carries out the cleavage of the
eIF4G homologues, is a papain-like cysteine proteinase (19, 20); it is
the first protein encoded on the FMDV polyprotein (Fig. 1, B
and C). Lpro frees itself by cleavage between
its own C terminus and the N terminus of VP4. As the initiation of
protein synthesis on the FMDV RNA can occur at one of two AUG codons
lying 84 nucleotides apart, two forms of the Lpro are
synthesized (designated Labpro and Lbpro). The
reason for this is not clear, as both forms appear to have the same
enzymatic properties (21). All work described here was carried out with
the Lbpro form.
Recognition and cleavage of the sites on the polyprotein and on the
eIF4G homologues present a number of questions. Cleavage at the
junction of Lbpro and VP4 occurs at
Lys-Leu-Lys201- A further open question is the mechanism of self-processing (20, 21,
24). X-ray structural data provided evidence for an intermolecular
mechanism, as the CTE of one Lbpro molecule was bound by
the active site of the neighboring molecule (20). However, modeling
studies using this structure suggested that a CTE would be able to
reach into the active site of the same molecule, hinting that an
intramolecular reaction was possible. Finally, it is not known whether
self-processing is a prerequisite for cleavage of the eIF4G homologues.
In this paper, we examine these questions by expressing various mutants
of Lbpro in RRLs and investigating the kinetics of cleavage
of the polyprotein and eIF4GI.
Plasmids--
The plasmid pet8cFMDV Lbpro (encoding
the mature Lbpro that consists of amino acids 29-201
of the FMDV polyprotein) contains the FMDV nucleotides 892-1413 of the
FMDV O1k cDNA (25) followed by two stop codons, cloned
into the NcoI and BamHI restriction sites of the
T7 polymerase expression vector pET8c (Novagen). The plasmid pet8cFMDV
LbproVP4VP2 (encoding the mature Lbpro, all 85 amino acids of VP4 and 78 amino acids of VP2) contains the FMDV
nucleotides 892-1896 followed by two stop codons similarly cloned into
pet8c (13). In pet8cFMDV LbproC51A VP4VP2, the active site
cysteine 51 is replaced by an alanine.
The plasmid pet8c
To construct pCITE LbproVP4VP2, the LbproVP4VP2
expression block was excised from pet8cFMDV LbproVP4VP2
using the XbaI site of pET8c and the BamHI site
at the 3' end of the expression block and subcloned into pBluescript. The LbproVP4VP2 was then introduced as an NcoI
PstI fragment into pCITE1 which had been cleaved by
NcoI and PstI. In this construction, translation
initiates at an AUG codon three codons upstream from the first
Lbpro codon. On linearization with SalI and
transcription with T7 polymerase, an RNA is produced containing the
encephalomyocarditis virus IRES which subsequently encodes the
Lbpro with three extra amino acids at the N terminus, all
85 amino acids of VP4 and 23 amino acids of VP2.
In Vitro Mutagenesis--
Restriction sites for
Bpu10I and SacI were introduced into pCITE
LbproVP4VP2 at the positions indicated in Fig.
2A using standard methods of
PCR mutagenesis; the mutations do not change the amino acid sequence.
Replacement of the 38-base pair Bpu10I SacI
fragment with synthetic oligonucleotides enables the amino acid
substitutions indicated in Table I to be introduced at the
Lpro VP4 junction. Lbpro C-terminal variants
were generated in pet8cLbpro by replacing the 47-base pair
C-terminal BsiWI BamHI fragment with the required
synthetic oligonucleotides as described previously (23). All mutations
were confirmed by sequencing.
In Vitro Transcription and Translation--
Plasmids were
linearized with the indicated restriction enzyme and transcribed as
described (18). In vitro translation reactions (typically 50 µl) contained 70% RRL (Promega), 20 µCi of
[35S]methionine (1000 Ci/mmol, American Research
Company), and amino acids (except methionine) at 20 µM.
After preincubation for 2 min at 30 °C, translation was started by
addition of RNA. RNA concentrations (typically about 10 ng/µl) were
adjusted so that the Lbpro concentration reached between 15 and 20 pg/µl after 8 min of incubation, unless otherwise stated.
Aliquots (10 µl) were removed at the designated time points, and the
reaction was stopped by immediate transfer to ice and the addition of
unlabeled methionine and cysteine to a final concentration of 2 mM and Laemmli sample buffer.
Electrophoresis and Immunoblotting--
The PAGE system of Dasso
and Jackson (26) was used for separation of translation products (gels
contained 15% acrylamide) and for monitoring the state of eIF4GI (gels
contained 6% acrylamide). Translation products were detected by
fluorography; the state of eIF4GI was determined by immunoblotting
using the anti-eIF4GI peptide 7 antiserum (27) as described (18).
Quantification of Protein Synthesis in RRLs--
Quantification
of protein synthesis using an Instant Imager (Canberra Packard) was as
described previously, except that calculations were adjusted for the
use of [35S]methionine alone (18). Briefly, radioactivity
in a particular band in the dried polyacrylamide gel was counted. By
taking into account the counting efficiency (0.8%), the specific
activity of the methionine in the assay (163 dpm/fmol), the
number of methionines in Lbpro, and the molecular weight of
Lbpro (19.8 kDa), the amount of protein present in a
particular band can be estimated.
What Is the Relationship between Self-processing of
Lbpro and eIF4GI Cleavage?--
To investigate whether
self-processing of the Lbpro from the growing polypeptide
chain was a prerequisite for the cleavage of eIF4GI, it was necessary
to produce mutant enzymes that could not process themselves from the
growing polypeptide chain but that still possessed an intact active
site and a correctly folded structure. Therefore, we introduced
specific mutations into the cleavage site between Lbpro
and VP4VP2. To simplify this, we noted that restriction sites for
Bpu10I and SacI could be introduced before and
after the site of Lbpro cleavage without affecting the
amino acid sequence (Fig. 2A). However, the previously
employed transcription vector pet8c also contained a Bpu10I
site, making it necessary to move the LbproVP4VP2
expression cassette into pCITE1 which has itself no restriction sites
for Bpu10I and SacI. The ensuing plasmid was
designated pCITE LbproVP4VP2 (Fig. 2A).
To examine Lbpro self-processing and eIF4GI cleavage using
pCITE LbproVP4VP2, a standard translation reaction was
performed (Fig. 2, B and C, left panels). The
cleavage products Lbpro and VP4VP2 are visible on this
fluorogram after 8 min. Lbpro encoded by pCITE
LbproVP4VP2 has four methionines, whereas VP4VP2 has only
two; thus, the intensity of the Lbpro band is about twice
that of VP4VP2. The high efficiency of self-processing is indicated by
the low amount of the uncleaved precursor (LbproVP4VP2).
Table I shows the extent of
self-processing at 12 min.
The cleavage of the endogenous eIF4GI in the RRL under these conditions
occurs rapidly (Fig. 2C, left panel); 50% cleavage was
observed between 4 and 8 min. The concentration of Lbpro at
8 min was determined to be 15 pg/µl by counting the band in the
Instant Imager. These results are almost identical to those previously
reported, using pet8c LbproVP4VP2 driven from an uncapped
RNA that did not contain an IRES (18).
The mutations indicated in Table I were then introduced at the
Lbpro VP4 cleavage site using synthetic oligonucleotides.
First, the P1 lysine was replaced by glutamine
(LbproVP4VP2(K201Q)), as the Lbpro
three-dimensional structure (20) had revealed that the P1 lysine residue is bound within a deep groove consisting of three glutamate residues. Their aliphatic side chains bind the lysine side chain, whereas the amino group of the lysine interacts with the carboxyl groups of the glutamates. We reasoned that the uncharged glutamine residue would lack the electrostatic interaction and that the shorter
side chain would further cause the amide group to clash with the
glutamate side chains. Indeed, the pattern of proteins synthesized from
this RNA is different from that of the wild-type RNA (Fig. 2B,
middle panel). After 12 min, self-processing drops from over 85%
in the wild-type to 37.5% in the mutant protein (Table I). Thus,
although the self-processing reaction of Lbpro is impaired
by this mutation, it is not completely inhibited.
The activity of this mutant protein on the cleavage of endogenous
eIF4GI in the RRL was then examined by immunoblotting (Fig. 2C,
middle panel). In contrast to the effect on self-processing, no
effect on the processing of eIF4GI was observed. Reduction of the
amount of RNA in the RRL also failed to reveal any differences in the
rate of eIF4GI processing between the wild-type and the mutant protein
(data not shown).
As the K201Q mutant protein still possessed some self-processing
activity, we wished to examine whether a complete inhibition of
self-processing would eliminate the mature Lbpro from the
reaction and thus affect the kinetics of eIF4GI processing. Therefore,
we substituted the P2 leucine with serine in addition to the P1 lysine
to glutamine substitution; the encoded mutant protein was designated
LbproVP4VP2(L200S,K201Q). As both the Lbpro and
eIF4GI cleavage sites have leucine at the P2 position (13) and the
Lbpro structure shows a deep hydrophobic pocket for the
binding of the P2 residue, we reasoned that a P2 serine residue would
not be accepted and that its presence in addition to the P1 mutation would inhibit any self-processing activity. Fig. 2B
(right panel) shows that this is indeed the case. After 12 min, only the uncleaved product was visible. Remarkably, however, no
effect on the cleavage of eIF4GI was observed (Fig. 2C, right
panel); the kinetics of cleavage are almost identical to those of
the wild-type enzyme.
The above changes in the amino acid sequence at the cleavage junction
clearly affected the self-processing reaction but not the cleavage of
eIF4GI by Lbpro. To produce a second mutant protein that
was handicapped in self-processing, we chose to replace the P1 lysine
with glycine (LbproVP4VP2(K201G); Table I) and then
examined the effect on eIF4GI cleavage. Fig.
3A (compare left
and center panels) shows that the absence of a basic group
before the scissile bond reduced self-processing to about the level
seen with the K201Q mutant protein (Table I). Once again, however, no
reduction in the activity of the Lbpro on eIF4GI was
observed (Fig. 3B, left and center panels).
Finally, to confirm the hypothesis that the presence of a basic residue
at either P1 or P1' was sufficient to allow cleavage by the
Lbpro, we used the oligonucleotide cassette to introduce an
arginine residue into the K201G mutant protein at the P1' position
(i.e. to replace residue glycine 1 of VP4 with arginine)
to give the mutant protein LbproVP4VP2(K201G, G202R)
(Table I). As can be seen in Fig. 3A (right panel), the presence of P1' arginine in the K201G,G202R mutant protein restored the cleavage to almost wild-type levels (compare Fig.
3A, left and right panels and Table I). Once
again, no effect on the cleavage of eIF4GI could be observed (Fig.
3B, right panel).
It might be argued that the enzyme is recognizing the newly introduced
Arg202 as the P1 residue, with cleavage occurring between
Arg202 and Ala203. However, we believe this to
be unlikely as this would imply that Leu200 would be the P3
residue and Gly201 the P2 residue. As the three-dimensional
structure of Lbpro shows that a basic residue is preferred
at P3 and a hydrophobic residue at P2, it is improbable that leucine
and glycine could provide sufficient binding energy at these positions.
In summary, these results support the predictions of the
three-dimensional structure about the substrate specificity of
Lbpro and demonstrate clearly that Lbpro which
is still covalently connected to the capsid proteins can efficiently
cleave eIF4GI. Thus, self-processing of the Lbpro is not a
prerequisite for the efficient cleavage of eIF4GI.
Role of the C Terminus on the Activity of the
Lbpro--
The above experiments confirmed the high
efficiency of eIF4GI cleavage in RRLs by Lbpro, regardless
of whether self-processing had occurred or not. What are the features
of the Lbpro that enable it to cleave eIF4GI so
efficiently? One unique feature of Lbpro, which is not
found in papain, is the presence of the 18-amino acid CTE in
Lbpro. We wished to examine whether this feature was
involved in some way in eIF4GI cleavage.
To investigate this, we used a second construction
pet8cLbpro that contains a synthetic stop codon after the
Lys201 residue, so that a mature Lbpro is
expressed without the need for self-processing (Fig.
4A). The kinetics of eIF4GI
cleavage are the same as those with pet8c LbproVP4VP2 (18).
C-terminal deletions were introduced into pet8cLbpro by
replacing the wild-type BsiWI BamHI fragment with
synthetic oligonucleotides. Initially, two deletions in
Lbpro were made; the first (designated
Lbpro-6; Table II) lacked the
six most C-terminal amino acids, i.e. just those which had
been found in the active site of the neighboring molecule in the
crystal structure. In the second (Lbpro-18), the entire 18 amino acids of the CTE were removed. RNAs were prepared from both
deletion mutants and were used to drive protein synthesis in RRLs;
synthesis of Lbpro (Fig. 4B) and cleavage of
eIF4GI (Fig. 4C) were analyzed as before. The mutant protein
Lbpro-6 cleaved eIF4GI with kinetics similar to that of the
wild-type enzyme (Fig. 4, B and C, left and
middle panels); however, the mutant protein
Lbpro-18 had a reduced ability to cleave eIF4GI, cleavage
was still incomplete after 12 min (Fig. 4, B and
C, left and right panels). This was
despite the fact that about twice as much Lbpro-18 had been
synthesized than in the wild-type reaction.
Fig. 4B shows that the deletion Lbpro-6 migrates
more slowly than the wild-type Lbpro. This is reminiscent
of an observation of Sangar et al. (28) who demonstrated
that prolonged incubation (i.e. longer than 1 h) of
Lbpro in RRLs led to a modification of the protein which
caused it to migrate more slowly on SDS-PAGE. This modified form
appeared to be deleted at its C terminus because it could also be
generated by carboxypeptidase A digestion (28). Thus, removal of a
small number of amino acids decreases the mobility of Lbpro
on SDS-PAGE. We believe that the missing 6 amino acids of the Lbpro-6 deletion are responsible for the reduced mobility
of this protein.
The above experiment suggested that the presence of the CTE was
required for full enzymatic activity. This notion was further strengthened by a serendipitous observation made during the synthesis of the above deletion mutants. A variant plasmid was obtained in which
an incorrect, truncated oligonucleotide was inserted between the
BsiWI site and the BamHI site at the 3' end of
the Lbpro (Fig. 4A and Table II). As a
consequence, the reading frame is shifted from amino acid 183 onwards
and there are only 8 amino acids before the BamHI site.
Thus, translation of an RNA transcribed from a template linearized with
BamHI produces a variant encoding a C-terminal extension of
8 amino acids, all differing from the wild-type (Table II). This
variant was designated Lbpro
Nevertheless, examination of these variants in the RRL system (Fig.
5) showed that both Lbpro The Lbpro C-terminal Variants Are Not Affected in Their
Ability to Process a Polyprotein Substrate--
In the above
experiments, the activity of the Lbpro C-terminal variants
was monitored only on the endogenous eIF4GI present in the RRL extract.
We wished to investigate whether these variants were also impaired in
their ability to recognize the polyprotein cleavage site. To generate a
suitable substrate, the construction
The protein produced in RRL from the pet8c
Cleavage of eIF4GI also takes place under these conditions. Examination
of the fate of the eIF4GI in the RRLs shows that over 50% is cleaved
using Lbpro before 8 min (Fig. 6C, left panel;
Table III); this is comparable with the
experiments in which only one RNA was translated. The Lbpro
concentration was estimated to be about 20 pg/µl by counting the
band. This agrees with previous observations in which 50% cleavage of
eIF4GI was achieved when the Lbpro concentration had
reached 15 pg/µl (18).
This experiment was then repeated using mRNAs encoding
Lbpro Comparison of Intra- and Intermolecular Lbpro
Cleavage--
In the experiment shown in Fig. 6, Lbpro can
only be cleaving
The result is shown in Fig. 7A. Four products are visible
during the time course. These are the uncleaved
The processing of the endogenous eIF4GI by Lbpro expressed
as LbproVP4VP2 is comparable with that observed when the
Lbpro is translated as a mature protein (compare Fig.
6C with Fig. 7B). The cleavage of
In summary, the reactions in order of efficiency catalyzed by the
Lbpro are the intramolecular processing at the
Lbpro VP4 junction and the cleavage of eIF4GI as part of
the eIF4F complex followed by the much less efficient intermolecular
cleavage at the Lbpro VP4 junction.
The experiments described here examine the relationship of the
self-processing reaction of the FMDV Lbpro to the cleavage
of the cellular substrate eIF4GI. By using mutagenesis to vary the
cleavage site at the Lbpro VP4 junction and to produce
C-terminal variants, factors affecting the rate of both reactions could
be ascertained. As the polyprotein and eIF4GI substrates examined are
those that are cleaved during replication of the virus in
vivo, these findings are directly relevant to the biological
activity of the Lbpro.
We began by investigating whether the inhibition of self-processing
affected the rate of eIF4GI cleavage. For this, we mutated the
Lbpro to prevent self-processing without affecting either
the catalytic residues or the structure of the enzyme. Initially,
mutations were introduced at the P1 position (Table I). The
substitution of the P1 lysine with either glycine or glutamine impaired
self-processing but did not inhibit it completely. Replacement of the
P1' glycine with arginine (thus mimicking the P1' residue in the eIF4GI
cleavage site) in the mutant protein containing glycine at P1 restored self-processing to wild-type levels, thus confirming the unusual requirement of this enzyme for a basic residue at either the P1 or P1' positions.
Complete inhibition of the self-processing reaction was obtained by the
introduction of a second mutation, namely leucine to serine at P2, in
the P1 lysine to glutamine mutant protein. This stresses the importance
of the presence of a hydrophobic residue at P2, a property that
Lbpro shares with most other papain-like enzymes (30).
In all of the above self-processing mutant proteins, however, a
reduction in the rate or extent of eIF4GI cleavage was not observed.
Thus, cleavage at the Lbpro VP4 junction is not an
essential prerequisite for cleavage of eIF4GI. This implies that the
enzyme is active as part of the growing polypeptide chain and that it
may be active while the polypeptide chain is still bound to the ribosome.
Why is the cleavage of eIF4GI so efficient, despite the lack of
self-processing? To begin, it can be assumed that the substrate eIF4GI
is in the optimal conformation, as the binding of eIF4E to eIF4GI
stimulates Lbpro cleavage of eIF4GI (31), and most of the
eIF4GI in RRLs is complexed to eIF4E (29). For the enzyme, the
C-terminal deletion experiments indicate a role for the C terminus.
Thus, the mutant protein lacking the entire CTE cleaved eIF4GI at a
significantly reduced rate; however, a much more drastic reduction in
the reaction rate could be seen with the variants possessing an
aberrant CTE. Nevertheless, all C-terminal variants recognized the
Lbpro VP4 cleavage site in the intermolecular cleavage at
the same rate. Thus, the CTE is required for efficient intermolecular
cleavage of eIF4GI but not for that on the polyprotein substrate.
Three explanations for this observation appear plausible. First, the C
terminus may be involved in a direct interaction with eIF4GI, perhaps
fitting specifically into a pocket, or even eIF4E; removal of the CTE
would prevent this interaction. However, it is not clear from this
theory why the presence of aberrant CTEs would have a further effect on
eIF4GI processing than the variants lacking a CTE. Second, the CTE
could stabilize the Lbpro in a conformation that favors
eIF4GI cleavage but that is not required for intermolecular polyprotein
processing. The lack of the CTE would obviate the stabilizing effect;
however, the presence of the aberrant CTE might destabilize this
conformation or even prevent it from being adopted. The third
possibility would require that binding of the Lbpro CTE to
eIF4GI induces a conformational change in this protein that exposes the
cleavage site. In this case, proteolysis would be the rate-limiting
step; in the CTE deletion mutants, the rate-limiting step would be the
exposure of the active site in the absence of the CTE.
It is interesting to note that, compared with papain, the
Lbpro achieves its active conformation without a pro-domain
and without activation at low pH (32). Indeed the experiments with the
CTE variants appear to suggest a much more important role for the C
terminus, a region of Lbpro that has no equivalent in
papain (20).
Finally, cleavage reactions of the Lbpro on intra- and
intermolecular polyprotein substrates were examined simultaneously.
Thus, RNAs were translated so that the rate at which both intra- and intermolecular self-processing occurred could be measured (Fig. 7).
These experiments were compared with one in which intramolecular self-processing was not required because the Lbpro
contained a stop codon at its C terminus (Fig. 6). No difference in the
rates of cleavage of eIF4GI or the polyprotein intermolecular substrate
were observed. In contrast, the kinetics of cleavage of the
Lbpro VP4 junction were different, depending on whether the
enzyme and substrate were part of the same polypeptide chain or not. The cleavage of the substrate when part of the same chain was much more
rapid, suggesting that this reaction is an intramolecular one, with the
C terminus of Lbpro folding into the active site of the
same molecule, as proposed by modeling studies based on the crystal
structure (20). This also implies that the In summary, we have shown that self-processing is not a prerequisite
for cleavage of eIFGI by Lbpro and have established the
hierarchy of the Lbpro cleavage reactions. The first two
are the extremely efficient intramolecular self-processing and the
intermolecular cleavage of eIF4GI followed by the much less efficient
intermolecular cleavage of the polyprotein sequence.
We thank Dieter Blaas and Joachim Seipelt for
critical reading of the manuscript.
*
This work was supported by the Austrian Science Foundation
Grant P-13667 (to T. S).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.
§
To whom correspondence should be addressed. Tel.: 43 1 4277 61620;
Fax: 43 1 4277 9616, E-mail: timothy.skern@univie.ac.at.
Published, JBC Papers in Press, July 17, 2001, DOI 10.1074/jbc.M104192200
The abbreviations used are:
eIF, eukaryotic
initiation factor;
CTE, C-terminal extension;
FMDV, foot-and-mouth
disease virus;
IRES, internal ribosome entry site;
Lpro, leader proteinase;
PAGE, polyacrylamide gel electrophoresis;
RRL, rabbit reticulocyte lysate;
PCR, polymerase chain reaction.
Foot-and-Mouth Disease Virus Leader Proteinase
INVOLVEMENT OF C-TERMINAL RESIDUES IN SELF-PROCESSING AND
CLEAVAGE OF eIF4GI*
,, and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Gly-Ala-Gly. Subsequently, the
Lpro impairs protein synthesis from capped mRNAs in the
infected cell by processing a host protein, eukaryotic initiation
factor 4GI, at the sequence Asn-Leu-Gly--Arg-Thr-Thr. A rabbit
reticulocyte lysate system was used to examine the substrate
specificity of Lpro and the relationship of the two
cleavage reactions. We show that Lpro requires a basic
residue at one side of the scissile bond to carry out efficient
self-processing. This reaction is abrogated when leucine and lysine
prior to the cleavage site are substituted by serine and glutamine,
respectively. However, the cleavage of eIF4GI is unaffected by the
inhibition of self-processing. Removal of the 18-amino acid C-terminal
extension of Lpro slowed eIF4GI cleavage; replacement of
the C-terminal extension by unrelated amino acid sequences further
delayed this cleavage. Surprisingly, wild-type Lpro and the
C-terminal variants all processed the polyprotein cleavage site in an
intermolecular reaction at the same rate. However, when the polyprotein
cleavage site was part of the same polypeptide chain as the wild-type
Lbpro, the rate of processing was much more rapid. These
experiments strongly suggest that self-processing is an intramolecular reaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The known biological activities of
Lpro. The role of eIF4 proteins in the initiation of
protein synthesis and the cleavage of eIF4G by Lpro are
shown in A. eIF4 proteins involved in protein synthesis and
the outline of the 40 S ribosomal subunit are named. The
m7GDP 5' cap structure of cellular mRNAs (open
circle) and the Lpro cleavage site (arrow)
are indicated. The eIF4G C-terminal domain still forms an initiation
complex with IRES-containing mRNAs. The RNA genome of FMDV is shown
in B. The single open reading frame is shown as an
open box (with the mature viral proteins indicated),
non-coding regions by a line, and the IRES as a closed
box. Lpro forms are checkered. Protein
synthesis on this mRNA is shown in C; Lpro
self-processing is indicated as an intra- or intermolecular event. The
Lpro CTE is indicated by an open box.
-Gly202-Ala-Gly,
whereas that on eIF4GI takes place at the sequence
Asn-Leu-Gly634--Arg635-Thr-Thr (13). The
sequence of Lbpro cleavage on eIF4GII has not yet been
determined, but it would appear by comparison to be between
Asn-Phe-Gly685--Arg686-Gln-Thr (22).
To achieve this unusual specificity, the Lbpro has evolved
to be able to bind basic residues at P1 or P1', while maintaining the
requirement of papain-like enzymes for a hydrophobic residue at P2 (20,
23). Nevertheless, the exact determinants of specificity are still not clear.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
LbproVP was constructed by using PCR
amplification to delete the amino acids 30-75 of Lbpro in
pet8cFMDV LbproVP4VP2 to give pet8cFMDV
LbproVP4VP2. To introduce a fragment extending the
number of VP2 amino acids encoded to 140, the required fragment was
amplified from the original FMDV cDNA subclone p735 (25). Two stop
codons followed by a BamHI site were also encoded by the 3'
PCR primer. This sequence was then introduced into pet8cFMDV
LbproVP4VP2 as an RsrII BamHI
fragment to give pet8c LbproVP. Following linearization
with BamHI, transcription of this DNA gives an RNA encoding
an inactive Lpro lacking 46 amino acids at the N terminus
followed by all 85 amino acids of VP4 and 140 amino acids of VP2.
View larger version (21K):
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Fig. 2.
Effect of mutations at the Lbpro
VP4 junction on self-processing and eIF4GI cleavage. The structure
of the expression block LbproVP4VP2 is shown in
A. Restriction enzyme sites mentioned in the text are shown.
The sites Bpu10I and SacI were introduced by
site-directed mutagenesis without changing the amino acid sequence. The
arrowhead depicts the cleavage site of
Lbpro. Oligonucleotide cassettes were then used to mutate
the amino acids shown in bold. RRLs were incubated with or
without the indicated mRNAs (10 ng/µl; transcribed in
vitro following linearization with SalI) as described
under "Experimental Procedures"; protein synthesis was terminated
at the times given by placing the samples on ice followed by the
addition of unlabeled methionine and cysteine to 2 mM and
Laemmli sample buffer. Aliquots were analyzed on 15% polyacrylamide
gels followed by fluorography to detect the synthesis of
LbproVP4VP2 (B) and on 6% polyacrylamide gels
followed by immunoblotting for the status of eIF4GI (C).
Fluorographs were exposed for 15 h for LbproVP4VP2 and
LbproVP4VP2(L200S,K201Q) and 20 h for
LbproVP4VP2(K201Q) to enable self-processing at 8 min
to be seen. The positions of uncleaved LbproVP4VP2 and the
cleavage products Lbpro and VP4VP2 are marked in
B; C, intact eIF4GI and the N-terminal cleavage
product cpN are marked. Protein standards (in kDa) are
indicated in both panels.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Effect of amino acid substitutions at the Lbpro VP4
junction on self-processing
View larger version (18K):
[in a new window]
Fig. 3.
A basic residue before or after the cleavage
site is essential for efficient self-processing. The indicated
mRNAs (10 ng/µl; transcribed in vitro following
linearization with SalI) were used to program RRLs. Analysis
of protein synthesis (A) and the state of eIF4GI
(B) was as in Fig. 2. Fluorographs were exposed for 15 h for LbproVP4VP2 and
LbproVP4VP2(K201G,G202R) and 20 h for
LbproVP4VP2(K201G) to enable self-processing at 8 min
to be seen.
View larger version (21K):
[in a new window]
Fig. 4.
Effect of C-terminal deletions in the
Lbpro on the processing of eIF4GI. The structure of
the expression block Lbpro is shown in A. Restriction enzyme sites mentioned in the text are indicated. The
indicated mRNAs (5 ng/µl for Lbpro and 10 ng/µl for
the deletion mutants; transcribed in vitro following
linearization with BamHI) were used to program RRLs.
Analysis of protein synthesis (B) and the state of eIF4GI
(C) are as in Fig. 2. All fluorograms were exposed for
15 h.
Amino acid sequence of the wild-type Lbpro, CTE, and
deletion mutants
11*, as it lacks 11 amino
acids compared with the wild-type Lbpro; the asterisk
indicates the aberrant amino acid sequence of the CTE. In addition, RNA
encoding a second variant was produced by linearizing the same plasmid
with HindIII, which cleaves 481 nucleotides downstream of
the BamHI site; due to the position of the first stop codon
encountered, the CTE in this case has 28 amino acids, 9 longer than the
wild-type. Once again, they are of different sequence (Table II), so
that the variant was designated Lbpro + 9*. Although the
mutant sequences have a relatively high alanine content and are
somewhat hydrophobic, they are not otherwise unusual.
11* and Lbpro + 9* had an impaired ability to cleave
eIF4GI. With the Lbpro 11* variant, about 90% eIF4GI
cleavage is observed after 30 min of incubation, even though after 4 and 8 min similar protein concentrations to those of the wild-type
experiment shown in Fig. 4B are present. Processing with the
variant Lbpro + 9* (Fig. 5, right panels) was
reduced even more dramatically, as 50% eIF4GI cleavage did not occur
until 30 min after cleavage, even though the RNA concentration had been
adjusted to ensure that twice the amount of protein was synthesized in
this experiment compared with the wild-type Lbpro in Fig.
4.
View larger version (29K):
[in a new window]
Fig. 5.
The presence of heterologous sequences at the
C terminus of Lbpro severely disrupts eIF4GI cleavage.
The indicated mRNAs (transcribed in vitro following
linearization with BamHI for Lbpro 11* (10 ng/µl) and HindIII for Lbpro + 9* (20 ng/µl)) were used to program RRLs. Analysis of protein synthesis
(A) and the state of eIF4GI (B) was as in Fig. 2.
Fluorograms were exposed for 15 h.
LbproVP was
prepared (Fig. 6A). Following
linearization with BamHI, transcription generates an RNA
encoding an inactive Lbpro with a 46-amino acid deletion,
all 85 amino acids of VP4, and 140 amino acids of VP2. The deletion in
the Lbpro part and the extension of the VP2 part make the
cleavage products of LbproVP4VP2 and LbproVP
distinguishable from each other on PAGE.
View larger version (30K):
[in a new window]
Fig. 6.
Intermolecular cleavage of the
Lpro VP4 junction by Lbpro wild-type and
C-terminal variants. The structure of the expression block for the
intermolecular substrate LbproVP is shown in
A. Restriction enzyme sites mentioned in the text are shown.
RNA was synthesized from LbproVP after cleavage with
BamHI. RRLs were programmed simultaneously with the
indicated mRNAs (10 ng/µl each) and incubated for the indicated
times. Analysis of protein synthesis (B) and the state of
eIF4GI (C) was as in Fig. 2. The positions of uncleaved
LbproVP, the cleavage product VP, and the
Lpro variants are marked in B; C,
intact eIF4GI and the cleavage product cpN are
marked.
LbproVP
construction is of apparent molecular mass of 46 kDa (Fig.
6B, far right lane) and is proteolytically inactive (Fig. 6,
B and C, far right lanes). The ability of
LbproVP to serve as substrate was examined by
simultaneously translating its RNA with the pet8cLbpro RNA
(Fig. 6B, left panel). Initially, after 8 min of incubation, two products of translation were observed, the mature Lbpro
and the uncleaved substrate LbproVP (Fig. 6B, left
panel). Subsequently, a third product of apparent molecular mass
of 34 kDa becomes visible, representing the VP part of the substrate
following cleavage at the junction between Lbpro and VP.
50% cleavage of LbproVP is achieved between 15 and 30 min. Eventually, all LbproVP is converted to product, as
sufficient proteinase is presumably present to cleave rapidly any newly
synthesized substrate so that it is no longer observed. The
Lbpro part of the substrate has a molecular mass of 14.2 kDa; despite this, however, it is not resolved by this gel system.
Comparison of cleavage efficiency of wild-type Lbpro
and its C-terminal deletions
18 and Lbpro + 9*. The kinetics of
cleavage of LbproVP by these two variants are very
similar to those of the wild type (Fig. 6B, compare the
middle and right panel with the left one; Table III), indicating that the absence of the CTE or the presence of an aberrant one do not affect the ability of the variants to process the polyprotein substrate in an intermolecular reaction. In
contrast, cleavage of eIF4GI by the C-terminal variants was once again
delayed (Fig. 6C, middle and right panels; Table
III). For the variant lacking the CTE, Lbpro 18, 50%
cleavage was achieved between 8 and 15 min at a proteinase concentration between 20 and 52 pg/µl; with the variant with the aberrant C terminus, Lbpro + 9*, 50% cleavage was obtained
after about 45 min at a concentration of 75 pg/µl.
LbproVP and eIF4GI intermolecularly. It
has been a long-standing question, however, as to whether the initial
cleavage of the Lbpro from the growing polyprotein
(i.e. from LbproVP4VP2) is also an intra- or an
intermolecular event (20, 21, 24). To investigate this, we translated
the LbproVP RNA together with that of
LbproVP4VP2 to see whether the cleavage kinetics of
LbproVP or eIF4GI were changed (Fig.
7). This can be achieved because, as
stated above, all the cleavage products from LbproVP4VP2
and LbproVP can be separated from one another by
SDS-PAGE.
View larger version (37K):
[in a new window]
Fig. 7.
Comparison of intermolecular and
intramolecular Lbpro cleavage. RRLs were programmed
simultaneously with the indicated mRNAs (10 ng/µl
each) and incubated for the times shown. As a control for the mobility
of unprocessed LbproVP4VP2, the mRNA encoding the
inactive LbproC51A VP4VP2 was also translated
simultaneously with LbproVP (far
right lane). Analysis of protein synthesis
(A) and the state of eIF4GI (B) was as in Fig. 2.
A, the positions of the uncleaved intermolecular
substrate LbproVP and the cleavage product VP are marked
as are the positions of intramolecular cleavage products
Lbpro and VP4VP2. B, intact eIF4GI and the
cleavage product cpN are marked.
LbproVP (46 kDa) and the VP cleavage products (34 kDa) (the 14.2-kDa Lbpro cleavage product is once again
not resolved) as well as the mature Lbpro (19.8 kDa) and
the VP4VP2 (15 kDa) cleavage product. However, the uncleaved protein
LbproVP4VP2 is not a major product of the reaction; the
uncleaved precursor is essentially only visible with the inactive
leader proteinase mutant LbproC51A VP4VP2 (Fig. 7A,
far right lane). Therefore, the processing of the
Lbpro from the growing LbproVP4VP2 polypeptide
chain must be extremely efficient, strongly implying that this reaction
is an intramolecular one.
LbproVP with Lbpro expressed from
LbproVP4VP2 is also comparable to that with
Lbpro (compare Fig. 6B with Fig. 7A)
and is therefore not delayed by self-processing. Furthermore, the
self-processing of LbproVP4VP2 occurs much more efficiently
than the cleavage of LbproVP, even though the sequence
of the cleavage site is the same. This strongly implies that
Lbpro frees itself from the growing polypeptide chain by an
intramolecular cleavage.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
LbproVP
substrate adopts a conformation competent for the intramolecular reaction and must unfold so that it can be processed in
trans.
ACKNOWLEDGEMENTS
FOOTNOTES
Both authors contributed equally to this work.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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