Identification and Characterization of Human Rhinovirus-14 3C
Protease Deamidation Isoform*
Gregory A.
Cox,
Robert B.
Johnson,
James A.
Cook,
Mark
Wakulchik,
Melvin G.
Johnson,
Elcira C.
Villarreal
, and
Q. May
Wang
From the Lilly Research Laboratories, Eli Lilly and Company,
Indianapolis, Indiana 46285
 |
ABSTRACT |
A purified recombinant human rhinovirus-14 3C
protease preparation contained only ~50% active enzyme as titrated
using specifically designed irreversible 3C protease inhibitors.
Analysis of the purified 3C protein by isoelectric focusing showed
differently charged 3C isoforms that had isoelectric points (pI) of 8.3 (55%) and 9.0 (45%), with the latter one being consistent with the
predicted pI of the human rhinovirus-14 3C protein. Further analysis
indicated that the pI 8.3 protein was the deamidated form of 3C, and it displayed ~10-fold reduced cleavage activity relative to the original 3C protease sample. Peptide mapping followed by sequence analysis revealed that a single asparagine, Asn-164, was deamidated to aspartic
acid in the pI 8.3 isoform. Converting Asn-164 to Asp by site-directed
mutagenesis resulted in a mutated 3C protease with extremely low
activity, as seen with the pI 8.3 isoform, indicating a role of Asn-164
in substrate recognition and binding. In addition, the deamidated 3C
protease was found to be present in vivo, and its abundance
was related to the viral replication cycle. Moreover, mutant virus
carrying Asp-164 showed reduced viability in infected cells. Taken
together, our data suggest that 3C protein deamidation plays a role in
the regulation of its enzymatic activity.
 |
INTRODUCTION |
Human rhinovirus (HRV)1
infections are considered to be the most frequent causative agents of
the common cold and various other upper respiratory tract infections
(1). Rhinoviruses are members of the picornavirus family, which also
includes the apthoviruses (foot-and-mouth disease virus), cardioviruses
(encephalomyocarditis virus), enteroviruses (poliovirus and Coxsackie
virus), and hepatitis A virus. All picornaviruses have a
positive-sense, single-stranded RNA genome that is translated into a
single polyprotein precursor. In the case of HRVs, the viral
polyprotein is mainly processed by the viral proteases 2A and 3C to
generate functional proteins and enzymes (2, 3). The 2A protease
catalyzes the first cleavage between the structural and nonstructural
proteins, whereas the 3C protease catalyzes most of the subsequent
internal cleavages (2, 3).
The availability of active recombinant 3C protease greatly facilitated
its biochemical characterization. Purified recombinant viral 3C
protease was able to cleave different proteins and peptides at the bond
formed between glutamine and glycine (4, 5). It has been found that 3C
protease could regulate host cell function by cleaving important
cellular proteins during infection (6, 7). In addition to its
proteolytic activity, viral 3C protease has been shown to be a
RNA-binding protein and may be involved in formation of the viral
replication complex (8). As illustrated by its crystal structure, HRV
3C protease represents a novel class of cysteine protease that contains
a cysteine as the active site nucleophile but is structurally like a
serine protease (9-11). It has been considered to be an ideal target
for antiviral intervention due to its essential role in viral
replication and its unique protein structure. Various 3C protease
inhibitors have been synthesized and evaluated in recent years (for a
review, see Ref. 12).
Although extensive studies have been carried out on HRV 3C protease
cleavage specificity and assay development, little is known about the
regulation of viral 3C protease activity. Previous studies from our
group showed that active recombinant HRV14 3C protease could be
purified to homogeneity as shown by SDS-polyacrylamide gel
electrophoresis and was able to cleave various synthetic peptides (13,
14). In this report, we describe the identification of a deamidated
isoform of HRV14 3C protease present in the purified enzyme preparation
and in the infected cells. Because the deamidated form of 3C protease
displayed reduced cleavage activity as compared with the native form,
we propose that 3C protease deamidation may be involved in the
regulation of its cleavage activity.
 |
MATERIALS AND METHODS |
Preparation and Identification of Recombinant HRV14 3C
Protease--
HRV14 3C protease was expressed in bacterial cells and
purified as described previously (13). The identity of the purified HRV14 3C protease was confirmed by a combination of analyses including N-terminal amino acid sequencing, ion spray mass spectrometry, and
SDS-polyacrylamide gel electrophoresis followed by Western blot using
polyclonal antibodies raised in rabbits against the purified
recombinant HRV14 3C protease. IEF gel electrophoresis was performed
using precasted IsoGel Agarose IEF plates, pH 3-10, (FMC BioProducts)
with the anode enriched with 0.5 M acetic acid, pH 2.6, and
the cathode enriched with 1.0 M sodium hydroxide, pH 13. Protein concentration was determined by the Bradford method using
bovine serum albumin as the standard. Densitometric analysis of the
protein isoforms was performed with the Coomassie Blue-stained IEF gels
using a Bio-Rad GS-700 imaging densitometer.
Inhibitor Titration of Active HRV 3C Protease--
LY387838
(Boc-E(tBu)VLFvQ-OMe) is an irreversible peptidyl 3C protease inhibitor
prepared as described previously (15). Purified 3C protease (150 µl
at a concentration of 0.2 mg/ml) was pre-incubated with LY387838
(concentration range: 0-0.9 nmol) in a total volume of 900 µl
containing 50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 3% Me2SO. After 30 min at room
temperature, the remaining 3C protease activity was determined by a
colorimetric assay, as described previously, using peptide
EALFQ-p-nitroanilide (250 µM) as a substrate
(14).
Preparation and Isolation of Deamidated HRV14 3C
Protease--
The purified 3C protease preparation containing the two
differently charged 3C isoforms was further fractionated onto a Mono S
HR 5/5 column (Pharmacia Biotech Inc.). After washing the column with
Buffer A (25 mM Tris, 1 mM EDTA, and 5 mM dithiothreitol, pH 8.0), the bound protein was eluted
with a linear gradient of 0-500 mM KCl in Buffer A. To
obtain a large amount of the deamidated HRV 3C isoform, we performed
chemical deamidation of 3C protein as described previously (16).
Briefly, purified 3C protease (5 mg) was incubated at 37 °C for
72 h in the presence of 100 mM ammonium bicarbonate,
pH 9.0. After centrifugation at 16,000 × g for 10 min,
the clarified 3C protein was then dialyzed against Buffer A. The
converted pI 8.3 isoform was confirmed by IEF gel analysis of the
treated 3C protein sample.
Measurement of HRV14 3C Protease Activity--
The fluorogenic
peptide (aminobenzoic
acid)-Thr-Leu-Phe-Gln-Gly-Pro-Val-Phe(p-nitro)-Lys has been
shown to be cleaved between glutamine and glycine by HRV14 3C protease
(13). The cleavage reaction was performed at 30 °C for 60 min in a
400-µl mix containing 50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 200 µM
peptide substrate, and 0.3 µM 3C protease. The reaction
was started with the addition of the enzyme and monitored by the
fluorescence signal increase using a Perkin-Elmer LS50B luminescence
spectrophotometer at an excitation of 340 nm and an emission of 415 nm.
In some cases, 3C protease activity was determined using the
chromogenic peptide substrate EALFQ-p-nitroanilide as
described previously (14).
Identification of the 3C Protein Deamidation Site--
Peptide
mapping of native and deamidated 3C protein using trypsin was performed
at 37 °C for 18 h in 100 mM Tris-HCl, pH 8.5, at an
enzyme:substrate ratio of 1:20 (w/w). Digestion of viral 3C with V8
protease (Roche Molecular Biochemicals) was performed for 18 h at
25 °C in 25 mM ammonium bicarbonate, pH 7.8, with an
enzyme:substrate ratio of 1:25 (w/w). Reactions containing the cleaved
3C proteins were directly applied to a reverse-phase HPLC for peptide
separation. Peptide products were eluted by an acetonitrile gradient of
10-60% in 20 min and 60-90% in 6 min in 0.1% trifluoro-acetic acid
with a flow rate of 1 ml/min using a C18 column (4.6 × 250 mm;
5-mm particle size). Separated peptides were then subjected to
N-terminal amino acid sequencing.
HRV14 Infection of Cultured Cells--
Confluent monolayers of
cloned H-1 HeLa cells were infected with HRV14 (a gift from R. Rueckert, University of Wisconsin, Madison, WI) at a multiplicity of
infection of 100 plaque-forming units/cell. After an adsorption period
of 30 min at 25 °C, the infected cells were incubated at 35 °C
for 6, 8, 12, and 20 h before harvesting. The collected cell
pellets were washed twice with cold phosphate-buffered saline,
resuspended in 200 µl of phosphate-buffered saline and 0.5% Nonidet
P-40 containing protease inhibitors, incubated on ice for 15 min, and
spun for 5 min at 1,000 × g. 10 µg of the crude
supernatant extract from each sample were loaded onto IEF gels, and the
level of 3C protein was determined by Western blot. To determine virus
plaque formation, transfected cells were incubated at 35 °C for
48-72 h, fixed, and stained with crystal violet, and the numbers of
plaques formed were counted (17). The transfections were done in
duplicate and repeated four times. As controls, mock-infected cells
were treated as described above, but excluded the addition of virus.
Mutant Virus and 3C Protein Preparation--
Site-directed
mutagenesis was performed at the 3C deamidation site by combinatorial
polymerase chain reaction mutagenesis (18). The mutation of Asn-164 to
Asp was generated in the context of the whole HRV14 cDNA or in the
plasmid used to produce recombinant 3C protein in bacterial cells. To
examine the effect of this mutation on viral replication, mutant HRV14
RNA was transfected into HeLa cell monolayers under the conditions
described above, and the viral plaque formation was determined. For
generation of mutant 3C protein, expression vector carrying the Asp-164
mutation was used to transform competent bacterial cells under the
identical conditions used for wild type recombinant 3C protein
expression (13).
Other Analytical Methods--
CD spectra were recorded by a
JASCO J-600 spectropolarimeter using cylindrical cuvettes with a
pathlength of 0.05 cm (19). Measurement of native 3C protein was
performed in Buffer B (25 mM MES, 1 mM EDTA, 5 mM dithiothreitol, and 10% glycerol, pH 6.5), and
measurement of the deamidated 3C isoform was performed in Buffer B plus
100 mM ammonium bicarbonate at pH 9.0. The CD spectra were
collected in both the far (250-190 nm) and the near UV range (240-350 nm).
 |
RESULTS |
Identification of Different HRV 3C Protease
Isoforms
HRV14 3C protease has been found to cleave the viral
polyprotein precursor mainly at the Gln-Gly bond (4, 5). On the basis
of the 3C protease cleavage specificity, peptidyl derivatives of
vinylogous glutamine ester were designed and synthesized as described
previously (15). These Michael acceptors were found to inhibit HRV14
viral 3C protease selectively by forming stable 1:1 enzyme-inhibitor adducts (15). We used LY387838 (Fig.
1A), one of the most potent Michael acceptors against 3C protease, to titrate the active 3C enzyme
present in the preparation. Incremental addition of LY387838 to 1.5 nmol of purified 3C protein preparation resulted in proportional decreases of 3C cleavage activity. A complete loss of the enzyme activity was reached when ~0.7 nmol of LY387838 was added into the 3C
protein sample (Fig. 1B). Because the inhibitor binds 3C protease irreversibly in a 1:1 ratio, these results indicated that only
~47% of the purified 3C protein was active. Interestingly, when the
sample was subjected to IEF gel electrophoresis, two bands were
identified, 55% of which had a pI of 8.3, and the remaining 45% had a
pI of 9.0. (Fig. 2, lane
2).

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Fig. 1.
Active site titration of HRV14 3C protease by
LY387838. An aliquot of purified recombinant HRV14 3C protein
sample (total, 1.5 nmol) was mixed for 30 min with the indicated
amounts of compound LY387838. The remaining 3C protease activity was
determined as described under "Materials and Methods."
A, chemical structure of LY387838; B, HRV14 3C
protease activity titration by LY387838.
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Fig. 2.
Separation of deamidated 3C protease.
The purified recombinant 3C protein sample was further fractionated on
a Mono S column, and the separated 3C isoforms were analyzed on an IEF
gel as described under "Materials and Methods." Protein standards
are shown with their isoelectric points marked on the left
(lane 1); lane 2, 3C protein loaded onto the
column; lane 3, the pI 8.3 isoform in the first peak;
lane 4, the second peak eluted from the column. Each lane
contains ~100 ng of total proteins.
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To further elucidate the identities of the proteins present in the 3C
preparation, several experiments were performed. Western blot analysis
indicated that both bands separated by IEF gels could be recognized by
the polyclonal antibodies against 3C protein (data not shown). Amino
acid sequence analysis of the two proteins revealed an identical
15-residue sequence at the N terminus, consistent with the predicted 3C
protein sequence. Further analysis by ion spray mass spectrometry
yielded the expected molecular weight of ~19,998 (data not shown).
Taken together, these data indicated that purified viral 3C protein
existed in two differently charged forms with very similar molecular
mass. The calculated pI of the recombinant 3C protein is 9.0;
therefore, we assumed that the pI 9.0 isoform was the native one. The
pI 8.3 isoform showed decreased pI, and we reasoned that it was due to
either a loss of positively charged groups or a gain of negatively
charged amino acids in the protein. Considering the fact that the two
isoforms had very similar molecular weight, we thought that the pI 8.3 isoform was generated from deamidation at the amide group of certain
asparagine and/or glutamine residues of the native 3C protein because
deamidation could convert an uncharged amino acid to a negatively
charged residue with only a 1 Da mass difference.
To verify whether the pI 8.3 isoform was caused by deamidation,
purified recombinant 3C protein sample was treated with ammonium bicarbonate to promote deamidation chemically as described previously (16). Analysis of the treated sample by IEF gels indicated that the pI
9.0 isoform disappeared along with an increase of the pI 8.3 isoform,
which was shown as the major band covering over 90% of the total
protein on the gels (data not shown). These data strongly suggested
that the pI 8.3 isoform was generated from deamidation at certain
residues of the HRV14 3C protease.
Identification of the 3C Protein Deamidation Site--
HRV14 3C
protease contained 182 amino acids along with 12 asparagine and 7 glutamine residues. To identify the 3C deamidation sites, ammonium
bicarbonate-treated and untreated 3C protein samples were digested with
trypsin or V8 protease, and the resulting peptides were separated by
reverse-phase HPLC. As seen in Fig. 3,
the HPLC elution profiles of the tryptic peptides generated from both
treated and untreated 3C protein were similar, except that peak
patterns eluted at ~19.7 min from the two samples were different.
Amino acid sequencing data showed that peak 36 of the treated 3C
protein corresponded to amino acid residues 156-166
(Ile-Phe-Gly-Ile-His-Val-Gly-Gly-Asn-Gly-Arg); however, the predicted
asparagine (Asn-164) was found to be its deamidated form, Asp-164
(Table I). In contrast, the corresponding peptide from the untreated 3C protease eluted as a doublet peak (peaks 30 and 31 in Fig. 3) with retention times
of 19.6-19.8 min. Amino acid sequencing of peak 30 identified the
expected Asn-164, but the peptide from peak 31 was found to contain
only Asp-164 (Table I).

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Fig. 3.
Identification of the HRV14 3C protein
deamidation site. Trypsin digestion of wild type and chemically
deamidated 3C protease was performed as described under "Materials
and Methods." The resulting peptides were separated by reverse-phase
HPLC. The HPLC elution profiles of the peptides of the original
(B) and the deamidated (A) 3C protein samples are
shown. Peaks with changed retention time are marked.
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Table I
Amino acid sequence of isolated peptides derived from HRV14 3C protein
Bold amino acid residues represent a change from the predicted
sequence.
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The molecular mass of these peptides was examined by mass spectrometry
(Table I). The molecular mass of the peptides in peaks 30, 31, and 36 was 1126.6 and 1127.3 Da, respectively, consistent with the mass change
resulting from one asparagine deamidation to aspartic acid. These data
suggested that Asn-164 was deamidated in the 3C sample treated with
ammonium bicarbonate, and it was also present in the untreated 3C
protease sample (Table I). Using trypsin and Staphylococcus
aureus V8 protease digestion, we were able to identify 17 of the
19 total potential deamidation sites in 3C protein, with two exceptions
for Asn-80 and Gln-182. Of the 17 residues examined, Asn-164 was the
only one found to be deamidated.
Cleavage Activity of Different 3C Isoforms--
Considering that
only half of the 3C protein present in the preparation was active as
shown by the inhibitor titration, we decided to separate the two
isoforms, the pI 8.3 and pI 9.0 forms, to examine their protease
activity. Separation was performed by fractionating the mixed 3C
protein on an ion-exchange column as described under "Materials and
Methods," through which two peaks were eluted from the column.
Analysis by IEF gels revealed that the first peak was free of the pI
9.0 protein, and it contained only the pI 8.3 form (Fig. 2, lane
3); however, the second peak contained both isoforms (Fig. 2,
lane 4). As seen in Fig.
4A, the purified pI 8.3 isoform showed ~10-fold reduced cleavage activity against a peptide
substrate as compared with the original enzyme preparation containing
both isoforms, whereas the second peak sample showed a very similar
activity to the original 3C protease. Numerous attempts to isolate just
the pI 9.0 form proved difficult, always contaminating with a small
amount of the pI 8.3 isoform (Fig. 2, lane 4).

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Fig. 4.
Cleavage activity of deamidated 3C protease.
A, cleavage activity of the 3C isoforms. The purified
recombinant 3C protein sample was further fractionated on a Mono S
column, and the separated 3C isoforms (0.3 µM) were
assayed for cleavage activity against the fluorescent peptide substrate
as described under "Materials and Methods." The enzymatic activity
of the control 3C protease pre-Mono S column ( ), the pI 8.3 isoform
in the first peak ( ), and the second peak sample containing both
isoforms after the Mono S column ( ) is shown. B,
proteolytic activity of mutant Asp-164 3C protein ( ) as compared
with the wild type enzyme ( ). 3C protease (0.2 µM)
activity was assayed using the colorimetric peptide
EALFQ-p-nitroanilide as the substrate under the conditions
described previously (14). Absorbance (A405 nm)
of the released yellow-colored p-nitoaniline was recorded by
a spectrophotometer.
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Examination of the chemically deamidated 3C sample for peptide cleavage
activity was also performed. It showed approximately 10-fold reduced
cleavage activity as compared with the enzyme treated in the absence of
the chemical (data not shown). In addition, CD spectra indicated that
the secondary structure of the chemically deamidated 3C protease was
very similar to that of the native 3C protease (data not shown),
suggesting that the reduced activity of the treated 3C sample was not
the result of a denatured 3C protein. Furthermore, we generated the
mutant 3C protein containing Asp-164 using site-directed mutagenesis.
Analysis of the purified recombinant Asp-164 mutant 3C protein on IEF
gels indicated that the native pI 9.0 form disappeared, and the mutant
protein appeared as a single band with a pI of approximately 8.3 (data
not shown). This mutant 3C protease showed reduced protease activity as
compared with the wild type 3C protein (Fig. 4B).
Presence of the HRV14 3C Deamidation Isoform in Vivo--
To
determine whether or not the HRV14 3C protease was possibly deamidated
in vivo, we infected HeLa cells with HRV14 and examined the
extracts at different time points after infection via IEF gel
separation followed by Western blot analysis. The 3C protease was
detected at 6 h after infection, mostly in the native pI 9.0 protein form (Fig. 5, lane 1).
However, the pI 8.3 isoform started to show at 8 h and reached
approximately equal intensity compared with the pI 9.0 isoform at
12 h after infection (Fig. 5, lane 3). These results
suggested that HRV14 3C protease existed as two isoforms in
vivo (native and deamidated), consistent with the in
vitro data.

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Fig. 5.
Existence of different 3C protease isoforms
in HRV14-infected HeLa cells. IEF gel separation and Western blot
analysis of the infected HeLa cell lysates were performed as described
under "Materials and Methods." Protein extracts from HRV14-infected
HeLa cells at 6, 8, and 12 h after infection are shown in
lanes 1, 2, and 3, respectively. Lane
4 is the extract from the control mock-infected HeLa cells.
Isoelectric points of the standard proteins are shown on the
left of the gel.
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Next, we constructed a mutant HRV14 virus carrying the Asp-164 mutation
to examine the potential effect of the deamidation of 3C on viral
replication. This single mutation was introduced into the HRV14
cDNA clone, transcribed into RNA, and then transfected into HeLa
cells as described. The plaques formed by the wild type and mutant
virus were compared. It was found that the mutant virus showed ~71%
reduction in viral plaque formation as compared with the wild type, and
the mutant plaques were smaller than the wild type ones. These results
indicated that the viability of the virus was directly linked to the 3C
function and support our finding that Asn-164 might play an important
role for regulation of 3C protease activity in vivo.
 |
DISCUSSION |
Determination of the concentration of active protease present in a
protein sample is very important for studying protease cleavage
activity or inhibition kinetics. This is often done using specific
burst titrants, for example, irreversible protease inhibitors. Many
cysteine proteases can be titrated using readily available E-64, an
epoxysuccinyl derivative that specifically inhibits papain-like cysteine proteases (20). However, the viral cysteine protease 3C has
been found to be insensitive to E-64 (5, 14), perhaps due to the unique
active site conformation and protein architecture of HRV 3C. Recently,
we developed a series of irreversible peptidic inhibitors targeting the
HRV14 3C protease (15). These compounds, mimicking the 3C peptide
substrate sequences, have been shown to be potent 3C protease
inhibitors exhibiting specific antiviral activity in cell culture
assays (15). Because these compounds could rapidly form 1:1 covalent
complexes with the enzyme, we have explored the possibility of using
these inhibitors for titration of active 3C protease present in the
sample or preparation. Our data clearly show that these Michael
acceptors, such as LY387838, can be used as active site titrants for
rhinovirus 3C protease. These results, in combination with the IEF gel
analysis data, led to the identification of the deamidated 3C protease
isoform present in the recombinant protein preparation and in the
HRV-infected cells.
It is worthy to note that the sequence flanking the 3C deamidation site
is very conserved among different HRV serotypes as well as
polioviruses. Table II shows the amino
acid sequence alignment across the deamidated site Asn-164 of HRV14 3C
protein among the other HRV serotypes. As revealed by the 3C NMR model
formed between the protease and a peptide substrate (14), this region
is involved in substrate binding, especially the interaction with the
hydrophobic P4 amino acid present in the substrate. Deamidation of
Asn-164 would convert the uncharged asparagine to a hydrophilic Asp
residue, which would result in a loss of the efficient hydrophobic
interaction between the protease and its substrate and could explain
the reduced peptide cleavage activity associated with the Asp-164
mutant 3C protease.
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Table II
Sequence alignments of HRV 3C protease flanking Asn-164
The amino acids corresponding to residues 135-182 of HRV14 3C protein
are shown. The conserved residues at and around the putative deamidated
asparagine are underlined.
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Regulation of enzymatic activity by post-translational modifications
has been widely described. The most well-known examples include the
activation or inhibition by phosphorylation on some of the hydroxyl
groups of serine/threonine and/or tyrosine residues of cellular enzymes
involved in signal transduction pathways. Protein deamidation has been
reported previously (21); however, its impact on enzymatic activity has
rarely been investigated. Until recently, deamidation of the Rho
protein, a GTP-hydrolyzing enzyme, as a mechanism controlling its
GTPase activity has been reported (22, 23). In the case of Rho protein,
a single glutamine residue can be deamidated by the bacterial toxins
termed cytotoxic necrotizing factors, and this selective deamidation
results in an inhibition of the Rho GTPase activity (22, 23).
Consistent with this observation, we have found that deamidation of a
single asparagine could generate negative impacts on viral 3C protease activity. This is the first demonstration that a viral enzyme activity
might be subject to regulation by a selective deamidation at the site
implicated in substrate recognition and binding.
A key question raised here is how the 3C deamidation happened in
vitro as well as in vivo. Deamidation of glutamine or
asparagine can be a consequence of enzymatic or nonenzymatic activity
(for a review, see Ref. 24). In the case of the Rho GTPase, selective deamidation is catalyzed by the bacterial toxin (22, 23). It has been
speculated that nonenzymatic deamidation occurs most frequently at the
sites containing downstream glycine or serine residues such as Asn-Gly
or Asn-Ser (24). Interestingly, in the case of HRV14 3C protease,
Asn-164 is indeed followed by a glycine residue, which is also
conserved in the 3C proteins of other HRV serotypes, as seen in Table
II. However, deamidation did not occur to another Asn-Gly pair present
in the HRV14 3C protein (data not shown). Thus, at this time, it is
unclear whether the selective deamidation of Asn-164 in the 3C protein
is catalyzed by a specific enzyme or is generated through a
nonenzymatic process. Meanwhile, based on the fact that Asn-164 is
located in the 3C enzyme active center, it is also possible that 3C
deamidation is an autocatalytic reaction. Deamidation is a hydrolytic
reaction of an amide bond, which in principal is similar to the peptide amide bond cleavage reaction catalyzed by the 3C protease.
Nevertheless, elucidation of this deamidation process will contribute
to a better understanding of the 3C protease regulation.
In summary, this study reports a method to titrate active HRV 3C
protease and provides evidence that a deamidation isoform of HRV14 3C
protease was present in the purified 3C protein preparation and in
HRV-infected cells. The deamidation site has been located at an
asparagine residue involved in substrate binding; thus, this
modification dramatically decreased the 3C protease activity in
vitro and in vivo. Based on these results, we propose
that HRV 3C deamidation at Asn-164 might play a role in the regulation of its enzymatic activity.
 |
ACKNOWLEDGEMENTS |
We thank Gary Birch, Michele Smith, Leroy
Baker, Gerald Becker, Joe Colacino, Allen Kline, and Rick Loncharich at
Lilly Research Laboratories for helpful discussions and John Richardson
for the ion spray mass spectrometry analysis. We are grateful to R. Hanzlik (University of Kansas) for synthesis of the protease inhibitors and Ann Palmenberg (University of Wisconsin, Madison, WI) for providing
us with the amino acid sequence of picornaviral 3C proteins.
 |
FOOTNOTES |
*
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: Drop Code 0438, Eli
Lilly and Company, Indianapolis, IN 46285. Tel.: 317-277-6975; Fax:
317-276-1743; E-mail: qmwang{at}Lilly.com (for Q. M. W.). Tel.: 317-276-5160; Fax: 317-276-1743; E-mail: Villarreal_Elcira_C{at}Lilly.com (for E. C. V.).
 |
ABBREVIATIONS |
The abbreviations used are:
HRV, human
rhinovirus;
IEF, isoelectric focusing;
pI, isoelectric points;
HPLC, high pressure liquid chromatography;
MES, 4-morpholineethanesulfonic
acid.
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 1999 by the American Society for Biochemistry and Molecular Biology.