(Received for publication, May 22, 1995)
From the
We report here the production of active recombinant single-chain
human cytomegalovirus protease in Escherichia coli and
development of a continuous assay for this protease. In order to
produce the human cytomegalovirus (HCMV) protease for structural
studies and accurate kinetic analysis, mutation of alanine 143 at an
internal cleavage site was introduced to prevent autoproteolysis. The
resulting soluble 29-kDa A143Q protease was purified to homogeneity as
a stable single-chain protein by hydrophobic interaction and
ionic-exchange chromatography. The in vivo protein substrate,
assembly protein precursor, was also expressed and purified for
activity studies. To develop a continuous protease assay, fluorescent
synthetic peptide substrates similar to the cleavage sequence P5 to P5`
of the maturation site containing anthranilic acid and nitrotyrosine as
a resonance energy transfer donor-acceptor pair were designed. Purified
HCMV A143Q protease cleaved the recombinant assembly protein precursor
with K and k
values of 3.0 ± 1.0 µM and 13.3 ± 1.6
min
. The K
for peptide
substrates is at least 45-fold higher than for the natural protein
substrate, but the k
values are similar. A
sensitive assay was developed using fluorescent peptide substrates,
which can detect nM HCMV protease activity.
The Herpesviridae family includes several human
pathogenic species such as herpes simplex virus 1 and 2 (HSV-1 and -2), ()cytomegalovirus (CMV), Epstein-Barr virus, and
varicella-zoster virus. Viral infection by HCMV is very common, and
40-80% of population becomes infected by HCMV before
adulthood(1) . HCMV is a serious pathogen in immunocompromised
individuals, especially those patients with AIDS, receiving organ or
bone marrow transplants, or undergoing cancer chemotherapy or steroid
therapy. CMV can cause damage in many organs, including the lung,
retina, liver, and gastrointestinal tract. Ganciclovir and foscarnet
are inhibitors of viral DNA polymerase and have been used to treat HCMV
infections; however, they have the undesired side effects of nucleotide
analogs(2) .
All members of the Herpesviridae family are similar at both the morphological and genomic levels. Herpesviruses contain a DNA genome of over 100 kilobases, an icosahedral capsid, and a lipoprotein envelope. The viral genome replicates inside the nucleus of infected cells and is then packaged into an intermediate capsid and followed by the acquisition of a nuclear membrane envelope and release of the virion from the infected cell. The HSV-1 assembly protein precursor, ICP35, is a major component of the intermediate capsid but is absent in mature virions(3, 4, 5) . During virion maturation, ICP35 undergoes proteolytic processing to generate the mature assembly protein that lacks approximately 20 amino acids from the carboxyl terminus. An HSV-1 temperature-sensitive mutant (ts1201) that it is defective in the processing of ICP35 has been reported by Preston et al.(6) . This mutant virus fails to package progeny viral DNA into virions at the nonpermissive temperature, and only empty capsids accumulate. This ts lesion was mapped to the UL26 ORF. The UL26 ORF overlaps with the ICP35 producing UL26.5 ORF, and these two ORFs are 3`-coterminal(6) . Liu and Roizman (7) reported that the product of the UL26 ORF is an 80-kDa protease that is responsible for the proteolytic processing of ICP35 protein. The UL26.5 promoter lies within the UL26 ORF. Translation initiation of ICP35 begins at methionine codon 307 of the 80-kDa protease. The amino acid sequence of the ICP35 is therefore identical to the carboxyl-terminal region of the 80-kDa protease. During viral replication, the 80-kDa protease cleaves both itself and ICP35 at a common maturation site approximately 5 kDa from the shared carboxyl terminus. The 80-kDa protease also undergoes a unique autoproteolytic cleavage at a release site between amino acid residues 246 and 247, resulting in the release of a 29-kDa catalytic domain. These findings, together with the recent work done with the UL26 null mutant virus (8) suggested that herpes proteases play a critical role in viral particle maturation and are attractive targets for anti-viral chemotherapy.
The human CMV UL80 ORF was reported to share homology
with HSV-1 UL26 ORF (9) as well as with the assembly protein
nested genes ORF of the simian
cytomegalovirus(10, 11) . Gibson and co-workers (12, 13) have demonstrated that the carboxyl-terminal
region of the HCMV UL80 encodes a protein that is proteolytically
processed in a similar fashion as the HSV-1 ICP35. By transient
transfection assays, the simian CMV assembly protein nested gene-1 ORF
was shown to encode a 590-amino acid protease whose amino-terminal 249
residues possessed proteolytic activity. This region also contains
sequence motifs that are highly homologous to proteases of other
herpesviruses(14) . More recently, Baum et al.(15) used [S]methionine labeling of Escherichia coli harboring expression plasmids containing UL80
ORF sequence and found an 85-kDa protease that underwent
autoproteolysis in vivo and produced a set of proteins with
molecular masses of 50, 29, 16, 13, and 5 kDa(15) . The three
cleavage sites of the 85-kDa protease were identified as follows: (i) a
maturation (M-) site at the carboxyl terminus, which shares sequence
identity with the cleavage site of assembly protein precursor, (ii) an
internal release (R-) site, which yielded the 29-kDa amino-terminal
catalytic domain and a 50-kDa carboxyl-terminal fragment, and (iii) an
internal (I-) site that lies within the 29-kDa catalytic domain between
Ala-143 and Ala-144.
The design of inhibitors for the HCMV protease requires a detailed understanding of its structure and catalytic mechanism. The major difficulty in obtaining pure and stable enzyme for such studies has been due to autoproteolysis, which results in cleavage within the catalytic domain. We have expressed the stable single-chain active HCMV A143Q protease, purified it to homogeneity for crystallographic studies, and developed a sensitive continuous protease assay using fluorogenic peptides as substrates for the kinetic studies.
Initially, the expression of the wild-type HCMV protease was attempted with the T7 expression phagemid pMGH4 (17) in the E. coli strain BL21(DE3). By using this expression system, wild-type HCMV protease could only be accumulated as an insoluble intact 29-kDa protein (Fig. 1A) at 42 °C. Upon refolding, proteolytic degradation of the 29-kDa protease into 16- and 13-kDa fragments started to occur as the protease concentration was increased to 2 mg/ml (data not shown). The same 16- and 13-kDa proteins were observed in induced E. coli lysate prepared from 37 °C cultures. These degradation fragments were previously seen in metabolically labeled E. coli lysates by Baum et al.(15) , and they were identified as the products of a cleavage event occurring between Ala-143 and Ala-144 at the I-site. Other groups have recently reported expression and purification of the wild-type HCMV protease catalytic domain either from inclusion bodies (25, 26) or as a two-chain protease due to autoproteolytic processing at the I-site (27) . As a consequence of this autoproteolytic breakdown, preparation of active single-chain wild-type HCMV protease at high concentrations required for crystallographic studies is not feasible.
Figure 1:
Expression of the
HCMV protease catalytic domain in E. coli. Each sample of
total crude lysate was prepared from E. coli equivalent to 0.2 A, boiled in the reducing sample buffer, and
loaded on a 16% SDS-polyacrylamide gel that was stained with Coomassie
Brilliant Blue after electrophoresis. + and - denote the
induced and uninduced culture lysates, respectively. Arrowhead marks positions of two proteolytic fragments and the intact HCMV
protease catalytic domain. LaneM, molecular mass
markers. Molecular masses of markers are indicated in kDa. A,
expression of the wild-type HCMV protease. Lanes1 and 2 and lanes 3 and 4 are uninduced (-) and induced (+) cultures bearing pCMVP1-5
grown at 37 and 42 °C after induction, respectively. B,
expression of the HCMV proteases with various substitutions of Ala-143
at 37 °C. Lane1 is the lysate from an induced
culture bearing pCMVP1-5 with the A143Q mutation. Lanes2-11 are pairs of uninduced and induced cultures
with A143G, A143N, A143S, A143T, or an A143V mutation. Lane12 is the induced culture with the wild-type Ala-143
residue.
Ala-143 of the I-site
lies between the conserved sequence motifs CD1 and CD2 among herpes
proteases. Deletion of the corresponding region in the simian CMV
resulted in a stable protease and autolytic cleavage at the I-site was
not observed. This mutated protease could still cleave its own assembly
protein precursor at the M-site with approximately the same efficiency
as the wild-type enzyme(28) . In light of this observation, we
mutated the alanine 143 residue of the HCMV protease to test if this
mutation might confer resistance to autoproteolysis but still retain
wild-type proteolytic activity. Mutations at Ala-143 to Gly, Gln, Asn,
Ser, Thr, and Val were made. Based on their autoproteolytic activities
in crude lysates (see Fig. 1B), mutants can be divided
into two groups. Group I mutants, A143G and A143S, still produced 16-
and 13-kDa fragments. Autoproteolytic activities observed with the
group I mutants suggests that the I-site prefers amino acid residues
with small side chains at P1 position. All group II mutants: A143Q,
A143N, A143V, and A143T, produced intact 29-kDa protease abundantly at
37 °C in E. coli. During the preparation of this
manuscript, a similar approach was reported by Holwerda et
al.(27) . They disrupted the I-site by changing valine 141
to alanine (VEAA to AEA
A) and recovered active 30-kDa
one-chain enzyme. In our expression construct, the histidine tag used
by Holwerda et al.(27) for purification purposes was
avoided so as to obtain crystallographic information reflective of the
native HCMV protease without unnecessary changes.
We chose to purify the HCMV A143Q protease and to test whether this mutant protease retains proteolytic activity. Purification included phenol-Sepharose, Q- and S-Sepharose chromatographic steps (Fig. 2). HCMV A143Q protease was stable during purification and at 4 °C for up to 1 month at less than 10 mg/ml. The amino-terminal amino acid sequencing of purified A143Q showed the correct sequence of HCMV protease with 80% of the protease retaining methionine as the first residue, while the remaining 20% had the initiation methionine cleaved off and thus started at the second residue threonine. Ellman titration showed that only three of the five sulfhydryl groups in the native A143Q protease were reactive against 5,5`-dithionitrobenzoic acid. After denaturation, the number of sulfhydryl groups/protease molecule accessible to 5,5`-dithionitrobenzoic acid reaction increased from 3 to 4.5. These results indicate that no disulfide bonds exist in the HCMV protease catalytic domain and that two of the five cysteines may be buried in the hydrophobic core of the native protein. The reported thiol content of wild-type HCMV protease after exposure to 2% SDS is 4.9 ± 0.5 (26) , which is similar to the 4.5 thiol groups found in the denatured A143Q protease. It was therefore concluded that as an intracellular enzyme, all five cysteines in the catalytic domain of HCMV protease are kept in the reduced form. HCMV protease, unlike papain, does not have cysteine as an active site residue. However, it was observed by us and also reported by others that the HCMV A143Q protease loses activity rapidly in the absence of DTT (26) and that DTT prevented the loss of activity. It is clear that the cysteine residues need to be reduced for maximal activity. Therefore, during experiments of extended duration, DTT was supplied exogenously at 1-10 mM. However, for experiments with short incubation time, the DTT at 8-12 µM carried over from the enzyme stock was adequate.
Figure 2: Purification of the HCMV protease catalytic domain A143Q from the E. coli lysate. LaneM, molecular mass markers. Lanes1 and 2, total lysates from uninduced(-) and induced (+) E. coli grown at 37 °C; lane3, the soluble fraction of induced E. coli lysate; lanes4 and 5, Q- and S-Sepharose pool containing the A143Q protease.
The same expression phagemid pMGH4 was
used to express the HCMV assembly protein precursor in E.
coli. The recombinant assembly protein precursor was soluble in
the lysate. Purification included Q-Sepharose, hydroxylapatite column
chromatography, and salt precipitations, and yielded assembly protein
precursor with sufficient purity for enzymatic studies. During
purification, it was observed that assembly protein precursor requires
5-10 mM DTT to maintain its solubility. Amino-terminal
sequencing confirmed the 45-kDa assembly protein precursor identity and
showed that its initiation methionine was removed. The enzymatic
activity of purified A143Q was initially demonstrated using the
assembly protein precursor. As shown in Fig. 3, 2.5 µg (1.08
µM) of substrate could be completely processed within 10
min by 0.5 µg (0.29 µM) of purified A143Q protease. In
the assay buffer, 6 mM DTT was included to optimize the A143Q
protease activity and to keep the assembly protein precursor soluble.
Cleavage of the assembly protein precursor by the A143Q protease was
quantitated by densitometric tracing of the full-length 45-kDa and
processed 40-kDa protein bands on Coomassie Blue-stained gels after
SDS-PAGE electrophoresis. The K and k
were determined to be 3.0 ± 1.0
µM and 13.3 ± 1.6 min
,
respectively. The large deviation in K
was mostly
due to the experimental limit inherent to this discontinuous gel-based
assay. The concentration range of substrate was also limited since the K
was so low. In vitro proteolytic
processing of the HCMV assembly protein precursor at the M-site by a
recombinant mutant V141A single-chain and wild-type two-chain HCMV
protease was also recently reported by Holwerda et
al.(27) . The processing of protein substrate was,
however, only about 50% complete in the presence of 20% glycerol and 10
mM DTT after a 19-h incubation. The authors suggest that the
incomplete turnover is due to a time-dependent loss of protease
activity. The HCMV A143Q is significantly more stable than the mutant
V141A and wild-type enzyme as observed both under assay conditions and
during storage.
Figure 3: Time course of assembly protein precursor cleavage by the purified HCMV A143Q protease. Cleavage were carried out by mixing 2.5 µg of purified assembly protein precursor and 0.5 µg of purified A143Q protease in a final volume of 60 µl of 10 mM HEPES, pH 7.0, and 6 mM DTT, and incubated at 37 °C. LaneM, molecular mass markers. Lane1, substrate alone incubated for 90 min; lane2, cleavage reaction stopped immediately by boiling after mixing substrate with protease; lanes3-13, time points incubated for 1, 2, 3, 4, 5, 10, 15, 20, 30, 60, and 90 min, respectively. Samples were boiled in reducing sample buffer, and subjected to 16% SDS-PAGE and staining. Molecular mass of markers is indicated in kDa.
In order to develop a sensitive continuous
peptide-based assay for the HCMV protease, three peptide substrates (Table 1) mimicking the maturation cleavage site of the
full-length HCMV protease were made. Various synthetic peptides were
previously used by other groups to measure the HCMV protease
activity(25, 26, 27, 28, 29) .
The sequence of the peptide substrate A6376 was the same as the one
used by Burck et al.(26) . This peptide sequence
differs from the native M-site sequence at P6, P2`, and P6` positions.
These changes were maintained in A6376 for its ease of synthesis and
higher solubility as compared with its native sequence. After cleavage
of A6376 peptide by the HCMV protease, the amino-terminal peptide
product was readily resolved by reverse-phase HPLC. Cleavage at the A/S
scissile bond within the A6376 peptide was confirmed by the same
retention time observed by HPLC analysis of the synthetic peptide
containing only P6 to P1 residues. The terminal sequences of the A6376
peptide were also modified to produce the resonance energy transfer
fluorogenic peptides PK03 and AM1013. The carboxyl-terminal glycine in
PK03 and lysine in AM1013 was chosen solely for synthetic reasons. We
chose anthranilic acid and nitrotyrosine as the donor and acceptor pair
mainly because they showed the sufficient quenching efficiency needed
for long range resonance energy transfer over 10-16 amino
acids(24) . With 10 amino acids corresponding to roughly 30
Å distance between the two chromophores, only 25% of the initial
fluorescence (or 75% quenching efficiency) was observed with PK03 and
AM1013. After cleavage of these fluorogenic peptide substrates, the
fluorescence of the anthranilic acid increases roughly 4-fold. Part of
the starting fluorescence is due to free anthranilic acid contaminating
the peptide sample (data not shown). However, the incomplete quenching
of the anthranilic acid by the nitrotyrosine is compensated for by the
very high quantum yield of anthranilic acid. Reliable initial rates
were generated by turning over as little as 100 nM, or
equivalent to 0.5% of the fluorogenic substrate during the reaction.
There is no measurable rate of substrate hydrolysis in the absence of
HCMV protease. The values of k/K
for these substrates and assembly protein precursor are shown in Table 2. The fluorogenic peptides have larger k
/K
values than the A6376
peptide presumably due to the removal of the K
-increasing arginine at the amino terminus rather
than the addition of the nitrotyrosine and anthranilic acid moieties.
This presumption is supported by AM1013 where the positions of the two
moieties are switched with little effect on k
/K
. From this data, we see
that PK03 is a better substrate than A6376 by a factor of 4.3-fold.
This continuous fluorescent assay is very sensitive and able to detect
the HCMV protease activity in nanomolar concentration over a short
reaction time.
After obtaining the k and K
values of purified HCMV A143Q protease for
different substrates, we examined if the natural protein substrate
would be turned over with a much higher k
than
the peptide substrate due to possible conformational changes occurring
around the catalytic residues upon substrate binding. We were also
interested in establishing that protease activity measured with
synthetic peptides reflects its proteolytic activity in cleaving its in vivo protein substrate. Data presented in Table 2show that the k
values are all very
similar; regardless of the substrate used. This suggests that the
geometric arrangements of catalytic residues in the Michaelis complexes
formed with different substrates are very similar. Different K
values indicate that the HCMV protease has a
binding affinity toward its in vivo protein substrate at least
45-fold stronger than for synthetic peptide substrates presumably due
to the larger surface binding areas as compared with peptides. The
binding of the HCMV protease to residues outside of the M-site of the
assembly protein precursor, however, does not cause significant change
in k
. Based on the similar k
values in this comparative analysis, activity measured with the
synthetic peptides does reflect its proteolytic activity in cleaving
its in vivo protein substrate.
In order to establish that
the mutant HCMV A143Q protease has the same activity as the wild-type
enzyme, a comparison of the enzymatic activity of the HCMV A143Q mutant
protease was made against the activities previously reported by various
other groups (25, 26, 27, 28, 29) for
the wild-type counterpart (Table 3). This analysis clearly
demonstrates that mutant A143Q protease has very similar activity
against peptide substrate when compared with the wild-type HCMV
protease. In one case, when identical synthetic peptide was used as the
substrate for both enzymes, the A143Q protease has k and K
values of 18.5 min
and 476 µM (or k
/K
of 0.038
µM
min
) at 25 °C
as compared with 16.0 min
and 586 µM (or k
/K
of 0.027
µM
min
) for the
refolded wild-type enzyme at 37 °C(26) . The slight
differences in data generated with two enzyme forms are likely due to
different temperatures used for cleavage reactions. It was observed
that HCMV protease had maximal activity at 27 °C (data not shown).
Based on such analysis presented in Table 3, we conclude that the
mutant A143Q protease possesses the same, or similar, enzymatic
activity as its wild-type counterpart against synthetic peptide
substrates. We have not compared the activity of the mutant A143Q and
wild-type protease against the natural protein substrate directly due
to difficulty inherent in the gel-based assay and the reported
instability of the wild-type enzyme. The discontinuous gel-based assay
may also lack the sensitivity and accuracy to detect potential subtle
differences between the two enzymes. However, since the protease
activity measured with the A143Q against the synthetic peptides does
reflect its proteolytic activity in cleaving in vivo protein
substrate as discussed in the previous section, it can be inferred that
the mutant A143Q protease also has the same, or similar, activity
against its in vivo substrate as compared with the wild-type
enzyme. This argument is strengthened by the fact that (i) alanine 143
of the HCMV protease lies in a variable region and close to a six-amino
acid insertion present only in HCMV and Epstein-Barr virus proteases,
but not HSV-1 and varicella-zoster virus proteases, and (ii) that
alanine 143 may reside in a flexible loop and thus becomes accessible
to proteolytic attack and is unlikely to serve any significant role in
substrate binding or catalysis.
Many factors affected the activity
of this protease. Both the k and the K
were affected by changes in the glycerol,
Me
SO, and buffer concentrations. Increasing concentrations
of glycerol enhance the formation of peptide cleavage products by
20-fold (Fig. 4). Moreover, this increase in activity can be
even greater than the observed 20-fold enhancement since the amounts of
product turned over at higher concentrations of glycerol do not
represent initial rates of product formation. In the absence of
glycerol, increasing concentrations of Me
SO in the reaction
mixture up to 25% enhance the activity (Fig. 5). However, in the
presence of 50% glycerol the addition of Me
SO is harmful to
activity. An increase in the concentration of NaCl also decreases the
A143Q protease activity. This NaCl-inhibition can be fit to an
IC
of 25 mM, and such inhibition is likely due to
ionic strength since many divalent cation salts also inhibit A143Q
activity (data not shown). The effect of ionic strength on k
and K
for A6376 is
demonstrated in Fig. 6, where these values were determined in 10
and 100 mM MOPS, pH 7.2, in the presence of 50% glycerol.
These data are presented as a double reciprocal plot, but the k
and K
were determined by
fitting the data to the nonlinear form of the Michaelis-Menten equation
as described under ``Experimental Procedures.'' At 10 mM MOPS, the k
was 5.57
min
, and the K
was 152
µM. At 100 mM MOPS, the k
was 9.2 min
, and the K
was 1500 µM. The measured values of k
for this experiment are low since 4%
Me
SO was included in the assay. Increases in
Me
SO concentration seem to have a detrimental effect on k
. The effect of the MOPS concentration is
largely on K
rather than k
,
and this is reflected in the 6.2-fold difference in the k
/K
value.
Figure 4: Effect of glycerol on the HCMV A143Q protease activity. HCMV A143Q protease was assayed at 30 °C for 15 min in 10 mM MOPS, pH 7.2, 8 µM DTT with increasing concentrations of glycerol. Activity was determined with A6376 peptide by the HPLC-based assay. The percent activity was calculated from the maximal activity, which measured the amount of product formed over the 15-min incubation.
Figure 5:
Effect of MeSO on the HCMV
A143Q protease activity. HCMV A143Q protease was assayed at 30 °C
in 50 mM MOPS, pH 7.2, 10 µM DTT in the presence
or absence of 50% glycerol with increasing concentrations of
Me
SO. Activity was determined with A6376 by the HPLC-based
assay. Relative activities versus Me
SO
concentrations are plotted, and 100% activity represents the maximal
activity calculated from the amount of product formed during a 15-min
incubation. Maximal activity in 50% glycerol was 7-fold greater than in
its absence at this buffer concentration.
Figure 6:
Effect of buffer concentration on k and K
for A6376.
HCMV A143Q protease activity was measured by the HPLC assay at 30
°C for 15 min in 10 or 100 mM MOPS, pH 7.2, 8 µM DTT, 50% glycerol, and 4% Me
SO. The concentration
range of A6376 was 50-400 µM and 100-2000
µM; the concentration of HCMV protease was 0.16 and 1.1
µM for the 10 mM MOPS and 100 mM MOPS
data sets, respectively. The data are shown as a double-reciprocal plot
where V
represents the amount of product formed
per minute during a 15-min incubation.
In summary,
we have demonstrate that (i) by one single amino acid substitution, the
A143Q mutant can be purified from E. coli as a stable
single-chain 29-kDa protein, (ii) purified A143Q protease can recognize
and cleave its in vivo protein substrate efficiently and is
sufficient in cleaving the assembly protein precursor in vitro without other cellular or viral factors, (iii) a sensitive,
continuous peptide-based fluorescent assay has been developed for the
HCMV protease to detect activity in nM concentration of enzyme (Fig. 7), and activity detected with peptide substrates has k values similar to that measured with the
protein substrate, and (iv) A143Q mutant enzyme has the same, or
similar, activity as the wild-type enzyme.
Figure 7:
Progress curve of product formation for
PK03 in the RET-fluorogenic assay. This time course was collected at 25
°C in 25 mM MOPS pH 7.2, 1 mM DTT, 50% glycerol,
and 2% MeSO. The concentrations of PK03 and HCMV A143Q
protease were 20 and 0.67 µM, respectively. For clarity,
only 5% of the collected data points are shown. The data were fit to a
single exponential equation with k
= 0.11
min
. The value k
/K
was calculated
as described under ``Experimental
Procedures.''
The HCMV protease has recently been demonstrated by Holwerda et al.(27) to be a serine protease, and Ser-132 was identified as the active site nucleophile based on the irreversible modification by the serine protease inhibitor diisopropyl fluorophosphate. Similar irreversible modification of the HSV-1 protease at Ser-129 by diisopropyl fluorophosphate was reported by DiIanni et al.(29) . And, mutation of the equivalent Ser residue in the simian CMV protease to Ala also eliminated the enzyme activity(28) . Since HSV and CMV are members of the Herpesviridae family, and their proteases share sequence homology, it suggests that both HCMV and HSV-1 protease could belong to the serine protease class. Inhibition of the HCMV protease by N-ethylmaleimide and iodoacetamide, at high inhibitor/enzyme molar ratio, was also documented (26) and confirmed in this laboratory (data not shown). However, this inhibition may be due to nonspecific alkylation of Cys residues leading to inactivation of the enzyme.
Interestingly, the recently determined human rhinovirus 3C protease structure reveals that its three-dimensional structure possesses a trypsin-like polypeptide fold, and its catalytic residues Cys-146, His-40, and Glu-71 have an overall geometry similar to that of the Ser-His-Asp catalytic triad found in trypsin-like serine proteases (30) . The human rhinovirus protease structure demonstrates that this trypsin-like viral protease has a cysteine in place of serine to serve as an active site nucleophile. For the HCMV protease, His-63 and Glu-122 were recently identified as two other residues in the active site triad(31) . The different spacing of the HCMV protease active site residues: His-63, Glu-122, and Ser-132 and the lack of sequence homology with other known serine proteases together suggest that herpes proteases belong to a new class of serine proteases and their structures may be completely different from any solved serine protease structure. The structure solution of the HCMV A143Q protease is currently being pursued. Three-dimensional structure of the HCMV protease will undoubtedly reveal if the Ser-132 is indeed the active site residue and its geometric arrangement within the catalytic triad of this new class of viral protease.