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(Received for publication, July 5, 1995) From the
The catalytic efficiency of the mature HSV-1 protease has been
examined as a function of solvent composition. With the peptide
substrate HTYLQASEKFKMWG-amide, the specificity constant (k
The catalytic activity of the herpes simplex virus type 1
(HSV-1) ( ICP35 is present in an immature form
of HSV capsids, known as B capsids, during capsid assembly within
infected cell nuclei. The proteolytic conversion of ICP35 from this
immature form (ICP35cd) to the shorter form found only within cell
nuclei (ICP35ef) is concomitant with the conversion of B capsids to
capsids that contain viral DNA, known as C capsids. Thus, HSV protease
action occurs within the cell nucleus and possibly within the viral
capsid itself during a morphological transformation of
capsids(8) . The necessity of HSV protease activity for the
infectivity of HSV has prompted recent in vitro characterization of this enzyme and the related protease from
human cytomegalovirus
(HCMV)(9, 10, 11, 12, 13) .
Inactivation with diisopropyl fluorophosphate was used to identify
these enzymes as serine proteases(10) , but sequence analyses
have not revealed obvious homologies with the well characterized groups
of serine or other proteases. Peptides containing the sequences found
in natural protein substrates are cleaved between a characteristic
Ala-Ser sequence. Using peptide substrates, we have reported a k
The effect of various
salts on activity was examined in assays in which the HSV-1 protease
and substrate concentrations were maintained constant. As shown in Table 1, many salts increased the activity of the protease,
particularly those with multivalent anions. In the case of sodium
citrate at 0.8 M, for instance, the observed hydrolytic rate
was enhanced 202-fold. Phosphate and sulfate were also potent
activators of the protease, increasing activity 118- and 110-fold,
respectively. Chloride had little effect, while bromide, iodide, and
perchlorate were all inhibitory. The order of anion effectiveness for
activation on a molarity basis (as opposed to ionic strength) is
Br Activation was also observed with the multiply hydroxylated
alcohols, glycerol, and sorbitol, as shown in Table 2. Other
cosolvents of more hydrophobic character, such as ethanol and
dimethylformamide, were strongly inactivating, even at 2%. We note,
however, that inhibition by these solvents could be overcome by the
activating cosolvents. For example, while 2% dimethyl sulfoxide reduced
HSV protease activity 37% (Table 2), it only reduced the activity
8% with 0.8 M sodium citrate present (data not shown). The
stability of the protease activity in the presence of various additives
was examined. In all cases, including no additive, 0.8 M citrate, 10% glycerol, or 0.2 M bromide, the accumulation
of product was linear over a 60-min period. Hence, the wide differences
in enzyme activity seen when various salts were added were not due to
time-dependent differences of protease stability in the assays. The
activation of the HSV-1 protease by increasing citrate and phosphate
concentrations was progressive and did not saturate at the highest salt
concentration tested, as shown in Fig. 1. The greatest effects
for either citrate or phosphate appeared at concentrations of 0.4 M and above, although some activation was also observed at a
concentration as low as 0.05 M (Fig. 1, inset). In the presence of activators, substrate saturation
was observable within the limits of peptide solubility, so k
Figure 1:
Relative activity of the HSV-1 protease
as a function of sodium citrate or sodium phosphate concentration.
HSV-1 protease activity at 37 °C was measured with the peptide
HTYLQASEKFKMW-amide as described under ``Materials and
Methods.''
Figure 2:
Fluorescence emission changes in the HSV-1
protease or N-acetyltryptophanamide in the presence of
activating concentrations of citrate. Spectra were acquired as
described under ``Materials and Methods.'' A, the
HSV-1 protease (0.94 µM); B, N-acetyl-L-tryptophanamide (0.94 µM). In A and B, solvents were as follows: no citrate
added(--), 0.4 M sodium citrate(- - -),
and 0.8 M sodium citrate (--). In C, the
HSV-1 protease fluorescence emission intensity at 343 nm for spectra
obtained with various sodium citrate concentrations is plotted as a
function of citrate concentration.
To further probe possible
effects of activators on the HSV-1 protease itself, sensitivity to
trypsin was examined. Following treatment of the enzyme with trypsin
for 60 min at 37 °C, SDS-PAGE was performed to qualitatively
examine the extent of trypsin digestion of the HSV protease as a
function of citrate concentration. As shown in Fig. 3, 0.8 M citrate protected the enzyme against trypsin digestion. The
activity of trypsin itself was shown to not significantly change,
either by incubation in the high citrate buffer or by incubation with
the HSV-1 protease under these activation conditions (data not shown).
Figure 3:
Susceptibility of the HSV-1 protease to
trypsin proteolysis in the presence of sodium citrate. The HSV-1
protease was incubated for 60 min at 37 °C with or without trypsin,
either with or without 0.8 M sodium citrate present, as
described under ``Materials and Methods.'' Samples were
analyzed by SDS-PAGE (16% gels) and stained with Coomassie Brilliant
Blue. Lanes1 and 2, no trypsin added; lanes3 and 4, trypsin added at 0.1
µg/ml. Lanes1 and 3, no citrate added; lanes2 and 4, sodium citrate present at 0.8 M during incubation. The arrow indicates the position
of the 27-kDa undigested HSV-1 protease. Molecular mass markers are in
the firstlane, with their corresponding molecular
masses marked in kilodaltons.
In this work, we have described solutes that enhance the
specificity constant for the HSV-1 protease (k The anion activation data reported herein for
the HSV protease are not characteristic of a site-specific binding
event as no saturation of the activation was observed (Fig. 1).
In addition, a site-specific activation would be expected to display
chemical structure correlations, but in the examples shown here (Table 1), phosphate, citrate, and isocitrate exhibited similar
effects on activity. The observed order of potencies for anion
activation of the HSV protease correlates with the Hofmeister series of
anions, such that the effectiveness for activation is
Br A comparison of the k The
activation described here raises important questions regarding the
conditions under which protease catalysis occurs in vivo during virus assembly. Is the nucleus an activating
microenvironment, with its abundance of nucleotide polyanions, so that
the HSV protease is activated only after transport into the nucleus? Is
the mature form of the HSV protease the biologically relevant form in
terms of in vivo proteolytic events? Our examination of some
likely nuclear activators did reveal activation by selected
triphosphate nucleotides (Table 3), but in view of the inhibition
by other nucleotides, the meaning of these activity effects is unclear.
It is important to note, however, that the DNA concentration in the
vicinity of a developing capsid is much higher than we were able to
test. In an HSV C-type capsid in which the DNA packaging is complete,
the concentration of DNA phosphate diesters can be calculated to be
The proteases of the
herpes group of viruses are synthesized as precursor proteins. In the
case of HCMV, Jones et al.(22) have found that the
HCMV protease precursors appear to be as active as the mature form of
the HCMV protease in a bacterial expression system. It remains to be
determined which of the protease forms of either HSV or HCMV, precursor
or mature, actually perform the cleavages during capsid maturation.
Since the mature HSV protease appears to have less activity than the
mature HCMV protease in in vitro assays containing no special
additives (5.2 versus 404 M
Volume 270,
Number 39,
Issue of September 29, pp. 22697-22700, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/K
) at pH 7.5
for cleavage is 5.2 M
s
.
This value increases to 38 M
s
when 25% glycerol is present in the reaction mixture. It was
found that glycerol activation is but one case of the general
phenomenon of HSV-1 protease activation by kosmotropes, or water
structure-forming cosolvents. For example, an 860-fold increase in the
protease activity (k
/K
= 4500 M
s
) occurs in the presence of 0.8 M sodium citrate. Similarly, the presence of 0.8 M sodium
phosphate activates the catalytic efficiency by 420-fold (k
/K
= 2200 M
s
). The extent of
HSV-1 protease activation by various anions correlates with the
Hofmeister series. Both the susceptibility to proteolysis by trypsin
and the protein fluorescence spectra of the HSV-1 protease change in
the presence of activating solvents, suggesting a conformational change
accompanying activation.
)protease is essential for viral nucleocapsid
formation and for viral replication(1, 2) . The
proteases of the herpesviruses are synthesized as precursor proteins
that undergo autoproteolytic processing during viral assembly. The
protease catalytic domain is localized in the N terminus of the
precursor, which in the case of HSV is the N-terminal 247 amino acids
of the 635-amino acid precursor
protein(3, 4, 5) . The natural substrates for
the HSV protease are the viral protease precursor and the viral
assembly protein known as VP22a or ICP35 (infected cell protein 35).
The protease precursor and ICP35 are encoded by the Ul26 and Ul26.5 genes of HSV-1, respectively. The open reading frames
of these genes overlap such that the smaller open reading frame of Ul26.5 is identical to the C-terminal end of the open reading
frame of Ul26, the protease precursor
gene(6, 7) ./K
for the
purified mature HSV protease of 38 M
s
, and DiIanni et al. reported a k
/K
of 37 M
s
with a similar assay
condition that included 25% glycerol(9, 12) . These
values are many orders of magnitude lower than found for other serine
proteases such as chymotrypsin and thrombin (10
M s
) (14, 15) and much lower than observed for other viral
proteases such as rhinovirus 3C protease (1440 M
s
) (16) and human immunodeficiency
virus protease (13,000 M
s
)(17) . While it is conceivable that
the low catalytic efficiency of the HSV protease observed in vitro may be sufficient to account for its essential physiological role
in nucleocapsid assembly, the low activity prompts the consideration
that some factor might enhance catalytic efficiency, such as an
accessory cellular or viral component or appropriate solvent
conditions. This report describes our findings of a large activation
effect of kosmotropes, or water structure-forming substances, on the
catalytic efficiency of the HSV-1 protease. The results suggest that a
different conformation of the enzyme in the presence of kosmotropes is
a factor affecting its action in catalyzing amide hydrolysis.
Bacterial Expression and Enzyme Purification
A
306-amino acid precursor form of the HSV-1 protease was expressed in Escherichia coli cells and purified with a modified version of
the protocol described previously(12) . Briefly, soluble enzyme
precursor from lysed E. coli cells was chromatographed on
Fractogel-SO
(Merck) at pH 7.0 with elution by a gradient
of sodium citrate. Autoprocessing to the mature 246-amino acid form of
the enzyme was achieved by the addition of 1.8 M sodium
citrate to peak fractions to a final concentration of 0.5 M,
followed by incubation at 4 °C for 2 h. The processed sample was
desalted on Sephadex G-25 (Pharmacia Biotech Inc.). Rechromatography on
Fractogel-SO afforded a preparation of the mature enzyme
that was >95% pure as judged by SDS-PAGE with Coomassie Brilliant
Blue staining. N-terminal sequencing demonstrated that the N terminus
began with alanine (codon 2), such that the first encoded amino acid,
methionine, had been removed. Thus, the protein is one amino acid
shorter (246) than the encoded length (247 amino acids). Protein
concentrations were determined by quantitative amino acid analysis.Cosolvent Effects
For experiments examining anion
effects, buffer solutions were prepared with the sodium salt of the
anion, and the pH was adjusted to 7.5 with concentrated HCl where
necessary (i.e. citrate, glutamate, and malate). An exception
was phosphate, where NaH
PO
was prepared in the
buffer, and the pH was adjusted upward with NaOH.Activity Measurements
Enzyme activity was measured
with the peptide substrates HTYLQASEKFKMW-amide and
HTYLQASEKFKMWG-amide using high pressure liquid chromatography for the
separation of products from substrates and for
quantitation(12) . C-terminal cleavage product detection was
with a Hewlett-Packard Model 1046A fluorescence detector (excitation at
280 nm and emission at 350 nm), giving a 10:1 signal-to-noise ratio for
45 pmol of product peptide. Reactions were performed at 37 °C in pH
7.5 buffer containing 52 mM MES, 52 mM TAPSO, 100
mM diethanolamine, 1 mM EDTA, 1 mM dithiothreitol, 0.1% bovine serum albumin, and the various
additives listed below and in Table 1Table 2Table 3.
Reactions were initiated by the addition of a stock HSV-1 protease
solution (0.625 mg/ml) in the above buffer to a final concentration of
0.025 mg/ml (0.94 µM) and quenched with the addition of 5%
HPO. Assays comparing various anion and
cosolvent effects were conducted with a substrate concentration of
either 53 or 210 µM.
Proteolysis of the HSV Protease
Trypsin treatment
of the HSV-1 protease (0.15 mg/ml) was performed with 0.1 µg/ml
trypsin at 37 °C for 60 min. Trypsin activity measurements with the
fluorogenic substrate t-butoxycarbonyl-Val-Pro-Arg-7-amino-4-methylcoumarin were
performed at 37 °C. After trypsin treatment, samples were boiled
with SDS gel sample buffer, and small volumes of sodium citrate were
added to some samples to equalize the extent of band spreading on the
gel. SDS-PAGE was performed with 16% gels, and proteins were visualized
by Coomassie Brilliant Blue staining.Steady-state Fluorescence
Measurements
Fluorescence emission spectra of the HSV-1 protease
were obtained with a Spex Fluorolog spectrophotometer, with excitation
at 280 nm and all slit widths at 4 mm. Samples were in the activity
assay buffer without bovine serum albumin and were held at 37 °C
during data acquisition. HSV-1 protease concentration was 0.025 mg/ml.
Activation by Anions
The HSV-1 protease had a
specificity constant (k/K
)
of 5.2 M s
at 37 °C
at pH 7.5 with no glycerol present using a peptide substrate that
represents the cleavage site that generates the mature protease C
terminus, HTYLQASEKFKMW-amide. Under initial velocity conditions, where
the reaction velocity was shown to be constant for 60 min, the rate was
linearly proportional to substrate concentration from 0.1 to 2
mM. Thus, the K
was well above the
highest substrate concentration tested, and k/K
was calculated from the
slope of the velocity-concentration line., I
< Cl
< CH
COO < F
< SO
,
PO
<
citrate
. This is the same order as the Hofmeister
series of anions(18) . The activation of the HSV-1 protease by
anions was insensitive to stereochemical configuration, as evidenced
when isocitrate was compared with citrate or when L-malate and L-glutamate were compared with their D-isomers (Table 1). The effect of the counterions on HSV protease activity
was relatively insignificant, with (NH
)SO being slightly less activating relative to the Na and K
sulfate salts (data not shown).
and K
parameters in the
presence of 0.2 M sodium citrate were determined to be 3
min and 1.32 mM (k
/K
= 40 M s
), respectively, and
those in the presence of 0.8 M citrate were determined to be 4
min
and 0.016 mM (k
/K
= 4500 M s
), respectively.
Relative to the case where no activator is present (k
/K
= 5.2 M s
), k
/K
increased 860-fold in
the presence of 0.8 M citrate. Similarly, using a peptide
substrate representing the cleavage site within ICP35,
ALVNASSAAHVDV-amide, k/K
increased from 0.45 to 488 M s
(1085-fold) by changing citrate from 0 to
0.8 M, demonstrating that activation is not
substrate-specific.
, citrate; 
,
phosphate.
Nucleotide Effects on Activity
Nucleotides and
polynucleotides were examined for possible effects on HSV protease
activity. The results are shown in Table 3. While some activation
can be seen with triphosphate nucleotides such as CTP and dGTP, other
nucleotides were inhibitory. The concentrations of polynucleotides
examined were limited due to solubility. Within the range of
polynucleotide concentrations examined, the effects are minor in
comparison with those seen with much higher concentrations of anions (Table 1).Anion Effects on the HSV-1 Protease
Alone
Fluorescence emission spectra of the HSV-1 protease are
shown in Fig. 2A. The emission maximum is 354 nm in the
absence of any activators. In the presence of citrate, the emission
spectra shift toward shorter wavelength maxima with markedly greater
intensity (Fig. 2). Phosphate was also observed to produce a
similar shift (data not shown). In contrast, the same solvent changes
produced a red shift in emission maximum for N-acetyltryptophanamide, as shown in Fig. 2B.
Short peptides containing tryptophan also exhibited a red shift in the
higher salt buffers (data not shown). Titration of wavelength shifts
and intensity changes as a function of citrate concentration produced
minimal effects on the HSV-1 protease at concentrations <0.4 M, but were much more pronounced at higher concentrations, as
shown in Fig. 2C. Interestingly, the trend of solvent
effect on fluorescence shown in Fig. 2C is similar to
activation effects shown in Fig. 1.
/K
) orders of magnitude
over that found in simple aqueous buffers. The k/K
of 4500 M s
reported here in
activity assays containing 0.8 M sodium citrate is
860
times greater than in the absence of citrate (5.2 M
s
) using the substrate
HTYLQASEKFKMW-amide. We have previously reported a specificity constant
of 38 M
s
using the same
substrate in 25% glycerol(12) , a cosolvent somewhat less
activating than citrate. The specificity constants listed here for the
HSV protease can be contrasted with that found for the closely related
recombinant HCMV protease in the absence of any of activators (404 M
s
)(11) . Some
increase in the activity of the HCMV protease has been noted in the
presence of glycerol(19) , although the kinetics have not been
examined in detail.
, I
< Cl
< CH
COO < F
< SO
,
PO
<
citrate
. HSV-1 protease activation is thus a general
solvent effect. Indeed, the activation data described here have the
characteristic Hofmeister effect attributes: the effects become
apparent at moderate concentrations (0.01-1 M); the
effects are dominated by anions; and there is a sign inversion of
effect at NaCl (reviewed in (18) ). The anions producing
activation of the HSV-1 protease are also those that have been termed
kosmotropes, or water structure-forming solutes.
and K
values for the
substrate HTYLQASEKFKMW-amide using different sodium citrate
concentrations revealed that the kinetic parameter most changed by the
kosmotropes is the K, such that 1.32 and 0.016
mM were obtained as K values for 0.2 and
0.8 M citrate, respectively. Since kosmotropes are also
solutes that produce ``salting-out'' and aggregation effects
for proteins, it is conceivable that the lowering of K observed here is a result of anion destabilization of the free
peptide substrate in solution, producing a relative stabilization of
the enzyme-substrate complex and a lowering of the observed K(20) . While there may be some
contribution of anion destabilization of the unbound substrate to the
lowering of K, the loss of susceptibility of the
HSV-1 protease to trypsin digestion in 0.8 M citrate and
spectral changes evidence a conformational change in the HSV-1 protease
( Fig. 2and Fig. 3). Hence, kosmotropes may promote a
conformational state of the mature HSV-1 protease with a greater
affinity for substrate than exists in simple aqueous solution.1.5 M, or 450 mg/ml(21) .
s
), it may be that the most important
functional form of the HSV protease is an activated form, possibly a
precursor. Kosmotropes induce in the mature HSV protease a more active
conformation of the catalytic domain, possibly one occurring naturally
when the domain is in association with other viral components. It
remains for the precursor forms of the herpesvirus proteases to be
isolated and evaluated as catalysts.
)
We thank Dr. Victor M. Garsky for synthesis of
peptides and Dr. Mohinder K. Sardana for amino acid analyses. We also
thank Dr. Lawrence C. Kuo for support and useful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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