(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/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.
The catalytic activity of the herpes simplex virus type 1
(HSV-1) ()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) .
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/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.
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, 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).
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 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.
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.'' , citrate;
,
phosphate.
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/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.
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, 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.
A comparison of the k 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.
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
1.5 M, or 450 mg/ml(21) .
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 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.