(Received for publication, August 17, 1995; and in revised form, September 29, 1995)
From the
The herpes simplex virus type 1 protease is expressed as an
80,000-dalton polypeptide, encoded within the 635-amino acid open
reading frame of the UL26 gene. The two known protein substrates for
this enzyme are the protease itself and the capsid assembly protein
ICP35 (Liu, F., and Roizman, B.(1991) J. Virol. 65,
5149-5156). In this report we describe the use of a rapid and
quantitative assay for characterizing the protease. The assay uses a
glutathione S-transferase fusion protein containing the
COOH-terminal cleavage site of ICP35 as the substrate (GST-56). The
protease consists of N, the NH
-terminal 247
amino acid catalytic domain of the UL26 gene product, also expressed as
a GST fusion protein. Upon cleavage with N
, a single 25-mer
peptide is released from GST-56, which is soluble in trichloroacetic
acid. Using this assay, the protease displayed a pH optimum between 7
and 9 but most importantly had an absolute requirement for high
concentrations of an antichaeotrophic agent. Strong salting out salts
such as Na
SO
and KPO
(
1 M) stimulated activity, whereas NaCl and KCl had no effect.
The degree of stimulation by 1.25 M Na
SO
and KPO
were 100-150- and 200-300-fold,
respectively. Using the fluorescent probe 1-anilino-8-naphthalene
sulfonate, the protease was shown to bind the dye in the presence of
1.25 M Na
SO
or KPO
, but
not at low ionic strength or in the presence of 1.25 or 2.2 M NaCl. This binding was most likely at the protease active site
because a high affinity cleavage site peptide, but not a control
peptide, could displace the dye. In addition to cleaving GST-56, the
herpes simplex virus type I protease also cleaved the purified 56-mer
peptide. Circular dichroism and NMR spectroscopy showed the peptide to
be primarily random coil under physiological conditions, suggesting
that antichaeotrophic agents affect the conformation of the substrate
as well as the protease.
The existence of a herpesvirus-specific protease was first
reported in 1991, when the protein encoded by the UL26 gene of herpes
simplex virus I (HSV-1) ()was shown to process both itself
and the precursor form of a capsid scaffolding protein ICP35 ((1) ; also known as VP22a, p40). Shortly afterwards, other
reports demonstrated the existence of similar proteases in simian (2) and human (3) cytomegaloviruses (CMV). These
processing systems are reminiscent of those in bacteriophage, whereby a
specific phage protease processes a scaffolding protein during capsid
maturation(4, 5) .
In early studies involving
expression of recombinant protein in Escherichia coli, the
HSV-1 protease was shown to cleave two distinct sites, both contained
within the protease polypeptide itself (6, 7) . Both
occur between Ala and Ser residues, at positions 247 and 248 and
positions 610 and 611(7) . This specificity is shared by the
CMV enzyme(2, 3) . The carboxyl-terminal cleavage
occurs at a site common to both the protease and a capsid scaffolding
protein ICP35 (M site) ()and releases a 25-residue,
COOH-terminal peptide. ICP35 was recently shown to be a product of the
UL26.5 gene and to be in-frame and entirely contained within the
carboxyl-terminal 329 amino acids of the protease(9) .
Functionally, the processing of ICP35 by the protease appears to be an
essential viral event, because a ts mutant that fails to
process ICP35 at the nonpermissive temperature also fails to package
DNA(10) . Cleavage at the site proximal to the amino terminus
(R site) results in the release of N
, an
NH
-terminal, 247-residue polypeptide that contains the
proteolytic activity(11, 12) . This processed form of
the protease (also known as Prn or VP24) is known to be a constituent
of the viral capsid (13) and has been shown to be a serine
protease(14, 15) .
A major barrier in the study of
the HSV-1 protease has been the extremely low activity level of the
enzyme when assayed in vitro. In one study, DiIanni et al.(16) surveyed several peptide substrates and reported k and K
values of
0.2 min
and 190 µM, respectively, for
the cleavage of ALVNASSAAHVDV (M site peptide mP5-P8`). In a subsequent
study, Darke et al.(17) reported k
and K
values of 2.0
min
and 0.88 mM, respectively, for the
cleavage of the R site peptide HTYLQASEKFKMW-amide (rP6-P7`) and about
twice the activity as DiIanni et al.(16) for mP5`P8`.
In the present report we make use of a quick and sensitive assay for
the HSV-1 protease to characterize a unique interaction with
antichaeotrophic salts. Most significantly, we find that molar
concentrations of Na
SO
result in changes to
both the protease and substrate and stimulate activity over 100-fold.
Following the purification of an active form
of the protease(12) , a need arose for a rapid and quantitative
assay for activity. An acid solubilization assay was originally
developed in which metabolically labeled
[S]ICP35 could be cleaved by GST-N
to release a COOH-terminal, 25-amino acid peptide. This peptide
was soluble in 10% trichloroacetic acid. Because it contained two of
the protein's seven methionines, it could be easily quantitated
by liquid scintillation counting. An adaptation of this assay was to
use a fusion protein substrate in which the COOH-terminal 56-residue
peptide of ICP35 was fused to the COOH-terminal end of glutathione S-transferase (GST-56, Fig. 1). This protein was
expressed at high levels in E. coli and could be purified in
one step by glutathione agarose affinity chromatography in yields of
20-30 mg/liter of culture. The purified protein was free of
nonspecific proteolytic activity and was stable to prolonged
incubations at 30 °C. When purified from metabolically labeled
cells, [
S]IGST-56 was obtained at >95% purity
with yields of 0.15 mCi of labeled protein/mCi of labeled culture
(15-fold greater yield than [
S]ICP35).
Figure 1: Schematic of the fusion protein substrate GST-56, depicting GST (light box) and M site 56-mer peptide (dark box) domains. Cleavage of the 56-mer by the HSV-1 protease releases a TCA-soluble 25-mer peptide containing two of the protein's twelve methionines.
Figure 2:
HSV-1 protease assay, showing the salt
dependence for cleavage of 4 µM
[S]GST-56 by 0.3 µM GST-N
. A, release of the COOH-terminal,
25-mer peptide, as measured by liquid scintillation counting. B, analysis of the polypeptide cleavage by SDS gel
electrophoresis and autoradiography. Lane B, no enzyme was
present. C, quantitation of the polypeptides shown in B by two-dimensional radioactivity
detection.
In parallel studies using the homologous
protease from HCMV, a similar stimulation was seen (Table 1).
HSV-1 N and HCMV N
were stimulated 60- and
130-fold, respectively, by 1.25 M Na
SO
when using saturating levels of radiolabeled fusion protein
substrates. Under these conditions, the HCMV enzyme was about an order
of magnitude more active that the HSV-1 enzyme.
The stimulation of
HSV-1 N activity by Na
SO
was
further examined using a peptide substrate.
[
S]GST-56 from metabolically labeled cells was
treated with thrombin to produce GST and a 60-residue peptide. The
peptide, containing the 56-mer fused to a thrombin site linker
(Gly-Ser-Pro-Met) at its NH
terminus, was purified under
nondenaturing conditions. When assayed with HSV-1 N
in the
presence of 0, 0.5, and 1.25 M Na
SO
and analyzed by HPLC, cleavage was seen only in the presence of
Na
SO
(Table 2). The appearance of a
single radiolabeled proteolysis product (13.3 min) was consistent with
the occurrence of a single cleavage event in a peptide where the only
two methionines were at the extreme COOH terminus. However, when the
56-mer was examined for secondary structure, by either circular
dichroism (in 100 mM KPO
, pH 6.7) or NMR
spectroscopy (25 mM sodium acetate, pH 5.5), it exhibited
spectra characteristic of random coil (results not shown). Spectra were
not obtainable in 1.25 M Na
SO
.
Figure 3:
Comparison of different salts on the
stimulation of the HSV-1 protease. Assays were performed as described
under ``Materials and Methods'' using 1 µM [S]GST-56 and 0.1 µM N
. Indicated salts were present in the assays at the
concentrations shown: Na
SO
(
), KPO
(
), (NH
)
SO
(
),
and NaCl (
).
Given the lack of
activity by NaCl, additional antichaeotrophic or salting out agents (21, 22) were examined (Table 3). None of the
chlorides had any effect, even when added at 2.5 M. However,
all of the antichaeotrophs stimulated activity, in parallel with the
lyotrophic series of anions, the most potent being the phosphates,
followed by the sulfates and the more weakly antichaeotrophic acetates.
KPO and (NH
)PO
were about 75% more
potent than Na
SO
. (NH
)SO
and MgSO
were about 3-fold less potent, whereas
guanidine sulfate was nearly inactive. Among the acetates, only
Mg(OAc)
showed activity at 1.25 M. However,
because this salt contains two acetate anions/mol, the other acetates
were assayed at 2.5 M. All showed weak to moderate
stimulation.
In addition to salts, several other solutes that stabilize proteins were examined to look for HSV-1 protease stimulation(23) . Sucrose at 34% and glycerol at up to 62% produced marginal, if any stimulation, whereas results using larger polymers were inconclusive. Ficoll 400 and dextran sulfate rendered the solutions too viscous to permit pelleting of the substrate in the TCA assay, whereas polyethylene glycols (400-8000 molecular weight range) precipitated everything. However, when activity was measured using the coupled translation/cleavage assay, polyethylene glycols of average molecular weights 600-3350 produced a slight stimulation when present at 10-20% (results not shown).
Figure 4:
Fluorescence emission spectra of 0.5
µM HSV-1 N ± 10 µM ANS in
buffers of varying composition. A, 50 mM Tricine, pH
8.0. B, 50 mM Tricine, pH 8.0, 1.25 M
Na
SO
. C, 50 mM Tricine, pH
8.0, 2.2 M NaCl. D, same conditions as B. In
the experiment denoted by the dotted curve 50 µM
rP4-P8` was added to the reaction.
If the 465 nm fluorescence were due to the specific binding
of ANS to the substrate binding pocket of the protease, it might be
possible to quench this fluorescence through competition for this site
by a specific protease ligand. For this purpose, the R cleavage site
peptide YLQASEKFKMWG (rP4-P8`), previously shown to inhibit
[S]GST-56 cleavage (IC
=
5-10 µM)
, was used as a competitor. In Fig. 4D, the addition of rP4-P8` to a solution of
N
and ANS in 1.25 M Na
SO
buffer resulted in a large increase in emission from tryptophan
(peptide Trp at P7`) but a decrease of the 465 nm peak. Such an effect
was not seen using a control peptide, ASNAEAGALVNAS (mP12-P1`),
previously implied to be noncleavable (16) and shown to be
noninhibitory.
Thus, the presence of an antichaeotrophic
salt results in the formation of a hydrophobic area on the protease,
possibly the substrate binding site, which is not present at low ionic
strength or at high concentrations of a neutral salt.
In the initial characterization of the HSV protease, an in vitro translation and cleavage assay (1) surprisingly revealed that activity could be greatly
stimulated by high concentrations of NaSO
and
KPO
but not KCl. This finding was confirmed in a more
defined system, using purified proteins for both the protease and
substrate. Most striking, however, was the requirement for molar
concentrations of an antichaeotrophic salt; little effect was seen at
physiological ionic strength. In addition, NaCl did not affect activity
between 0 and 2.5 M (Fig. 3). These studies were
extended by surveying a variety of salts (Table 3), spanning the
lyotropic series of anions and cations. Most conclusive was the finding
that the potency of stimulation paralleled the order of stabilizing
anions, which preferentially hydrate
proteins(21, 22) : chloride
acetate < sulfate
< phosphate. The effect of cations was less pronounced. Sodium ion
appeared to be slightly more potent than ammonium, but guanidine, which
binds tightly to protein(25) , was inhibitory. Several other
reagents known to stabilize proteins were also examined. Glycerol (26) and sucrose(27) , both known to preferentially
hydrate proteins, were ineffective. This was somewhat surprising,
because glycerol was earlier shown to stimulate the activity of the
related HCMV protease(28) , which behaved like the HSV-1 enzyme
toward Na
SO
. Polyethylene glycols, which have a
more pronounced effect on water activity, were potent precipitants in
the TCA precipitation assay. They did, however, slightly stimulate
activity in the in vitro translation/cleavage assay.
The
above findings led to the question of whether the solvent effect might
be on the substrate or the enzyme. The substrate was initially examined
using spectroscopic methods to probe secondary structure. The 56-mer
domain of GST-56, which parallels authentic ICP35 in its cleavage
behavior, was isolated and purified under native conditions and then
submitted to analysis by circular dichroism and NMR spectroscopy. In
physiological concentrations of salt, the peptide was primarily random
coil. We were unable to obtain spectra in the concentrations of
NaSO
or KPO
required for optimal
cleavage by the protease. Nevertheless, these studies suggested that
one role of an antichaeotrophic agent might be to induce some unique
feature of secondary structure into the cleavage site region. Earlier
studies on peptides suggested a requirement for secondary structure on
the P` side of the scissile bond(16) .
The effects of salt
on the protease were also examined. Peptide cleavage experiments (not
shown) suggested that the K for substrates
decreased when assays were performed in high concentrations of
Na
SO
. This led to the notion that the effect
might involve changes in the structure at the active site. For this
purpose, the fluorescent dye ANS was used to probe protease topology.
Stryer (24) showed that it bound to apomyoglobin with a
dissociation constant of
10
M but not
to myoglobin. Furthermore, it could be displaced from the apoprotein by
the addition of hemin. When added to solutions of HSV-1 N
,
ANS bound protein only in the presence of 1.25 M
Na
SO
or KPO
but not NaCl (Fig. 4). These conditions paralleled those required for optimal
activity, suggesting that a conformational change had occurred upon
introduction of these antichaeotrophic salts. In contrast, carbonic
anhydrase did not bind ANS under any conditions.
To examine the
possibility that ANS was binding to the active site of N, a
high affinity substrate peptide, rP4-P8`, was used as competing ligand.
Addition of this peptide to the ANS bound protein solution resulted in
a decrease in ANS fluorescence, consistent with a model that the
salt-induced hydrophobic site was indeed the substrate binding site.
A consequence of our above results is the question of just why a protease should be stimulated by unusually high concentrations of antichaeotrophic salts. One explanation involves the local environment of the protease during cleavage. Recent work on the assembly and maturation of the HSV-1 capsid suggests that the uncleaved form of ICP35 (i.e. containing the COOH-terminal 25 amino acids) is required for the formation of ``sealed'' capsids(29, 30, 31) . Because the processed form of this protein is the predominant form found when immature B capsids are isolated from infected cells(32, 33, 34, 35) , cleavage by the protease most likely occurs within the capsid. Furthermore, based on the ultrastructure and protein stoichiometry calculations of Brown and colleagues(36, 37) , a major fraction of a B capsid volume can be accounted for by ICP35 protein. This suggests that the water activity (38) must be very low, a state that is perhaps approximated in an in vitro assay by the addition of molar concentrations of an antichaeotrophic salt.
Parts of this work were presented at the 18th International Herpesvirus Workshop, Pittsburgh, PA, July 25-30, 1993.