1 In transduced cells, the US3 protein kinase of herpes simplex 1 precludes activation and

1

In transduced cells, the US3 protein kinase of herpes simplex 1 precludes activation and

induction of apoptosis by transfected procaspase 3

Luca Benetti, and Bernard Roizman

The Marjorie B. Kovler Viral Oncology Laboratories,

The University of Chicago

Chicago IL 606037

*Corresponding author mailing address:

The Marjorie B. Kovler Viral Oncology Laboratories

The University of Chicago

910 East 58th Street

Chicago, IL 60637

Phone: (773) 702-1898

Fax: (773) 702-1631

E-mail: [email protected]

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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.00820-07 JVI Accepts, published online ahead of print on 18 July 2007

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Abstract

The US3 protein kinase of herpes simplex virus 1 blocks apoptosis induced by a

replication- incompetent virus mutants, pro-apoptotic members of the Bcl-2 family of protein

and by a variety of other agents that act at the pre-mitochondrial level in the pro-apoptotic

cascade. To define the role of the US3 in blocking apoptosis at post-mitochondrial level, we

investigated the US3 protein kinase in transduced cells that were either transfected with a

plasmid encoding procaspase 3 or superinfected with a pro-apoptotic mutant virus lacking the

gene encoding the infected cell protein No. 4. We show that: (i) US3 blocks the proteolytic

cleavage that generates active caspase 3 from the transfected zymogen procaspase 3,

concomitant with inhibition of apoptosis; (ii) studies based on detection of fluorescence emitted

upon cleavage of a synthetic caspase 3 substrate showed that expression of the US3 kinase and

appearance of the cleaved substrate were mutually exclusive. Lastly (iii) an affinity-purified

GST-US3 fusion protein, but not the inactive GST- US3K220N protein phosphorylated procaspase

3 in vitro. The studies published earlier on the effect of US3 on the upstream regulatory proteins

and current studies suggest that the US3 protein kinase may act on several proteins in the pro-

apoptotic cascade to enable the virus to complete its replication.

Introduction

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The studies described in this report rest on three observations. Foremost, at least two replication

defective herpes simplex virus mutants have been shown to activate a chain of events leading to

apoptosis in the infected cells. The two mutants lack the ability to express functional infected cell

proteins 4 (ICP4) or 27 (2, 23). Wild-type virus does not induce apoptosis, although

overexpression of Bcl-2 blocks or delays the appearance of cytopathic effects (10, 19).

The second fundamental observation is that wild-type virus blocks apoptosis by a vast

variety of pro-apoptotic stimuli. These include a wide range of drugs or physical conditions (9,

11, 17, 20, 23, 27). Included in the list are sorbitol – an agent capable of inducing osmotic shock

– and hyperthermia.

Lastly, at least four viral genes have been reported to block apoptosis. Of these UL39, the large

subunit of ribonucleotide reductase, blocks apoptosis by complementing corresponding deletion

mutants only (29) Glycoprotein D appears to act by blocking massive lysosomal discharges

following endocytic entry or egress (43) Glycoprotein J (17, 18, 43) has also been demonstrated

to block apoptosis, although the mechanism has remained unclear. Finally, the US3 protein

kinase has been shown to block apoptosis induced by a FICP4 virus mutant, by sorbitol or by

overexpression of pro-apoptotic members of the Bcl-2 family of proteins (3, 5, 6, 18, 24, 26, 27,

28). This report centers on the role of the US3 protein kinase in blocking apoptosis induced by

overexpression of the effector caspase 3. Relevant to this report are the following:

The US3 locus contains two overlapping transcriptional units encoding two protein

kinases (30, 33). The largest product (US3) is the 481-residue protein kinase shown to block

apoptosis (3, 6, 16, 24, 26-28), to play a role in the disruption or nuclear lamina to enable the

transit of capsids from nuclei to the cytoplasm (36), and to phosphorylate HDAC1 and 2,

essential for transactivation of genes introduced into cells by transduction (31, 32) and other

proteins. The smaller product, designated US3.5, initiates at methione 77. Its spectrum of

activities appears to be similar to that of US3 except that it does not block apoptosis and is

partially defective in restructuring nuclear lamina in the cell line tested (30). The substrate

specificity of both US3 and US3.5 kinases is virtually identical to that of protein kinase A (R-R-

X-S or R-X-X-T where X should not be an acidic amino acid residue (4, 30) and indeed an

antibody directed against phosphorylated protein kinase A target sequences reacts with proteins

phosphorylated by US3 or US3.5 protein kinases. The evidence suggests that protection from

apoptosis is most likely the result of direct phosphorylation by US3 of a cellular protein

containing a PKA-like target sequence (30) inasmuch as activation of PKA by forskolin blocked

apoptosis in a manner similar to that of US3 protein kinase (4). However, the relevant

antiapoptotic targets of the US3 protein kinase have remain elusive. The evidence that US3 can

block apoptosis induced by several regulatory proteins led us to explore the possibility that US3

acts in a redundant fashion both upstream on pre-mitochondrial regulatory factors and

downstream at the level of effector caspases. For this reason we examined the ability of US3 to

block activation of caspase 3 which would lead to cell death.

The various branches of the pro-apoptotic pathways converge at the point of activation

of effector caspases 3, 6 and 7 by caspases 8 and 9 (8, 12, 40). In particular, proteolytic cleavage

of procaspase-3 (the zymogen form of caspase-3) is a central event in the apoptotic process,

since it generates active caspase-3, the major effector of the cascade, responsible for

implementing the cell death program (34).

Although procaspase 3 can be auto activated by self cleavage - most likely under non

physiologic conditions in which the zymogen is overexpressed (13-15, 25), the activation of the

enzyme is tightly regulated. Activation can be blocked by survivin or p21Cip

(39, 41, 42), by

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phosphorylation (1) or by S-nitrosylation (38). Central to the function of caspase 3 is its role in

amplifying death signals. For example, active caspase-3 can cleave the proapoptotic Bcl-2

family member BAD and the resulting cleavage product exhibits a stronger proapoptotic activity

than the full length protein (7). The relevance of positive feedbacks in programmed cell death is

further exemplified by recent reports that caspase-3 and -7 –deficient fibroblasts were highly

resistant to various apoptotic stimuli and showed defects and delays in early apoptotic events at

the mitochondrial level (21, 22).

In the studies reported here, we transduced cells with baculoviruses encoding the US3

kinase driven by the human cytomegalovirus immediate-early promoter in order to express the

protein kinase in all or most cells in a dose dependent manner. We selected U2OS cells because

in these cells the baculovirus-dependent transduction of the US3 protein kinase does not require

the use of inhibitors of histone deacetylases (31) The FICP4 mutant d120 served as a positive

control since it does not express the US3 protein kinase and the enzyme readily blocks apoptosis

induced by the mutant virus (24, 26). We report that (i) the US3 protein kinase blocked the

activation of procaspase-3; (ii) activated caspase-3 could not be demonstrated in cells expressing

the US3 protein kinase and (iii) the US3 protein kinase phosphorylated in vitro recombinant

procaspase-3.

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Materials and Methods

Cell lines and viruses. U2OS (human osteosarcoma cells) were obtained from the

American Type Culture Collection (ATCC, Manassas, VA) and were grown in Dulbecco’s

modified Eagle’s medium supplemented with 10% fetal bovine serum.

The insect cell line Sf9 (Spodoptera frugiperda) was obtained from PharMingen (San

Diego, CA) and was maintained in Grace's medium supplemented with 10% fetal bovine

serum.HSV-1 strain F [HSV-1(F)], a limited-passage isolate, is the prototype HSV-1 strain used

in this laboratory. The HSV-1(KOS) d120 mutant, a kind gift from N. DeLuca (University of

Pittsburgh, Pittsburgh), lacks both copies of the 4 gene and was grown in

a Vero-derived cell

line (E5) expressing the 4 gene. The recombinant virus R7041, lacking the US3 gene, is

described elsewhere (35).

Plasmids, antibodies and reagents. The pORF-Casp3 plasmid, which expresses human

procaspase 3 under the control of a composite promoter, consisting of the elongation factor 1

alpha (EF-1g) core promoter combined to the 5'UTR of the human T cell leukemia virus (HTLV)

type 1 long terminal repeat, was purchased from Invivogen, San Diego, CA. pcDNA 3.1 was

purchased from Invitrogen, Carlsbad, CA. The MTS-ICP27 plasmid and the corresponding

baculovirus expressing ICP27 (G.Zhou and B. Roizman, unpublished studies) were made by

standard procedure described elsewhere (27). Rabbit antibody against caspase 3 was obtained

from Cell Signaling Technology, Berwyn, CA and used at a 1:500 dilution. Rabbit antibody

against PARP was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a

1:700 dilution. Monoclonal antibody against infected cell protein no. 27 was purchased from the

Goodwin Institute, Plantation, FL. Rabbit antibody against US3 was described elsewhere (26),.

The irreversible caspase 3 inhibitor Z-DEVD-fluoromethylketone (Z-DEVD-fmk) were

purchased from Calbiochem (La Jolla, CA). Purified recombinant human procaspase 3 and

human histone H1 were purchased from Calbiochem and Roche (Indianapolis, IN), respectively.

The NucView reagent was purchased from Biotium, Inc. (Hayward, CA).

Baculoviruses. Construction of control “empty” baculovirus (MTS-BAC) and US3-

expressing BAC (US3-BAC) (27). The US3(K200N)-BAC was made in parallel with the US3-

BAC; it expresses the enzymatically inactive, full length protein US3 protein (J. Munger and B.

Roizman, unpublished studies). Recombinant baculoviruses were generated using the

PharMingen baculovirus expression system as described previously. Briefly, plasmid DNA

containing wild-type or mutant US3 coding sequence cloned into baculovirus transfer vector

pAc-CMV was cotransfected into Sf9 insect cells, together with the BaculoGold baculovirus

DNA (PharMingen), according to the manufacturer’s instructions. Supernatant containing the

recombinant virus was collected and cleared by centrifugation at 2,500 rpm for 10 min 4 to 6

days after transfection, and virus was amplified in Sf9 cells grown in a 150-cm2 flask.

Transfection and transduction protocol. Replicate cultures of U2OS in 25 cm2 flasks

cells were exposed to approximately 10 PFU of the indicated baculoviruses per cell At 5 h after

transduction, the cells were transfected with 2og of pORF-CAsp3 plasmid. Alternatively, 2og of

empty pcDNA or MTS-1-ICP27 plasmids were used as transfection efficiency controls. The cells

were subsequently maintained at 37C for 24 h and then at 34C for an additional 16 hours in order

to avoid overgrowth.

Immunoblot Assays. Cells were harvested at the indicated times after treatment, rinsed

three times with PBS, and solubilized in radioimmunoprecipitation assay buffer in

the presence

of phosphatase inhibitors (10 mM NaF/10 mM -glycerophosphate/0.1 mM sodium vanadate)

and protease inhibitors (Complete, Roche). Lysed cells were stored on ice for 10 min before

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centrifugation at 14,000 rpm for 10 min. The protein concentration of the supernatant

fluids was

determined with the aid of a Bio-Rad protein assay. Protein samples denatured in disruption

buffer (50 mM Tris, pH 7.0/2.75% sucrose/5% 2-mercaptoethanol/2% SDS) were heated

at 95°C

for 5 min, electrophoretically separated in denaturing

polyacrylamide gels, electrically

transferred to a nitrocellulose sheet, blocked, and reacted with primary antibody followed by

appropriate secondary antibody conjugated to alkaline phosphatase (Bio-Rad, Hercules, CA).

Protein bands were visualized with 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium

(Denville Scientific, Metuchen, NJ).

DEVDase Activity Assay. Caspase 3 activity was assayed by using a tetrapeptide

conjugated to phenylnitraniline (DEVD-pNA) (Calbiochem).

Cells were harvested at the

indicated times after infection with 10 PFU of virus per cell, rinsed three times

with PBS,

resuspended in 75 µl of lysis solution A {0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-

propanesulfonate/50 mM Hepes, pH 7.4/1 mM DTT/0.1 mM EDTA}, held on ice for 10 min,

and

centrifuged at 14,000 rpm for 10 min at 4°C. Equal amounts of protein in supernatant fluids were

tested for DEVDase activity according to the manufacturer's instructions.

Purification of GST-US3 Protein. Construction of the baculovirus expressing GST-US3

in Sf9 insect cells has been reported elsewhere (4). Baculovirus expressing the inactive mutant

US3(K220N) amino-terminally tagged with GST was constructed with the same strategy (J.

Munger and B. Roizman, unpublished studies). Infected Sf9 cultures were harvested, rinsed

twice with PBS (0.14 M NaCl/3 mM KCl/10 mM Na2HPO4/1.5 mM KH2PO4) and lysed

in

radioimmunoprecipitation assay buffer (1% Nonidet P-40/0.5% sodium deoxycholate/0.1% SDS

in PBS) in the presence of protease inhibitors (Complete, Roche) as recommended by the

manufacturer. Lysed cells were held on ice for 10 min before brief sonication

and centrifugation

at 14,000 rpm for 10 min in an Eppendorf 5415 C centrifuge. The GST-chimeric proteins were

bound to glutathione Sepharose beads (Amersham Biosciences Piscataway, NJ), rinsed four

times with PBS, and stored at 4°C.

Kinase Assays. One microgram of GST or GST-US3 attached to glutathione Sepharose

beads was reacted with 2.5 µg of procaspase 3 or Histone H1 in 50 µl of kinase buffer (20 mM

Tris·HCl, pH 7.5/50 mM KCl/10 mM MgCl2/10 mM 2-mercaptoethanol), supplemented with 100

µM ATP and 20 µCi (1 Ci = 37 GBq) of [ -

32P]ATP. The samples were reacted at 30°C for

30

min. The beads were pelleted by low-speed centrifugation and gel-loading buffer was added to

the supernatants, which were subsequently heated to 95°C for 5 min, resolved by PAGE,

transferred to nitrocellulose membrane, and analyzed by autoradiography.

Quantification of

32P

phosphorylation of the substrates was done with the aid of a Molecular Dynamics Storm 860

PhosphorImager. Immunofluorescence analysis. U2OS cells were seeded on 4-well slides, incubated with

MTS-BAC or either ~10 or ~0.5 PFU/cells of US3-BAC, and after 6 hours exposed to mutant

virus d120. 17 hours after infection the cell culture medium was replaced by fresh medium

containing the NucView reagent, according to the manufacturer’s instructions. After one

additional hour cells were fixed with 4% PFA in PBS for 15 minutes, permealized with 0.1%

Triton X-100 in PBS for 2 minutes, incubated in blocking solution (10% fetal bovine serum in

PBS), for 2 hours at 4C and then with anti US3 Ab (1:500, 1h 30 minutes) and secondary anti-

rabbit conjugated with Texas Red fluorescent dye (1:400, 55 minutes). The slide cultures were

then mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The

slides were examined in Zeiss (Thornwood, NY) confocal microscope. Digitized images of the

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fluorescent antibody-stained cells were taken with a Zeiss camera (AxioCam) and were

acquired

with software provided by Zeiss.

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Results

The activation of procaspase 3 is blocked the US3 protein kinase. The objective of

these experiments was to determine the effect of the US3 protein kinase on the activity of

procaspase 3 in transduced cells. Three series of experiments were done to test the hypothesis

that the US3 protein kinase blocks the activation of procaspase 3.

In the first, U2OS cells were transduced with approximately 10 PFU of empty

baculoviruses (MTS-Bac) or 10 PFU of recombinant baculoviruses expressing either wild-type

US3 ORF or the inactive kinase mutant K220N per cell. After 5 h, the cells were transfected

with 2 og of pcDNA or with the pORF-caspase 3 plasmid, encoding human procaspase 3. The

MTS1-ICP27 plasmid, encoding HSV-1 ICP27 was used as a control for transfection efficiency.

The cells were maintained for 24 h at 37C and for an additional 16 h at 34C to reduce cell

overgrowth. The cells were then harvested, solubilized, subjected to electrophoresis on a

denaturing polyacrylamide gel, transferred to a nitrocellulose sheet, and reacted with antibodies

against PARP, ICP27 and Caspase 3, respectively. The results shown in panel A of Figure 1

were as follows:

Procaspase 3 was cleaved in cells transduced with MTS-BAC and transfected with

procaspase 3 (lane 3) and to a lesser extent in cells transduced with K220N and transfected with

procaspase 3 (lane 7) but not in cells transduced with functional US3 and transfected with

procaspase 3 (lane 5). The cleavage product had apparent Mr of 17,000, consistent with that of

the active form of the enzyme.

PARP was cleaved in cells transfected with caspase 3 in the absence of functional US3

kinase (lanes 3 and 7). The accumulating fragment had apparent Mr of 85,000 as would be

expected for the product of caspase 3-mediated cleavage.

ICP27 was expressed to the same extent in cells transduced with US3 or K220N mutant

(lanes 6 and 8).

The objective of the second series of experiments was to ascertain that the procaspase 3

expressed in the absence of the US3 protein kinase was activated and expressed DEVDase

activity. U2OS cells were transduced with approximately 10 PFU of MTS-BAC, or of

recombinant baculoviruses expressing wild-type US3 per cell. After 5 h the cells were transfected

with 2og of pcDNA or the plasmid pORF-caspase 3 and maintained as described above. The

cells were harvested 40 h after transfection, solubilized and assayed for DEVDase activity as

described in Materials and Methods. The results (Figure 1 Panel B) show that cells transduced

with MTS-BAC and transfected with procaspase 3 exhibited a marked increase in DEVDase

activity compared to those of control cells, whereas cells transduced with US3 BAC and

transfected with procaspase 3 did not.

The objectives of the third series of experiments were to further characterize the activity

induced by procaspase 3. U2OS cells were transduced with approximately 10 PFU of MTS-Bac,

or with a recombinant baculoviruses expressing wild-type US3 per cell. After 5 h the cells were

transfected with 2og of pcDNA or the plasmid pORF-caspase 3, expressing human procaspase 3.

The irreversible caspase-3 inhibitor Z-DEVD-fmk (50 oM) was added 14 h after transfection. At

40 h after transfection, cells were harvested, solubilized, subjected to electrophoresis in a

denaturing polyacrylamide gel, transferred to a nitrocellulose sheet, and reacted with antibodies

against PARP or caspase 3. The salient features of the results (Figure 1C) were as follows:

Antibody to caspase 3 reacted with a protein band characteristic of procaspase 3 and with

a fast migrating polypeptide in cells transduced with MTS-Bac and transfected with caspase 3

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(Figure 1 Panel C lane 3). This polypeptide was not detected in lysates of cells transduced with

US3-Bac and transfected with procaspase 3 (Lane 6) or in lysates of cells transduced with MTS-

Bac, transfected with procaspase 3 and treated with the Z-DEVD-fmk inhibitor (Lane 4).

We conclude for this series of experiments that in transfected cells procaspase 3 is

activated concurrently with the intracellular accumulation the Mr 17,000 polypeptide, that the

activated caspase 3 expresses DEVDase activity, cleaves PARP and is inhibited by Z-DEVD-

FMK as would be expected of bona fide activated caspase 3. The results also show that US3

protein kinase blocks the cleavage of procaspase and its proteolytic activity.

The caspase 3 activity and US3 expression are mutually exclusive. The objective of

thee studies was to determine whether US3 protein kinase decreases or blocks caspase 3

activation in individual cells. In this series of experiments U2OS cells were mock-treated,

transduced with 10 PFU of MTS-Bac (Figure 2 panel A and C) or with either 10 PFU (Figure 2

panel D and E) or 0.5 PFU per cell (Figure 2 panel F) of US3-BAC. After 5 h the cells were

exposed to 10 PFU of d120 mutant virus per cell. After 17 h of incubation the cell cultures were

replenished with medium containing 5mM NucView for 1 h. The cells were then fixed (4% PFA

for 15 minutes), permealized (0.1% Triton X-100 for 2 minutes), incubated in blocking solution

(10% fetal bovine serum, 2h at 4C) and then with anti US3 Ab (1:500, 1h 30’) and secondary

anti-rabbit conjugated with Texas Red fluorescent dye (1:400, 55’). The NucView reagent is

inert and cytoplasmic; it contains a DVED sequence that is a target of active caspase 3. The

product of such cleavage is a green fluorescent DNA dye that stains the nucleus of apoptotic

cells. The cultures were examined and images acquired with the aid of a Zeiss confocal

microscope. In all, for each experimental point, a total of 363 to 413 cells in adjacent fields were

counted in addition to a thorough examination of the cultures. The results, (Figures 2 and 3)

were as follows:

Examination of the mock-treated cultures (Figure 2 panel A) yielded one cell

spontaneously undergoing apoptosis among an excess of 400 cells. Cultures infected with d120

mutant virus only or transduced with MTS-Bac and infected with d120 mutant virus exhibited

green fluorescence indicative of active caspase 3 in 37% to 39% of cells. In cells transduced with

10 PFU of US3-Bac per cell, 98 percent of cells expressed US3 protein kinase. In these cultures,

the fraction of cells exhibiting active caspase 3 dropped to 1.7%. Among the cells counted, only

one exhibited both US3 and active caspase 3 activity. One possible explanation for the paucity of

cells exhibiting both active caspase and US3 protein kinase is that the multiplicity of US3-Bac per

cell (10) was too high. In cultures exposed to 0.5 PFU of US3-Bac and 10 PFU of d120 mutant

virus per cell we found 32% of cells exhibited active caspase 3 and 17% of cells exhibited US3

protein kinase. Again only one cell among the 365 cells counted exhibited both active caspase 3

and US3 protein kinase.

We conclude from these studies that US3 protein kinase blocks activation of procaspase

3.

The US3 protein kinase phosphorylates procaspase 3.in vitro. One microgram of

recombinant human procaspase 3 (Figure 4, lanes 3-4) or of purified histone H1 (lanes 5-6) were

reacted with 2.5 mg of purified GST -US3 or GST -US3 K220N attached to glutathione sepharose

beads. After a 30 minutes incubation at 30 C the reaction mixtures were electrophoretically

separated on a denaturing polyacrylamide gel, transferred to a nitrocellulose membrane and

either reacted with antibody to Caspase 3 (Figure 4 Panel A) or subjected to autoradiography

(Panel B). The results were as follows:

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The purified caspase 3 formed several bands that reacted with the anti-caspase 3 antibody

in reaction mixtures containing wild-type US3 protein kinase (Lane 3) or the inactive K220N

mutant kinase (lane 4). Procaspase 3 protein is identified by the arrowhead. The faster migrating

bands reactive with the anti caspase 3 antibody mort likely represent degradation products

copurified with the full length protein. Most of the bands in the reaction mixture containing

active US3 protein kinase were phosphorylated (Panel B lane 3) In contrast there was no

evidence of phosphorylation of the caspase 3 in mixtures containing K220N US3 mutated

protein. It is noteworthy that a small fraction of the phosphorylated procaspase 3 protein

migrated more slowly than the slowest migrating band in Figure 4 Panel B lane 3. Finally,

histone H1 was phosphorylated in mixtures containing the active kinase (Figure 4 Panel B lane

5) but not in mixtures containing the K220N protein (Figure 4 panel B lane 6).

These results indicate that at least in in vitro studies active US3 kinase, but not the

inactive US3 protein prepared under identical conditions, phosphorylated procaspase 3.

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Discussion

The fundamental finding that the US3 kinase blocks apoptosis emerged from studies on

the pro-apoptotic activities of FICP4 mutant d120 (23). Since then the list was extended to

include a variety of exogenous activator and pro-apoptotic proteins (6, 27, 28). A central

question - the target of the US3 protein kinase in blocking apoptosis - remains unresolved. The

impetus for the studies described here is the observation that US3 blocks the pro-apoptotic

proteins BAD, BAX and BID. The hypotheses that could explain these observation is that either

US3 targets all of these protein individually or that it targets a downstream effector. We expected

that under the non-physiologic overexpression of the downstream effector pro-caspase 3, an

inefficient but effective activation of caspase 3 could be attained by self cleavage of pro-caspase

3 leading to programmed cell death. In effect, what we have observed is a small amount of

cleavage of pro-caspase 3 to generate the small (MR 17,000) active caspase 3. We have also

documented evidence that in addition to the cleavage of pro-caspase 3 the transfected cells

exhibited activities attributed to and characteristic of caspase 3. Specifically we have shown that

the transfected cells exhibit DEVDase activity and that the inhibitor Z-DEVD blocks it. We

have also shown that cells transduced with a baculovirus encoding the US3 protein kinase

effectively block the cleavage of pro-caspase 3 and also block apoptosis whereas the

enzymatically defective K220N mutant failed to block both activation and the pro-apoptotic

activity of pro-caspase 3. A finding of particular interest is that in the system tested in this

report, expression of US3 protein kinase and cleavage of caspase 3 substrates, detected by

immunofluorescence, appeared to be mutually exclusive even under conditions of low

multiplicity transduction by the US3 baculovirus. We also show that pro-caspase 3 can serve as a

substrate of the US3 kinase. Taken together, the results unambiguously demonstrate the US3

protein kinase in the absence of other HSV-1 protein can block activation and pro-apoptotic

activity of caspase 3.

Notwithstanding the evidence reported here that US3 protein kinase effectively blocks an

effector caspase, we cannot exclude the possibility that the US3 protein kinase targets several

proteins in cellular pro-apoptotic machinery. The fundamental strategy of HSV-1 as it is

evolving from the studies of viral gene function is that key host defenses are blocked by multiple

gene products or by multiple functions expressed by a single protein (37) . The overall function

of US3 may emerge more clearly from the functional dissections of its domains.

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Acknowledgements These studies were aided by National Cancer Institute Grants CA115662, CA83939, CA71933,

CA78766, and CA88860.

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Figure Legends Figure 1. Effect of the US3 protein kinase on the processing and activity of transfected human

procaspase 3. Panel A: U2OS cells were exposed to 10 PFU of “empty” MTS-BAC, or of

recombinant baculoviruses expressing either wild-type US3 or the inactive mutant US3-K220N

per cell. After 5 h the cells were transfected with 2 og of pcDNA or the plasmid pORF-caspase

3, expressing human procaspase 3. The MTS-ICP27 was used as a control for transfection

efficiency. The cells were maintained at 37C for 24 h and at 34C afterwards, in order to avoid

overgrowth. At 40 h after transfection, the cells were harvested, solubilized, subjected to

electrophoresis in a denaturing polyacrylamide gel, transferred to a nitrocellulose sheet, and

reacted with antibodies against PARP, ICP27 or caspase 3. Panel B: U2OS cells were exposed

to 10 PFU of empty MTS-BAC, or of recombinant baculoviruses expressing wild-type US3 per

cell. After 5 h the cells were transfected with 2og of pcDNA or the plasmid pORF-caspase 3,

expressing human procaspase 3. At 40 h after transfection, the cells were harvested, solubilized

and assayed for DEVDase activity as described in Materials and Methods. Panel C. U2OS cells

were infected with approximately 10 PFU of empty MTS-BAC, or of recombinant baculoviruses

expressing wild-type US3 per cell. After 5 h the cells were transfected with 2 og of pcDNA or

the plasmid pORF-caspase 3, expressing human procaspase 3. The caspase-3 inhibitor Z-DEVD-

fmk (50 oM) was added 14 h after transfection. At 40 hours after transfection, cells were

harvested, solubilized, subjected to electrophoresis in a denaturing polyacrylamide gel,

transferred to a nitrocellulose sheet, and reacted with antibodies against PARP or caspase 3. The

arrowhead points to caspase 3, the cleavage product of procaspase 3.

Figure 2

Immune fluorescence analysis of U2OS cells transduced with MTS- or US3-BAC and infected

with d120 mutant virus. U2OS cells were transduced with 0.5 PFU of empty MTS (Panel C) or

US3-BAC (panel F) per cell or 10 PFU of empty MTS (Panel B) or US3-BAC (Panel E) per cell.

After 5 hours the cultures were exposed to 10 PFU of mutant d120 virus per cell and maintained

for 17 h. At that time the cells were incubated in fresh medium containing 5mM NucView for 1

h., then fixed (4% PFA for 15 minutes), permealized (0.1% Triton X-100 for 2 minutes), reacted

with blocking solution (10% fetal bovine serum for 2h at 4C) and then with anti US3 Ab (1:500,

1h 30’) and secondary anti-rabbit conjugated with Texas Red fluorescent dye (1:400, 55’).

Figure 3. Summary of the immunofluoresce analyses of U2OS cells transduced with empty

MTS or US3-BAC and infected with d120 mutant virus. The procedures are described in the

legend to Figure 2. The numbers at the top of the graph indicate the number of cells counted in

adjacent fields.

Figure 4 The US3 protein kinase can in vitro phosphorylate procaspase 3. One microgram of

recombinant human procaspase 3 (lanes 3-4) or of purified Histone H1 (lanes 5-6) were

incubated with 2.5 og of purified GST-US3 or GST-US3 K220N attached to glutathione

sepharose beads. After a 30 min. incubation at 30 C samples were subjected to polyacrylamide

gel electrophoresis, nitrocellulose membrane transfer and either immunoblotting with anti-

caspase 3 antibody (Panel A) or autoradiography (Panel B).

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