In transduced cells, the US3 protein kinase of herpes simplex virus 1 precludes activation and induction of apoptosis by transfected procaspase 3

Abstract

The US3 protein kinase of herpes simplex virus 1 blocks apoptosis induced by replication-incompetent virus mutants, proapoptotic members of the Bcl-2 family of proteins, and by a variety of other agents that act at the premitochondrial level in the proapoptotic cascade. To define the role of US3 in blocking apoptosis at the postmitochondrial level, we investigated the US3 protein kinase in transduced cells that were either transfected with a plasmid encoding procaspase 3 or superinfected with a proapoptotic mutant virus lacking the gene encoding the infected cell protein no. 4. (i) We show that 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. (iii) An affinity-purified glutathione S-transferase (GST)-US3 fusion protein, but not the inactive GST-US3(K220N) 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 proapoptotic cascade to enable the virus to complete its replication.

FIG. 1.

Effects of the U_S3 protein kinase on the processing and activity of transfected human procaspase 3. (A) U2OS cells were exposed to 10 PFU of “empty” MTS-BAC or of recombinant baculoviruses expressing either wild-type U_S3 or the inactive mutant U_S3-K220N per cell. After 5 h, the cells were transfected with 2 μg of pcDNA or the plasmid pORF-Casp3, expressing human procaspase 3. MTS-ICP27 was used as a control for transfection efficiency. The cells were maintained at 37°C for 24 h and at 34°C afterward 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. (B) U2OS cells were exposed to 10 PFU of empty MTS-BAC or of recombinant baculoviruses expressing wild-type U_S3 per cell. After 5 h, the cells were transfected with 2 μg of pcDNA or the plasmid pORF-Casp3, 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. (C) U2OS cells were infected with approximately 10 PFU of empty MTS-BAC or of recombinant baculoviruses expressing wild-type U_S3 per cell. After 5 h, the cells were transfected with 2 μg of pcDNA or the plasmid pORF-Casp3 expressing human procaspase 3. The caspase 3 inhibitor Z-DEVD-fmk (50 μM) was added 14 h after transfection. 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 or caspase 3. The arrowhead points to caspase 3, the cleavage product of procaspase 3.

FIG. 2.

Immune fluorescence analysis of U2OS cells transduced with MTS- or U_S3-BAC and infected with d120 mutant virus. U2OS cells were transduced with 0.5 PFU of U_S3-BAC (F) per cell or 10 PFU of empty MTS (A and C) or U_S3-BAC (E) per cell. After 5 h, 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 5 μM NucView for 1 h and then fixed (4% PFA for 15 min), permeabilized (0.1% Triton X-100 for 2 min), and reacted with blocking solution (10% fetal bovine serum for 2 h at 4°C) and then with anti U_S3 Ab (1:500; 1 h 30 min) and secondary anti-rabbit Ab conjugated with Texas Red fluorescent dye (1:400; 55 min).

FIG. 3.

Summary of the immunofluorescence analyses of U2OS cells transduced with empty MTS or U_S3-BAC and infected with d120 mutant virus. The procedures are described in the legend to Fig. 2. The numbers above the bars indicate the numbers of cells counted in adjacent fields.

FIG. 4.

The U_S3 protein kinase can in vitro phosphorylate procaspase 3. One microgram of recombinant human procaspase 3 (Casp. 3; lanes 3 and 4) or of purified histone H1 (Hist. H1; lanes 5 and 6) was incubated with 2.5 μg of purified GST-U_S3 or GST-U_S3 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 Ab (A) or autoradiography (B). Procaspase 3 protein and histone H1 protein are identified by the arrowheads.