INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Herpes simplex virus type 1 (HSV-1) releases viral progeny during productive infection in cultured cells, leading to lytic cell death. The expression of HSV-1 genes precedes infected cell lysis, and it occurs through a highly regulated cascade (28, 29) that begins with the production of the  (immediate-early [IE]) proteins. The  proteins, infected cell protein 0 (ICP0), ICP4, ICP22, and ICP27, have regulatory functions and cooperatively act to regulate the expression of all classes of viral genes (reviewed in reference 55). The  (early [E]) gene products, such as the viral thymidine kinase (TK), are synthesized next and are the proteins principally involved in viral DNA synthesis (reviewed in reference 11). The last set of viral proteins produced are the  (late [L]) proteins, which are mainly associated with virion structure and assembly, such as VP16 and vhs (7, 18, 51). The completion of the HSV-1 replication cycle leads ultimately to the destruction of the cells in culture, and this process is generally believed to occur through a necrotic route. Consequently, productive HSV-1 replication induces major biochemical alterations in the infected cells, including the loss of matrix binding proteins on the cell surface, membrane modifications, cytoskeletal destabilization, nucleolar alterations, chromatin margination, aggregation or damage, and a decrease in cellular macromolecular synthesis (6, 23, 26, 53-55). In addition, these morphological features observed with HSV-infected cells appear to be different from those associated with cells dying from apoptosis. In this case, the cells are characterized by morphological and biochemical changes that include cell shrinkage, membrane blebbing, nuclear condensation, and fragmentation of chromosomal DNA into nucleosomal oligomers (reviewed in reference 33). Therefore, it appears that a distinction exists between cytolysis due to viral replication and the apoptosis of cells.

Apoptosis, or programmed cell death, is a highly regulated process of cell suicide. It is activated during normal development and by various stimuli that disturb cell metabolism and physiology (73, 75). Apoptotic signals received through a death receptor pathway or a mitochondrial route converge to a central pathway involving a family of aspartate-specific cysteinyl proteases (cysteine aspartases, or caspases) which are activated by proteolytic cleavage during the process of cell death (2, 14, 22, 57, 69). Caspase activation leads to the processing of various cytoplasmic and nuclear targets by a subclass of caspases, called executioners, such as caspase-3, caspase-6, and caspase-7 (57, 74). Among the cleavage targets are the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), the DNA fragmentation factor (DFF), and scaffolding proteins such as lamins and actin (43, 57, 74). Thus, the process of apoptosis generally involves the processing of caspase-3, DFF, and PARP.

Due to the cell's innate ability to self-destruct, it is likely that apoptosis is also an important mechanism of host cell defense against viral infections. Accordingly, several distinct viruses appear to have developed mechanisms to block the premature apoptosis of infected cells (44, 66, 71). The most likely reason for this is to prevent the cellular apoptosis that occurs as a result of viral infection in order to prolong cell survival so that the production of the viral progeny can be maximized. Among the viral strategies to respond to programmed cell death pathways is the production of (i) antiapoptotic factors such as CrmA of cowpox virus (67, 68) and p35 and inhibitors of apoptosis from baculovirus (12, 13), which are viral inhibitors of caspases, and (ii) viral homologues of the cellular antiapoptotic protein Bcl-2, encoded by either Epstein-Barr virus (27), African swine fever virus (1), or herpesvirus saimiri (16). Recent reports (5, 19, 20, 30, 32, 35, 39, 70) showed that HSV-1 is also able to interfere with the process of apoptosis in infected cells. HSV-1 was able to prevent apoptosis which was externally induced by various stimuli including treatment with cycloheximide (CHX) (5, 20), ceramide, tumor necrosis factor, and anti-Fas antibody (20), osmotic shock using sorbitol (36) or ethanol (32), and hypothermia and thermal shock (19, 38, 39). It was also demonstrated that HSV-1 infection itself could induce apoptosis in cells. This conclusion was based on specific experiments in which cells were infected with mutant viruses (3, 5, 20, 39) or the infections occurred in the presence of the protein synthesis inhibitor CHX (5, 34). While the process of apoptosis in infected cells has now been clearly demonstrated, the specific mechanisms of its induction and prevention by the virus are not known.

In a previous report, we demonstrated that cultured epithelial cells of human origin infected with an ICP27 deletion strain of HSV-1 presented the characteristic features of apoptotic cells, including cell shrinkage, nuclear condensation, and DNA fragmentation (5). When the same cells were infected with a wild-type virus, they did not show these apoptotic features unless the infections were performed in the presence of CHX (5, 34). These results led us to conclude that both the wild-type and the ICP27-null viruses likely induce an apoptotic event in infected human cells, but the virus which lacks ICP27 protein is unable to prevent this process from killing the cells. To further investigate these findings, we used the ICP27-deficient virus (vBS27) as a tool for the study of the induction and inhibition of HSV-1-initiated apoptosis. In this report, we show that vBS27 induced apoptosis in HEp-2 cells through a pathway which involved the activation of the caspase-3 protease and the processing of the death substrates DFF and PARP. The induction of apoptosis by HSV-1 occurred prior to 6 h postinfection (hpi), and experiments involving temporal addition of CHX indicated that de novo viral protein synthesis was not required. The inhibition of apoptosis in HSV-1-infected HEp-2 cells was shown to be effective between 3 to 6 hpi, which corresponds to the transition period from the IE to E phase of viral replication. In addition, wild-type HSV-1 infection was able to block the apoptosis induced in cells by the addition of CHX staurosporine, and sorbitol. Both U_S3- and ICP22-deficient viruses showed a partial prevention of apoptosis, suggesting that these proteins may play a role in the inhibition process. In contrast, deletion of either the U_L13 or vhs gene product does not appear to affect the ability of HSV-1 to prevent apoptosis in infected cells. Finally, using UV-inactivated viruses, we demonstrated that binding and entry of the virus were not sufficient to induce apoptosis following HSV-1 infection. From these results, we conclude that either gene expression or other RNA metabolism processes likely play a role in the induction of apoptosis in HSV-1-infected cells.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Cell lines and viruses. All cells were maintained in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum. HEp-2 and Vero cells were obtained from the American Type culture Collection (Rockville, Md.). HSV-1(F) was obtained from Bernard Roizman (University of Chicago). Vero 2.2 cells, the HSV-1 (KOS1.1) virus, and vBS27 were generously provided by Saul Silverstein (Columbia University). Vero 2.2 is a derivative Vero cell line expressing ICP27 under its own promoter (62). F and KOS1.1 are the strains of wild-type HSV-1 used in this study. vBS27 (64) is the ICP27-null mutant virus used in this analysis; it contains a replacement of the 27 gene with the Escherichia coli lacZ gene and therefore must be propagated on an ICP27-complementing cell line, such as Vero 2.2 (62). The recombinant viruses R7041, R7356, and R325 [derivatives of HSV-1(F)] were obtained from Bernard Roizman; they contain, respectively, deletions of the genes encoding the protein kinase U_S3 (47), the kinase U_L13 (48), and the regulatory protein ICP22 (46). The vhs-Sma mutant virus, generously provided by G. Sullivan Read (University of Missouri, Kansas City), is a derivative of KOS1.1 carrying a deletion in the vhs gene (U_L41) (51). In all cases, the cell monolayers were infected at a multiplicity of infection of 10 PFU/cell and the infections were maintained at 37°C in Dulbecco's modified Eagle's medium with 5% newborn calf serum (NBCS) for the times indicated in the text. All tissue culture reagents were purchased from Life Technologies.

Biochemical and osmotic induction of apoptosis. Cell apoptosis was induced by the addition of staurosporine (Calbiochem, San Diego, Calif.) to the medium at a final concentration of 1 µM. Cells were maintained in the presence of staurosporine for 24 h. To inhibit protein synthesis in infected cells, and therefore also induce apoptosis (5, 34), CHX (Sigma) was added to the medium at a final concentration of 10 µg/ml for 24 h. This concentration of CHX was previously shown to be sufficient to completely block viral protein synthesis in HSV-1-infected HEp-2 cells (5). Osmotic shock by sorbitol treatment was used to induce apoptosis (34) as follows. At the times (hours postinfection) indicated, infected cells were incubated for 1 h with 5% NBCS containing 1 M sorbitol (Sigma) and then for an additional 3 h with sorbitol-free 5% NBCS before the protein extraction procedures. Confirmation of the induction of apoptosis by staurosporine and sorbitol was made by light microscopy to detect morphological changes as described previously (5).

Inhibition of caspases. The caspase-3 inhibitory peptide Z-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DEVD-fmk) and the caspase-1 inhibitory peptide N-acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO) were obtained from Calbiochem (San Diego) and used at final concentrations of 50 µM. Each inhibitor was added to the cell culture medium 1 h prior to infection and was present during the entire infection period (24 h).

Preparation of infected cell extracts, denaturing gel electrophoresis, and transblotting. Whole extracts of infected cells were obtained as previously described (5). Protein concentrations were measured using a modified Bradford assay (Bio-Rad) as recommended by the vendor. Equal amounts of infected cell proteins (50 µg) were separated in denaturing 12% N,N'-diallyltartardiamide-acrylamide gels (9) and electrically transferred to nitrocellulose membranes in a tank apparatus (Bio-Rad) prior to immunoblotting with various primary antibodies. Unless otherwise noted in the text, all biochemical reagents were obtained from Sigma. Nitrocellulose was obtained directly from Schleicher & Schuell. Prestained protein molecular weight markers were purchased from Life Technologies.

Immunological reagents. The following primary antibodies were used to detect viral proteins: (i) RGST22, rabbit polyclonal antibody specific for full-length ICP22 (10); (ii) 1113, mouse anti-ICP27 monoclonal antibody (Goodwin Institute for Cancer Research, Plantation, Fla.); (iii) 1114, mouse anti-ICP4 monoclonal antibody (Goodwin); (iv) 1112, mouse anti-ICP0 monoclonal antibody (Goodwin); (v) rabbit anti-thymidine kinase (TK) polyclonal antibody (gift of Bernard Roizman); and (vi) VP16 (1-21) mouse anti-VP16 monoclonal antibody (Santa Cruz Biotechnology, Inc.). Immunoblotting experiments were performed to detect cellular apoptotic proteins by using mouse anti-caspase-3 monoclonal antibody (Transduction Laboratories Inc.), mouse anti-PARP monoclonal antibody (Pharmingen), and goat anti-DFF polyclonal antibody (Santa Cruz Biotechnology). Secondary goat anti-rabbit, goat anti-mouse, and rabbit anti-goat antibodies conjugated with alkaline phosphatase were purchased from Southern Biotechnology (Birmingham, Ala.). Tetramethylrhodamine isothiocyanate (Texas red)-conjugated anti-rabbit immunoglobulin G (heavy plus light chain) [IgG (H+L) was purchased from Vector Laboratories (Santa Cruz, Calif.) and used at a dilution of 1:100 in 1% bovine serum albumin (BSA). Fluorescein isothiocyanate-conjugated anti-mouse IgG (H+L) was purchased from Boehringer Mannheim (Indianapolis, Ind.) and used at a dilution of 1:500 in 1% BSA.

UV inactivation of virus. Virus stocks (10⁷ to 10⁸ PFU) in 2 ml of 199V medium (Life Technologies) containing 2% newborn calf serum were placed in a 10-mm-diameter dish (Falcon) on ice at a distance of 10 cm from a germicidal lamp (model MR-4, 60 Hz; George W. Gates and Company, Franklin Square, N.Y.). Virus particles were exposed to UV light for 10 min with mixing every 2 min. Virus titers after UV treatment were determined on Vero cells for KOS1.1 or Vero 2.2 cells for vBS27.

Indirect immunofluorescence, microscopy, and computer graphics. The fixation and permeabilization of infected cells for indirect immunofluorescence studies were performed as described previously (45). Briefly, infections were terminated by fixing in 2% methanol-free formaldehyde (Polysciences, Inc.) for 20 min at room temperature. Cells were permeabilized with 100% acetone at 20°C for 3 to 5 min, rinsed twice in phosphate-buffered saline, and then blocked in 1% BSA containing 10 µg of pooled human immunoglobulin (mainly IgG) (Sigma) per ml at 4°C. Each primary antibody was added for 1 h. After extensive rinsing with phosphate-buffered saline, the appropriate secondary antibody was added and incubated for an additional 45 min. Finally, the cells were preserved in a 0.1% solution of Mowoil (Sigma) with 2.5% DABCO (Sigma) used as an antibleaching agent under a fresh coverslip and sealed with nail polish. Cells were visualized on a Zeiss Axiophot fluorescence microscope. Infected cell phenotypes were documented by phase-contrast light microscopy using an Olympus CK2/PM-10AK3 system with an attached 35-mm camera. Immunoblots and 35-mm slides were digitized at a resolution of 600 to 1,200 dots per inch, using an AGFA Arcus II scanner linked to a Macintosh G3 PowerPC workstation. Raw digital images, saved as tagged image files in Adobe Photoshop version 5.0, were organized into figures by Adobe Illustrator version 7.1. Grey-scale and RGB prints of figures were obtained with a Codonics dye sublimation printer.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

HSV-1-induced apoptosis involves a caspase-3-dependent pathway. The goal of this study was to analyze HSV-1-induced apoptosis in cells in tissue culture. In a previous report (5), we showed that a recombinant strain of HSV-1 possessing a deletion in the gene encoding the viral ICP27 regulatory protein (vBS27) induced apoptosis during the infection of human epithelial cells. In addition, no apoptotic features were seen during infection of the same cells with a wild-type virus strain (i.e., KOS1.1) unless the infections were performed in the presence of the protein synthesis inhibitor CHX. These results led to the conclusion that while both types of viruses were likely inducing apoptosis, the HSV-1 ICP27 deletion virus was incapable of preventing this process from killing the cells. In this study, we have expanded our system to discern the induction and inhibition of apoptosis during HSV-1 infections in human tissue culture cells.

View larger version (49K): [in this window] [in a new window]   FIG. 1.   Detection of PARP, DFF, and caspase-3 processing in infected HEp-2 cells at early times from 6 to 24 hpi (A) or late times from 11 to 48 hpi (B). Whole cell extracts prepared at various times postinfection from vBS27- or KOS1.1-infected cells were separated in a denaturing gel, transferred to nitrocellulose, and reacted with anti-PARP, anti-DFF, or anti-caspase-3 antibodies as described in Materials and Methods. "116" and "85" denote full-length (116,000-molecular-weight) and processed (85,000-molecular-weight) PARP, respectively. The locations of prestained molecular weight markers are indicated on the left.

Inhibition of caspase-3 processing restores IE synthesis in vBS27-infected HEp-2 cells. Previously, we showed that following vBS27 infection of human cells at 24 h, both IE and L viral proteins accumulated to levels lower than those observed with either human cells infected with the wild-type KOS1.1 virus or vBS27-infected Vero cells (5). In the latter two cases, no obvious morphological features of apoptosis were observed during infection. The goal of this experiment was to determine whether specific inhibition of caspase processing during infection would influence the accumulation of viral proteins in vBS27-infected human cells. HEp-2 cells were mock infected and infected with either the wild-type KOS1.1 virus or vBS27 in the presence and absence of (i) protein synthesis (CHX), (ii) caspase-1 (Ac-YVAD-CHO), or (iii) caspase-3 (Z-DEVD-fmk) inhibitor as described in Materials and Methods. The caspase-3 inhibitor prevents caspase-3 from digesting its substrates and proteolyzing itself. Each caspase inhibitor was used separately or in combination with CHX since we previously detected apoptotic features in HEp-2 cells when CHX was present throughout the course of wild-type virus infection (5). Following the preparation of whole cell extracts at 24 hpi, immunoblotting analyses were performed with anti-PARP, anti-DFF, anti-caspase-3, anti-ICP4, anti-ICP22, and anti-ICP27 antibodies. ICP4, ICP22, and ICP27 were representative IE proteins. The results from this experiment are presented in Fig. 2.

View larger version (57K): [in this window] [in a new window]   FIG. 2.   Detection of PARP, DFF, and caspase-3 (A) and ICP4, ICP22, and ICP27 (B) in infected HEp-2 cells. Total cell extracts were prepared from mock-infected cells or cells infected with KOS1.1 or vBS27 in the absence () and presence of the protein synthesis inhibitor CHX (10 µg/ml) or in the absence () and presence of caspase-1 or caspase-3 (1 or 3; 50 µg/ml) as described in Materials and Methods. Immunoblot analyses were done with the anti-PARP, anti-DFF, and anti-caspase-3 antibodies (A) or anti-ICP4 (1114), anti-ICP22 (RGST22), and anti-ICP27 (1113) antibodies (B). Bar at the left mark the distribution of the modified forms of ICP22. Minor background bands due to the anti-ICP22 antibody were observed in all of the CHX-treated cells.

Induction and inhibition of apoptosis by HSV-1 occurs prior to 6 hpi. The results presented in Fig. 1 and 2 demonstrate two things. First, HSV-1 infection induces apoptosis in cells. Second, following infection with wild-type HSV-1 but not vBS27, proteins which block apoptosis are synthesized. To determine the time period at which the protein-dependent inhibition of apoptosis was effective in HSV-1-infected cells, we designed the following strategy (Fig. 3A) based on a temporal addition of the protein synthesis inhibitor CHX. HEp-2 cell monolayers were infected with the wild-type KOS1.1 virus; at 1, 3, 6, 8, 12 hpi, CHX was added to the medium and maintained for 24 h. The 24-h CHX treatment was shown by cell morphology and chromatin degradation assays to be sufficient to enable the detection of apoptosis in infected cells (5). The morphologies associated with the induction of apoptosis included small, irregularly shaped cells suggestive of cell shrinkage and membrane blebbing (5). Each infection was performed in duplicate. One set was used to make cell extracts at the time of CHX addition, these extracts corresponded to 1, 3, 6, 8, and 12 hpi without treatment. In the second set, CHX was added at each designated time point postinfection and kept for 24 h before the extracts were prepared. Thus, viral protein synthesis was inhibited at different stages of the HSV-1 replication cycle. Three types of experiments were performed to characterize the apoptotic process in these infected cells. In the first series, phase-contrast microscopy was used to follow cell morphology changes; these results (Fig. 3B) were as follows.

View larger version (91K): [in this window] [in a new window]   FIG. 3.   Schematic of infections (A) and morphologies (B) of KOS1.1-infected HEp-2 cells with and without CHX treatment. A to L, phase-contrast images of mock-infected cells at 12 hpi (F) and KOS1.1-infected cells at 1, 3, 6, 8, and 12 hpi (A to E) and of mock-infected cells 24 h after the addition of CHX at 12 hpi (L) and KOS1.1-infected cells 24 h after the addition of CHX at 1, 3, 6, 8 and 12 hpi (G to K). Magnification, ×60. WCE, whole cell extract.

View larger version (83K): [in this window] [in a new window]   FIG. 4.   Detection of cellular (A and B) and viral (C and D) protein accumulation in KOS1.1-infected HEp-2 cells with and without CHX treatment. Whole cell extracts (WCE) prepared at 1, 3, 6, 8, or 12 hpi or at 24 h after the addition of CHX (10 µg/ml) at 1, 3, 6, 8, or 12 hpi from KOS1.1-infected cells were used for immunoblot analyses with anti-PARP, anti-DFF, and anti-caspase-3 antibodies (A and B) or with anti-ICP4, anti-ICP0, anti-ICP22, anti-ICP27 (IE proteins), anti-TK (E protein), and anti-VP16 (L protein) antibodies (C and D). M, mock-infected cells at 12 hpi; + CHX, addition of CHX for 24 h. In lanes 6 and 7 of panels B and D, the infected cells were incubated for an additional 24 h in the absence of CHX. "116" and "85" denote full-length (116,000-molecular-weight) and processed (85,000-molecular-weight) PARP, respectively.

Prevention of staurosporine- and sorbitol-induced apoptosis by HSV-1 occurs prior to 6 hpi. The previous experiments (Fig. 1 to 4) demonstrated that infection with the wild-type KOS1.1 virus was able to induce as well as prevent apoptosis. Our findings suggest that both induction and prevention occur prior to 6 hpi. The goal of this experiment was to determine whether HSV-1 infection was also effective in preventing apoptosis induced by other stimuli such as staurosporine, a protein kinase inhibitor (8) which induces apoptosis through a caspase-3 pathway (31), and sorbitol, which causes osmotic shock (34). KOS1.1-infected HEp-2 cells were treated with staurosporine or sorbitol at 1, 3, 6, or 12 hpi as described in Materials and Methods. To determine whether HSV-1 infected cells respond to these compounds in a manner similar to that for CHX, immunoblotting analyses were performed to detect PARP, DFF, and caspase-3 processing. As a control, staurosporine was also added to cells 30 min prior to infection.

View larger version (39K): [in this window] [in a new window]   FIG. 5.   Detection of PARP, DFF, and caspase-3 processing in KOS1.1-infected HEp-2 cells treated with staurosporine (A) or sorbitol (B). Whole cell extracts prepared from mock- or KOS1.1 (K)-infected cells untreated () or treated with staurosporine or sorbitol at 1, 3, 6, or 12 hpi were used for immunoblotting analyses with anti-PARP, anti-DFF, and anti-caspase-3 antibodies as described in Materials and Methods. "116" and "85" denote full-length (116,000-molecular-weight) and processed (85,000-molecular-weight) PARP, respectively.

Apoptosis occurs in cells infected with HSV-1 strains deleted for either ICP27, U_S3, or ICP22 but not with U_L13- or vhs-deleted virus and wild-type viruses. At the very early stage of HSV-1 infection, the virion host shutoff protein (vhs polypeptide) induces degradation of host mRNAs and the shutoff of most protein synthesis (17, 18, 37, 50, 51, 60, 65). This effect of the vhs protein on cellular mRNA stability and protein synthesis could be one of the mechanisms used by HSV-1 to prevent the infected cells from inducing the pathway of apoptosis. It was recently reported that the U_S3 gene of HSV-1 is involved in protecting cells from apoptosis during infection (39). The U_S3 protein kinase, along with the U_L13 gene product, was implicated in the phosphorylation of the ICP22 protein (48, 49). Since ICP22 was one of the IE proteins whose reduction correlated with increased apoptosis in Fig. 4, the goal of this experiment was to determine whether viruses possessing deletions in each of these genes could prevent cell death during infection. HEp-2 cells were mock infected or infected with vBS27, vhs-Sma, KOS1.1, R7041, R7356, R325, and HSV-1(F) for 24 hpi prior to performance of immunoblot experiments. HSV-1(F) and KOS1.1 were both used as wild-type controls as they are the parental strains of the recombinant viruses R7041, R7356, and R325 and the vhs-Sma and vBS27 viruses, respectively. vBS27-infected cells were used as a positive control for the presence of apoptotic features in infected cells (5).

View larger version (27K): [in this window] [in a new window]   FIG. 6.   Detection of PARP, DFF, and caspase-3 processing in HEp-2 cells infected with wild-type and mutant HSV-1. Whole cell extracts prepared from mock-infected cells (M) or cells infected with HSV-1 (F), vBS27 (27), R7041 (US3 deletion), R325 (ICP22 deletion), R7356 (UL13 deletion), vhs-Sma (vhs deletion), or KOS1.1 at 24 hpi were used for immunoblot analyses with anti-PARP, anti-DFF, and anti-caspase-3 antibodies as described in Materials and Methods. "116" and "85" denote full-length (116,000-molecular-weight) and processed (85,000-molecular-weight) PARP, respectively.

Binding and entry of HSV-1 are not sufficient to induce apoptosis. In Fig. 3 and 4, we showed that the KOS1.1 virus could induce apoptosis in the absence of detectable levels of viral proteins, suggesting that events prior to IE protein synthesis are sufficient to trigger cell apoptosis. The goal of this study was to confirm or eliminate the hypothesis that either the binding to or entry of the virion particle into the cell might facilitate the induction process. To focus on the first steps of viral infection, specifically virion binding, envelope fusion, and tegument/nucleocapsid entry into the cell, we prepared viruses which were inactivated by exposure to UV light. Since UV treatment damages the viral DNA genome in the virion, it was expected that viral transcription would be prevented and no IE gene products would be synthesized. In control experiments, KOS1.1 virus was UV inactivated as described in Materials and Methods. Under these conditions, we were able to reproducibly decrease the titer of the UV-treated virus (K_uv) by greater than 5 logs compared to untreated controls (data not shown). To confirm that the UV exposure did not perturb the ability of the treated virions to bind and enter the cells, we performed immunofluorescence experiments using anti-VP16 (tegument protein) and anti-ICP22 (IE protein) antibodies at 2 hpi in Vero cells as described in Materials and Methods. In this system, the ability of VP16 to translocate to the nucleus is used as a positive control for virion binding, fusion, and entry of the capsid and tegument proteins into the cytoplasm. ICP22 was chosen as a representative marker for IE protein synthesis. The results of this study (Fig. 7) indicated that only VP16 and not ICP22 could be detected in the nuclei of cells infected with K_uv whereas both proteins were observed with untreated virus. Based on these findings, we conclude that our UV exposure technique is efficient and that the inactivated viruses are able to enter cells since VP16 was translocated to the nucleus.

View larger version (14K): [in this window] [in a new window]   FIG. 7.   Indirect immunofluorescence of infected cells double labeled with antibodies specific for ICP22 (A to E) and VP16 (D to F). Vero cells were mock infected or infected (50 PFU/cell) with KOS1.1 or UV-inactivated KOS1.1 (KOS1.1uv) for 2 h and then subjected to formaldehyde-acetone fixation followed by immunostaining with anti-ICP22 (rabbit) and anti-VP16 (mouse) antibodies as described in Materials and Methods.

View larger version (33K): [in this window] [in a new window]   FIG. 8.   Detection of PARP, DFF, and caspase-3 processing in KOS1.1-infected HEp-2 cells untreated (A) or treated (B) with CHX. Whole cell extracts (WCE) were prepared from mock-infected cells (M) or cells infected with KOS1.1 (K) or UV-inactivated KOS1.1 (Kuv) virus at 6 or 24 hpi in the absence of CHX (A) and at 24 h after the addition of CHX (B) at either 1 hpi, 6 hpi, or 30 min before infection (0.5 hpi). Immunoblotting analyses were performed with anti-PARP, anti-DFF, and anti-caspase-3 antibodies. "116" and "85" are full-length (116,000-molecular-weight) and processed (85,000-molecular-weight) PARP, respectively.

View larger version (32K): [in this window] [in a new window]   FIG. 9.   Detection of PARP, DFF, and caspase-3 processing in vBS27-infected HEp-2 cells. vBS27 was inactivated by exposure to UV light as described in Materials and Methods. Whole cell extracts (WCE) prepared from mock-infected cells (M) or cells infected with vBS27 () and UV-inactivated vBS27 (uv) at 6 and 24 hpi in the absence of or at 24 hpi in the presence (+) of CHX were used for immunoblot analyses with anti-PARP, anti-DFF, and anti-caspase-3 antibodies. "116" and "85" denote full-length (116,000-molecular-weight) and processed (85,000-molecular-weight) PARP, respectively.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Previously, we showed that human cells died by apoptosis after infection with an HSV-1 ICP27 deletion virus and that the apoptotic features of these cells were identical to those of human cells infected with wild-type virus when total protein synthesis has been inhibited (5). From these results we concluded that both viruses likely induced apoptosis in human cells, but the mutant virus was not capable of preventing the apoptotic process from killing the cells (5). In this study, we used the ICP27 deletion virus vBS27 as a tool to further investigate the process of induction and prevention of apoptosis in HSV-1-infected human cells. The significant findings of our study can be summarized as follows.

(i) We showed that HSV-1-infected HEp-2 cells underwent apoptosis through a pathway which involved the activation and proteolysis of caspase-3, resulting in the processing of PARP and DFF. In the absence of any other treatment, these processing events were detected in the infected cells as early as 11 hpi in vBS27-infected cells. This corresponds to the time at which we originally observed the morphological and DNA laddering features associated with apoptosis in vBS27-infected HEp-2 cells (5). Although wild-type HSV-1 does not show obvious signs of apoptosis at 11 hpi, by 24 and 48 hpi, significant amounts of PARP, DFF, and caspase-3 processing could be detected.

One of the more intriguing findings of this study using the wild-type virus (Fig. 1B) is that although PARP and DFF processing was detected at 24 and 48 hpi, the caspase-3 level dropped between 6 and 11 hpi. This seems to infer the following model. Viral infection induces apoptosis via a pathway which activates and processes caspase-3. The virus also causes the synthesis of proteins which appear to prevent the processing of DFF and PARP until later in infection. Thus, the absence of ICP27 enables these two proteins to become processed much earlier, presumably because either ICP27 itself or another function dependent on ICP27 is the blocking activity. Taken together, these findings suggest that the inhibition of apoptosis by HSV-1 is only temporary rather than absolute. Accordingly, the CPE due to lytic viral replication in cells, traditionally assumed to proceed through a necrotic route, likely has an apoptotic component as well. While these studies focused on transformed human HEp-2 cells, we have observed similar apoptotic features following infection of primary human fibroblasts (4).

(ii) Furthermore, we demonstrated that the accumulation of the IE proteins in vBS27-infected HEp-2 cells, previously shown to be lower than in wild-type-infected cells (5), could be restored to wild-type levels when the infections were performed in the presence of a caspase-3 inhibitor. This result suggests that the inability of vBS27 virus to produce IE proteins is due to premature cell death involving the activation of the caspase-3. Therefore, possibly HSV-1 blocks apoptosis because of an increase of the amount of viral, and particularly IE, protein synthesized. The obvious consequence of this would be that the virus is then able to produce higher levels of infectious progeny. Consistent with this model is the fact that Vero cells, which seem unable to undergo apoptosis (5), yield higher-titer stocks of HSV-1 than do HEp-2 cells (21).

Our findings are consistent with recent studies which indicate that sorbitol induction of apoptosis involves caspase-3 and that wild-type HSV-1 is capable of blocking this process and preventing apoptosis which proceeds through the mitochondrial pathway (19). It is of interest that these researchers also observed (19) DNA fragmentation following infection of human neuroblastoma (SK-N-SH) cells with the d120 virus (15, 39), and they conclude that caspase-3 was not involved in d120-induced apoptosis.

(iii) The virus-dependent inhibitory function is effective in preventing apoptosis which is induced by exposure of cells to CHX, staurosporine, and sorbitol. These results suggest that the types of apoptosis induced by these treatments all proceed through a caspase-3 pathway. Therefore, it is quite likely that the viral blocking function acts at the same location in the pathway. Staurosporine is capable of inhibiting cytoplasmic and receptor tyrosine kinases (76). Recently, we demonstrated that several HSV-1 proteins, including ICP22, were tyrosine phosphorylated in infected cells. It will be of interest to determine whether viral protein phosphorylation might plays a role in the induction or prevention of apoptosis.

(iv) The HSV-1 ICP22 and U_S3 gene products are necessary but not sufficient for optimal prevention of apoptosis. The conclusion was based on the finding that partial PARP, DFF, and caspase-3 processing was observed during infections using viruses with deletions of either of these two genes. However, the role that these two proteins play in the prevention of apoptosis in cultured cells appears limited inasmuch as this level of caspase-3-dependent processing was negligible compared to that observed during infection with the ICP27 deletion virus. As ICP22 has been implicated in the neurogrowth of HSV-1 in mice (61), it is possible that ICP22's inhibitory potential is cell type dependent and therefore may be realized only in studies using whole animal models. ICP27 is a multifunctional regulatory phosphoprotein which can associate with other viral regulatory proteins and is required for optimal DNA synthesis and the expression of some late viral genes (41, 52, 56, 72). Recently, it was shown that ICP27 inhibits host cell splicing, redistributes splicing components throughout the nucleus, and aids in the export of RNA from the nucleus (24, 25, 42, 58, 59, 63, 64). Our current data do not indicate whether one of these known functions of ICP27 is also involved in the prevention of apoptosis. The fact that apoptosis is strongly induced when ICP27 is absent during HSV-1 infection suggests that these functions of ICP27 are not essential for the induction of apoptosis. In addition, we do not know whether ICP27 itself is directly involved in blocking apoptosis or whether another viral component, whose production or activity is dependent on ICP27, plays a role.

(v) The induction and prevention of apoptosis by wild-type HSV-1 infection occur prior to 6 hpi. This conclusion was based on results obtained with a protocol developed for this study involving temporal addition of CHX. This time period includes the transition from the IE to the E phase of replication. What was most intriguing was the fact that our detection of increased caspase-3-dependent processing correlated with reductions of the ICP0, ICP27, and ICP22 proteins. This is consistent with our findings described above for both the ICP27 and ICP22 deletion viruses which suggested that these two proteins are involved in the prevention of apoptosis. From our current data we cannot conclude whether ICP0 is directly required in the process. Also still unclear are the actual molecular basis for the virus-dependent inhibition of apoptosis as well as how many proteins are involved. For example, we cannot exclude the possibility that the virus can recruit cellular proteins to participate in the prevention process as well.

(vi) Virion binding and entry are not sufficient to induce apoptosis in HSV-1-infected cells. Neither the wild-type virus in the presence of CHX nor the ICP27 deletion virus could induce apoptosis following UV exposure. Control indirect immunofluorescence experiments confirmed that the UV treatment did not destroy the virus particles since virion-derived VP16 was detected in the nuclei of infected cells at 2 hpi.

Our earlier findings indicated that de novo viral protein synthesis is not required to induce apoptosis in infected cells (5). We now show that viral entry is also not sufficient to stimulate the apoptotic process. Taken together, the data lead us to conclude that either gene expression, presumably viral, or some other RNA processing event likely plays a role in the induction of apoptosis in HSV-1-infected cells. One possibility is that the decrease in RNA stability associated with the vhs activity (17, 50) acts as a stimulus of apoptosis. Consistent with this view was our initial observation that the vhs-Sma virus induced slightly less PARP processing than that seen with its parental KOS1.1 virus. However, since we were unable to detect PARP, DFF, or caspase-3 processing with UV-treated KOS1.1 and vBS27 viruses, both of which possess wild-type vhs protein, it appears that the vhs function may contribute little to the induction of cell death. Additional studies focusing on the activity of the vhs protein itself should help clarify this point.

If the general stability of transcripts in the infected cells does not play a role in the induction of apoptosis, other potential viral stimuli might involve any one of the steps associated with the expression of the IE genes. These potential induction mechanisms include transcription of the IE genes, splicing of the 22/47 and 0 genes, export of the IE RNAs from the nucleus, and the initial interactions of these RNAs with the translational machinery. The development of additional molecular genetic and biochemical systems is required to define the molecular trigger(s) for the induction of apoptosis in HSV-1-infected human cells.