INTRODUCTION

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The expression of herpes simplex virus type 1 (HSV-1) genes occurs through a highly regulated cascade (23) beginning with the production of the  or immediate-early (IE) proteins. The  regulatory proteins, infected cell proteins 0, 4, 22, and 27, cooperatively act to regulate the expression of all classes of viral genes (reviewed in reference 52). The  or 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 7). The last set of viral proteins produced are the  or late (L) proteins and are mainly associated with virion structure and assembly, such as the VP16, gD, and gC proteins (4, 15, 47). The  gene class is further subdivided into the ₁ and ₂ groups, where ₂ expression is absolutely dependent on viral DNA synthesis. 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 and morphological alterations (3, 18, 21, 52). However, features observed with wild type HSV-infected cells are different from those associated with cells dying from apoptosis. Apoptotic cells are characterized by cellular changes that include cell shrinkage, membrane blebbing, nuclear condensation, and fragmentation of chromosomal DNA into nucleosomal oligomers (reviewed in reference 28).

Previously, we reported that human epithelial cells present characteristic morphological and biochemical hallmarks of apoptotic cells when infected with an HSV-1 ICP27-null mutant virus (1, 2). These apoptotic features include membrane blebbing and the formation of apoptotic bodies. A time-lapse movie showing infected-cell apoptosis is available at our public domain website (http://www.mssm.edu/micro/blaho/webdata/maetaldns.shtml). In addition, we detected chromatin condensation, nuclear fragmentation, and nucleosomal laddering in ICP27-null-virus-infected cells. Finally, we determined that these defining features of apoptosis proceed through a pathway in which caspase-3 is activated and the death substrates DNA fragmentation factor (DFF) and poly(ADP-ribose) polymerase (PARP) are processed. These features are not observed with wild type-infected cells unless total protein synthesis is inhibited by the addition of cycloheximide during infection (1, 2, 30). From these results, we concluded that both wild-type and ICP27-null viruses induce apoptosis in human cells, but the ICP27-null virus is not able to prevent the apoptotic process from killing the cells.

ICP27 is a multifunctional phosphoprotein which is necessary for completion of the viral lytic cycle (32, 48, 53, 67, 69). During infection, ICP27 stimulates both E () gene and L () gene expression (19, 32, 48, 53, 55, 56, 65) and down-regulates IE () and E gene expression at late times (32, 48, 53). In addition, ICP27 significantly enhances the efficiency of viral DNA replication (32, 48), most likely by increasing the expression of viral DNA replication factors (33, 65). The ability of ICP27 to stimulate DNA replication is separable, by mutation, from its stimulatory effect on L genes (48, 49, 53). The regulatory effects of ICP27 can also be demonstrated in uninfected cells, as ICP27 can enhance or repress reporter genes in transient expression assays (19, 51, 59). Although the mechanism of action of ICP27 is as yet unknown, there is a great deal of evidence to suggest that it regulates gene expression posttranscriptionally through the modulation of mRNA processing and export. ICP27's known posttranscriptional activities include (i) modulation of pre-mRNA polyadenylation efficiency (33, 34, 58), (ii) binding and stabilization of the labile 3' ends of mRNA (8), (iii) redistribution of pre-mRNA splicing factors such as snRNPs (41, 57), (iv) inhibition of cellular and viral pre-mRNA splicing (20), and (v) nuclear retention of intron-containing viral transcripts (43, 56). Consistent with these activities is ICP27's ability to bind RNA (8, 25, 38) and to shuttle between the nucleus and cytoplasm (37, 42, 56, 60).

The timing of these well-documented regulatory functions of ICP27 is consistent with our observation that the apoptosis prevention function present during wild-type HSV-1 infection is detected after 3 h postinfection (p.i.) (2). The goal of this study was to define more precisely the features of viral replication which are involved in apoptosis prevention during HSV-1 infection of human epithelial cells. Using a series of recombinant HSV-1 viruses which possess deletion, nonsense, or point mutations in the gene encoding ICP27, we infected cultured HEp-2 cells and documented the extent of apoptosis for each virus. Analyses of cell morphology, cellular death factor processing, viral protein accumulation, viral DNA synthesis, and productive viral replication were performed. Our findings indicate that the accumulation of early () and leaky-late (₁), but not true late (₂), viral gene products correlates with apoptosis prevention during HSV-1 infection. Based on these results, we suggest that ICP27 regulates the synthesis of the viral proteins which participate in the blocking of apoptosis during HSV-1 infection.

	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.). V27 is a derivative Vero cell line which carries a stably integrated copy of the ICP27 gene (48). HEp-2 cell monolayers were infected at a multiplicity of infection (MOI) of 10 PFU/cell, and the infections were maintained at 37^oC in Dulbecco's modified Eagle's medium with 5% newborn calf serum for 24 h. For viral growth determination, Vero cells (2.2 × 10⁶) were infected at an MOI of 10 PFU per cell. The same inocula were used to infect parallel cultures of 2.5 × 10⁶ V27 cells. The cultures were harvested at 24 h p.i., and virus yield was determined by plaque assay on V27 cells. KOS1.1 (24) is the wild-type parental virus, and the features of all the recombinant viruses used in this study are described in detail in Table 1 and Fig. 1. The mutants d27-1, n59R, n263R, n406R, n504R, d1-2, d3-4, d4-5, d5-6, d1-5, M11, M15, and M16 have been described (36, 38, 49, 50). Construction of the d2-3 and d6-7 viruses was analogous to that of the d3-4 and d4-5 viruses (36). Two independent plaque isolates (a and b) of d1-2 and d4-5 were analyzed.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Schematic of the ICP27 protein showing locations of defined functional regions. These include NES, a putative nuclear export signal (open box); the acidic domain, involved in gene expression and DNA synthesis (striped box); NLS, nuclear localization signal (black box); NuLS, nucleolar localization signal (white box); RGG sequence, RNA binding motif (gray box); and activation and repression domains (arrows), which include a cysteine-histidine zinc-finger-like domain (checkered box). Italicized numbers define mutated sites (M11, M15, and M16) and the boundaries of in-frame deletions. Bold numbers correspond to relevant amino acid positions.

Infected cell extract preparations, denaturing gel electrophoresis, and transblotting. Whole extracts of infected cells were obtained as described previously (1). 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 15% N,N'-diallyltartardiamide (DATD)-acrylamide gels (1) 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. Protein molecular weight markers (not shown in figures) and all tissue culture reagents were purchased from Life Technologies.

Immunological reagents. The following primary antibodies were used to detect viral proteins: (i) RGST22, rabbit antibody specific for full-length ICP22 (6); (ii) rabbit anti-TK polyclonal antibody (gift of Bernard Roizman); (iii) VP16(1-21), mouse anti-VP16 monoclonal antibody (Santa Cruz Biotechnology, Inc.); (iv) H1119, mouse anti-ICP27 monoclonal antibody; (v) H1114, mouse anti-ICP4 monoclonal antibody; (vi) H1103, mouse anti-glycoprotein D (gD) monoclonal antibody; and (vii) H1104, mouse anti-glycoprotein C (gC) monoclonal antibody (Goodwin Institute for Cancer Research, Plantation, Fla.). Cellular proteins were detected 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, Inc). Mouse anti- tubulin monoclonal antibody (Sigma) was used to confirm equal loading of the cell extracts. Secondary (goat) anti-rabbit, anti-mouse, and (rabbit) anti-goat antibodies conjugated with alkaline phosphatase were purchased from Southern Biotechnologies Associates, Inc. (Birmingham, Ala.). Secondary (goat) anti-mouse antibody conjugated with horseradish peroxidase was obtained from Amersham and used for the VP16, gC, and  tubulin immunoblot analyses (chemiluminescence) presented in Fig. 4 and for gD in Fig. 7.

DNA replication assay. Approximately 10⁶ HEp-2 cells were either mock infected or infected with wild-type or mutant HSV-1 at an MOI of 10 PFU/cell. After a 1-h adsorption, the cells were overlaid with 199 medium containing 2% newborn calf serum. At 2 and 20 h p.i., total infected cell DNA was isolated by the following procedure. The medium was replaced by 3 ml of lysis buffer (10 mM Tris-HCl [pH 8.0], 10 mM Na₂EDTA, 2% sodium dodecyl sulfate, 100 µg of proteinase K/ml), and the flasks were incubated at 37°C for either 22 or 4 h. After the addition of 0.33 ml of 3 M sodium acetate, each lysate was extracted first with an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) and then with an equal volume of chloroform-isoamyl alcohol (24:1). After two sequential ethanol precipitations, the purified DNA was resuspended in TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM Na₂EDTA) and its concentration was determined by UV absorbance at 260 nm. Equal amounts of each DNA preparation (6.7 µg) were then digested with PstI, electrophoresed on a 1% agarose gel, and blotted to a nylon membrane. The membrane was hybridized with a ³²P-labeled 813-bp plasmid-derived EcoNI/EcoRV restriction fragment corresponding to the coding region of the HSV-1 U_L44 (gC) gene. This probe detects a 4.7-kb HSV-1 genomic PstI fragment. Hybridization, carried out overnight at 42°C in UltraHyb (Ambion), and washing were done according to the manufacturer's specifications. Quantitation of the radioactive bands was performed using a Molecular Dynamics phosphorimaging system.

Percentage of apoptotic cells. The percentages of apoptotic cells were determined as follows. HEp-2 cell monolayers grown on glass coverslips in 35-mm-diameter dishes were infected at an MOI of 5 PFU/cell in the absence or presence of the caspase inhibitory peptide Z-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk; Calbiochem, San Diego, Calif.) at a final concentration of 100 µM at 37^oC. At 15 h p.i., the cells were fixed with 2% formaldehyde in phosphate-buffered saline for 20 min, permeabilized with 100% acetone at 20°C for 4 min (44), and incubated with the DNA dye Hoechst 33258 (Sigma) at a final concentration of 5 µg/ml in phosphate-buffered saline for 10 min. The number of apoptotic cells with condensed chromatin DNA and fragmented nuclei (1), as well as the total number of cells in representative fields, were counted by using an Olympus model IX70 fluorescence microscope. The percentage of apoptotic cells was calculated as follows: (number of apoptotic cells/total number of cells) × 100. The data of Fig. 3 represent the mean of two independent experiments in which a minimum of 260 and a maximum of 325 cells were counted for each virus.

Quantitative measurements of processed proteins. The amounts of representative cellular and viral polypeptides were determined and used to define the percentage of PARP processing detected during infection as follows. Relative amounts of cellular (see Fig. 4) and viral (see Fig. 5) proteins and values of percent PARP processing (Table 2) were calculated using the public domain NIH Image program (developed at the National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). Immunoblots were digitized using an AGFA Arcus II scanner linked to a Macintosh G3 Power PC workstation. Raw digital images (2 in. horizontal) were saved as 8-bit gray-scale tagged image files (TIF) at a resolution of 300 dots per inch, using Adobe Photoshop version 5.0. Mean pixel measurements were calculated twice by using NIH Image software and averaged. Analysis areas were held constant and were 0.11 by 0.03 in. for PARP and processed PARP, 0.12 by 0.04 in. for caspase-3 and TK, and 0.12 by 0.07 in. for VP16. Percent PARP processing was defined as the percent conversion of full-length PARP (molecular weight, 116,000) to processed PARP (molecular weight, 85,000) {[processed PARP/(PARP + processed PARP)] × 100}. Amounts of PARP and caspase-3 were relative to that in mock-infected cells (100%). Processed PARP was relative to that measured with d27-1. TK and VP16 were relative to that measured with KOS1.1. As we reported previously, the observed amounts of unprocessed and processed PARP for any given infection are not molarly equivalent due to their differences in transfer efficiency (2). Thus, either the relative amounts of each form or the ratios of the two forms are comparable between each infection. Finally, all values were normalized to the amount of  tubulin present in each lane to eliminate any potential error due to differences in sample loadings.

Microscopy and computer graphics. Infected-cell phenotypes were documented by phase-contrast light microscopy using an Olympus CK2/PM-10AK3 system with an attached 35 mm camera. Immunoblots, autoradiograms, and 35 mm slides were digitized at a resolution of 800 to 1,000 dots per inch as described above. Raw digital images (TIF format) were organized using Adobe Illustrator version 7.1, and gray-scale prints of figures were obtained by using a Codonics dye sublimation printer. Time-lapse photography was performed on a Zeiss Axioskop phase-contrast microscope linked to a PowerMac G3. Digital images were captured at 5-min intervals, converted to TIFs using Photoshop, and organized into a movie using Quicktime 4.1. Certain results may be accessed through our website (http://www.mssm.edu/micro/blaho/webdata/maetaldns.shtml).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Recombinant viruses with defined mutations in ICP27 functional regions. Previously (1), we demonstrated apoptosis during the infection of human cells with a mutant virus possessing a complete deletion of the gene encoding ICP27 (i.e., vBS27). In this study, we set out to test whether different ICP27 mutant recombinant viruses carrying partial deletions, nonsense mutations, or point mutations in the ICP27 gene (Table 1) retain their ability to prevent apoptosis compared to the HSV-1 ICP27-null virus phenotype. Since infection with a virus containing a complete ICP27 deletion leads to apoptosis, all recombinant viruses tested in our study have the ability to induce this process. Therefore, we were interested in identifying which mutant viruses are unable to block the process as a consequence of their lesion in ICP27. Our goal is to use this series of recombinant viruses to identify the features of viral replication which participate in the prevention of apoptosis during HSV-1 infection of human epithelial cells.

HEp-2 cells infected with ICP27 mutant viruses present apoptotic phenotypes. HEp-2 cells infected with an ICP27-null virus show features characteristic of apoptosis, including membrane blebbing, small irregularly shaped cells, and the generation of apoptotic bodies (1, 2; http://www.mssm.edu/micro/blaho/webdata/maetaldns.shtml). As an initial step in the current analysis, we determined whether cells infected with viruses carrying mutations of specific regions in ICP27 present similar apoptotic phenotypes. HEp-2 cells were infected with the series of mutant viruses shown in Table 1. At 24 h p.i., the infected cell morphologies were documented by phase-contrast microscopy, as described in Materials and Methods, and compared to those observed for wild-type- and d27-1-infected cells. The results (Fig. 2) were organized into two groups: ICP27 mutations affecting the amino domains and those affecting the carboxy domains.

View larger version (124K): [in this window] [in a new window]   FIG. 2.   Morphologies of HEp-2 cells infected with all viruses used in this study. Cells infected with d27-1, KOS1.1, and mock-infected virus (A) or viruses infected with ICP27 with an amino domain deletion (B), ICP27 with a carboxy domain deletion (C), and ICP27 with a carboxy domain point mutation (D) were observed at 24 h p.i. by phase-contrast microscopy (magnification, ×20) as described in Materials and Methods.

Quantitation of apoptosis during ICP27 mutant virus infection of HEp-2 cells. The results in Fig. 2 indicate that infection of HEp-2 cells by various ICP27 mutant viruses can yield phenotypes identical to that observed with either KOS1.1 or d27-1 infection. However, certain viruses also generate cell morphologies which, while possessing features consistent with apoptosis, are not as extreme as that observed with d27-1. Therefore, the goal of this set of experiments was to quantitate the number of apoptotic cells detected during infection with each one of the viruses used for Fig. 2. HEp-2 cells were mock infected or infected with each of our series of viruses (Table 1). At 15 h p.i., the cells were fixed and stained with Hoechst DNA dye prior to visualization by fluorescence microscopy, as described in Materials and Methods. As a control, the z-VAD-fmk general caspase inhibitor was added throughout the course of a d27-1 infection. The percentage of total apoptotic cells detected was determined by counting the number of cells with condensed chromatin.

View larger version (53K): [in this window] [in a new window]   FIG. 3.   Visualization (A) and quantitation (B) of apoptotic HSV-1-infected HEp-2 cells. Mock- and HSV-1-infected HEp-2 cells were fixed at 15 h p.i., stained with Hoechst 33258, and visualized by fluorescence microscopy (magnification, ×60) as described in Materials and Methods. Panel A shows representative images of stained nuclei. d27-1-infected cells show classic condensed chromatin. The few apoptotic cells detected in mock-, KOS1.1-, and d27-1-infected cells in the presence of the general caspase inhibitory peptide (z-VAD-fmk) are marked by arrows. The percentage of apoptotic cells was determined by counting the number of infected cells with condensed chromatin. Gray, striped, and black histograms refer to apoptotic, partially apoptotic, and nonapoptotic viruses, respectively.

Detection of cellular death factor processing following infection with ICP27 mutant viruses. The experiments for which results are shown in Fig. 2 and 3 focused on cell morphologies during infection. The goal of this series of experiments was to measure the extent of proteolytic processing of representative cellular proteins associated with the apoptotic program in HEp-2 cells. We previously showed that during vBS27-induced apoptosis, certain cellular death factors such as PARP, DFF, and caspase-3 are processed (1). The possibility that wild type HSV-1 might degrade caspase-3 rather than activate it was excluded in experiments showing that full-length caspase-3 remains intact during productive HSV-1 infection when caspase inhibitors are present in the culture medium (1) (M. Aubert et al., data not shown). The processing of PARP, a 116,000-molecular-weight protein, generates an 85,000-molecular-weight product, and both forms were detected by the anti-PARP antibody used in this study (63, 67). Apoptosis-induced processing of DFF (molecular weight, 45,000) and caspase-3 (molecular weight, 32,000) resulted in the loss of reactivity with the anti-DFF and anti-caspase-3 antibodies. To analyze the processing of these death factors following infection with our series of viruses, HEp-2 cells were infected as for Fig. 2, whole extracts were made, and immunoblotting experiments were performed using anti-PARP, anti-caspase-3, and anti-DFF antibodies as described in Materials and Methods. To facilitate ease of comparison between each virus, relative amounts of processed PARP, unprocessed PARP, and caspase-3 were calculated. These values were determined after normalization to the amounts of  tubulin detected in each lane. The results showing PARP, caspase-3, and DFF processing are presented in Fig. 4.

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Immunoblot detection of cellular death factor processing in infected HEp-2 cells. Whole-cell extracts prepared at 24 h p.i. were used for immunoblot analyses with anti-PARP, anti-DFF, anti-caspase-3, and anti- tubulin antibodies. Amounts of full-length PARP, processed PARP, and caspase-3 were calculated relative to the amount of  tubulin as described in Materials and Methods. 116 and 85 refer to full-length and processed PARP, respectively. Lanes 4 and 7 are underlined to mark viruses analyzed in Fig. 7.

Viruses carrying mutations in ICP27's N-terminal acidic domain or the activation/repression domain are impaired for growth in Vero cells. The prior series of experiments demonstrated that viruses containing ICP27 mutations can be defined as either apoptotic, partially apoptotic, or nonapoptotic based on their properties observed during infection of HEp-2 cells. In order for us to use these viruses as tools to fully understand the aspects of HSV-1 infection which are required for the apoptotic prevention process, the goal of our remaining experiments was to compare the capacities of the viruses to block apoptosis with their abilities to productively replicate, synthesize viral DNA, and accumulate viral proteins. Traditionally, ICP27 functions have been assessed using Vero cells. Previously, we showed that the vBS27 ICP27-null virus does not induce apoptosis in Vero cells (1). We suggested that this may be because Vero cells have lost their ability to efficiently activate proapoptotic pathways in response to HSV-1 infection. In this portion of the study, we assessed the ability of our viruses to replicate under conditions in which apoptotic prevention functions are not required. The titers of each virus in Vero and V27 (ICP27-expressing Vero) cells were measured (Table 2). These data were compared (Table 2) to the number of apoptotic cells (Fig. 3) and the extent of PARP processing by comparing the relative amounts of processed and unprocessed PARP (Fig. 4).

Viral DNA synthesis in ICP27 mutant-infected HEp-2 cells. The goal of our final series of experiments was to determine as precisely as possible the feature of HSV-1 replication that correlates with the prevention of apoptosis in infected HEp-2 cells. The results presented in Table 2 show a comparison of the antiapoptotic potentials of our series of viruses with their abilities to efficiently replicate in Vero cells. Previously (48-50), the abilities of a subset of these viruses to synthesize their genomic DNA in Vero cells were determined (results summarized in Table 1). While DNA replication is a central event in the HSV-1 lytic cycle, ICP27 is not absolutely essential for this process in Vero cells (49). Viruses d27-1, d1-2, n406R, n263R, and n59R are defective for DNA replication in Vero cells, but M11, M15, M16, and n504R show levels of DNA replication that are comparable to that of the wild-type virus. Thus, it was of interest to measure the levels of viral DNA replication for each virus in HEp-2 cells to see if this process correlates with apoptosis prevention. At 2 and 20 h p.i., total infected cell DNA was isolated and digested with PstI. DNA hybridization was performed using a radioactive probe derived from the HSV-1 U_L44 gene (gC), and the amounts of viral DNA detected at the two time points were compared using a phosphorimager as described in Materials and Methods. For each infection, the radioactive signal at 20 h p.i. (replicated DNA) was divided by the signal at 2 h p.i. (input DNA) to generate values of fold DNA replication. The results (Fig. 5) were as follows.

View larger version (33K): [in this window] [in a new window]   FIG. 5.   Autoradiographic images of viral DNA. Total DNA was isolated from infected HEp-2 cells at 2 and 20 h p.i., digested with PstI, separated in an agarose gel, transferred to a nylon membrane, and hybridized with a radioactive probe derived from a portion of the HSV-1 UL44 gene, prior to autoradiography, as described in Materials and Methods. Amounts of DNA were quantitated using a phosphorimager, and values of fold-DNA replication were calculated by dividing the radioactive signal at 20 h p.i. (replicated DNA) by the signal at 2 h p.i. (input DNA).

Accumulations of TK, VP16, and gD but not gC, ICP22, or ICP4 correlate with prevention of apoptosis during HSV-1 infection. To assess the progression of viral replication, each infected cell sample for which data are presented in Fig. 4 was tested for the accumulation of representative viral proteins at 24 h p.i. Immunoblotting analyses were performed using antibodies specific for ICP27, ICP4, ICP22, TK, VP16, gD, and gC, and the results are presented in Fig. 6.

View larger version (66K): [in this window] [in a new window]   FIG. 6.   Immunoblot detection of viral protein accumulation in infected HEp-2 cells. Whole-cell extracts prepared at 24 h p.i. were used for immunoblot analyses with anti-ICP27, anti-ICP4, and anti-ICP22 (IE proteins), anti-TK (E protein), anti-VP16 and anti-gD (L proteins, 1), and anti-gC (L protein, 2) antibodies. Relative amounts of TK and VP16 were normalized to the amount of  tubulin and calculated as described in Materials and Methods. An asterisk marks n263R ICP27 protein since its accumulation is at low levels. Lanes 4 and 7 are underlined to mark viruses analyzed in Fig. 7.

View larger version (55K): [in this window] [in a new window]   FIG. 7.   Morphologies (A) and immunoblot detection of PARP and caspase-3 processing (B) and viral protein accumulation (C) in d1-2- and d4-5-infected HEp-2 cells. Cells infected with d27-1, KOS1.1, d1-2 (isolate a or b), and d4-5 (isolate a or b) viruses and mock-infected cells were observed at 24 h p.i. by phase-contrast microscopy (magnification, ×20). Whole-cell extracts prepared at 24 h p.i. were used for immunoblot analyses with anti-PARP and anti-caspase-3 antibodies or with anti-ICP27, anti-ICP4 (IE proteins), anti-TK (E protein), anti-gD (L protein, 1), and anti-gC (L protein, 2) antibodies as described in Materials and Methods. 116 and 85 refer to full-length and processed PARP, respectively.

Viral DNA synthesis and the production of true late gene products are not required for prevention of apoptosis in infected HEp-2 cells. The results in Fig. 5 indicate that apoptosis prevention by HSV-1 correlates with the infection proceeding to the viral DNA synthesis stage, but it is not necessary to generate wild-type levels of viral DNA replication. Furthermore, the results in Fig. 6 and 7 suggest that true late (₂) viral proteins are not required for the prevention activity. Since the expression of ₂ genes is absolutely dependent on viral DNA synthesis, the goal of this experiment was to determine whether HSV-1 could block apoptosis under infection conditions in which the process of viral DNA synthesis was specifically inhibited. In this experimental situation, productive viral replication will proceed to the point where the factors required for viral DNA replication are produced but where the polymerase activity is blocked. HEp-2 cells were mock, KOS1.1, and d4-5 infected in the presence or absence of 300 µg of phosphonoacetic acid (PAA) per ml. This amount of PAA was previously shown to be sufficient to completely block HSV-1(F) DNA synthesis in infected human cells (5). Whole-cell extracts were prepared at 24 h p.i. and used for immunoblot analyses with anti-PARP, -caspase-3, -ICP27, -ICP4, -TK, -gD, and -gC antibodies (Fig. 8).

View larger version (43K): [in this window] [in a new window]   FIG. 8.   Viral protein accumulation (A) and immunoblot detection of cellular death factor processing (B) in mock (M)-, KOS1.1-, and d4-5-infected HEp-2 cells in the presence (+) or absence () of PAA (300 µg/ml). Whole-cell extracts prepared at 24 h p.i. were used for immunoblot analyses with anti-PARP and anti-caspase-3 antibodies or with anti-ICP27 and anti-ICP4 (IE proteins), anti-TK (E protein), anti-gD (L protein, 1), and anti-gC (L protein, 2) antibodies as described in Materials and Methods. 116 and 85 refer to full-length and processed PARP, respectively.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We previously reported that a recombinant HSV-1 containing a complete deletion of the ICP27 gene was able to induce but not prevent apoptosis in human HEp-2 cells during infection (1). At least two models exist to explain these findings. (i) ICP27 itself may directly inhibit apoptosis. (ii) Alternatively, or additionally, ICP27 may induce the expression of other viral or cellular "preventers" of apoptosis. The goal of this project was to use a series of recombinant ICP27 mutant viruses to determine which features of the HSV-1 replication cycle are required for its inhibitory activity. In addition, these mutant viruses can provide novel information on the regulatory functions of ICP27 in infected human HEp-2 cells. The significant findings of our study can be summarized as follows.

Significant findings. (i) Recombinant ICP27 mutant viruses exhibit apoptotic, partially apoptotic, or nonapoptotic phenotypes in HEp-2 cells. Since ICP27-null viruses are completely unable to prevent apoptosis during infection, it was important to quantitatively determine whether various recombinant viruses mutated in specific regions of ICP27 could inhibit the process. To attain this goal, it was necessary for us to develop assays which would allow us to accurately quantitate the extent of this complex biological process. The phenomenon of apoptosis in human cells was originally defined in structural terms (29, 68). The defining features of the process are chromatin condensation (pyknosis), fragmentation of nuclei (karyorhexis), specific nucleosomal laddering, membrane blebbing, and the formation of apoptotic bodies. Apoptosis is currently recognized as resulting from regulated proteolysis (11, 17, 28, 31, 39, 54). When developing assays for measuring apoptosis during HSV-1 infection it was necessary to avoid potentially misleading results due to consequences of viral replication, such as the generation of substrates for terminal deoxynucleotidyl transferase (i.e., TUNEL) by random nicking of cellular DNA. For this reason we chose three complementary techniques based on changes in endogenous cellular components. We previously reported that microscopic inspection of ICP27-null-virus-infected HEp-2 cells reveals cell shrinkage, membrane blebbing, and the formation of apoptotic bodies (1). This technique was used to make preliminary classifications of the viruses. These original assignments were confirmed by quantitation of cells presenting condensed chromatin following infection: nonapoptotic, below 20%; partially apoptotic, between 20 and 50%; apoptotic, greater than 50%. Finally, our placement of the viruses in each group was substantiated by assays designed to detect the endogenous levels of cellular death factor processing.

(ii) Apoptosis prevention requires that HSV-1 infection proceed to the stage in which viral DNA replication takes place. Our experiments demonstrate that the viruses defined as nonapoptotic are those which are able to synthesize their DNA to some extent in HEp-2 cells. However, since there is at least a 10-fold variation in the amounts of the DNA replicated by these viruses, it appears that the magnitude of DNA synthesis is not a major determinant of apoptosis prevention. The more likely possibility is that apoptosis prevention requires that HSV-1 infection proceed to a point at which the factors necessary for viral DNA replication have accumulated. To confirm this hypothesis, we performed infections with the nonapoptotic KOS1.1 and d4-5 viruses in the presence of the DNA synthesis inhibitor PAA. The absence of apoptosis under these conditions confirmed that it is not the act of viral DNA synthesis itself which is required for apoptosis prevention. Since the expression of viral ₂ genes is absolutely dependent on viral DNA synthesis, the results also demonstrated that the accumulation of true late gene products is not required for the prevention of apoptosis.

(iii) Accumulation of viral early and leaky-late proteins correlates with prevention of apoptosis. Having grouped all viruses based on their apoptotic potentials, we attempted to correlate the apoptotic phenotypes of the viruses with their abilities to produce HSV-1 proteins. Our goal was to define more precisely which class of viral gene products are associated with apoptosis prevention. The apoptotic viruses d27-1, d1-5, d1-2, n263R, and n59R had reduced levels of almost all of the viral proteins examined. In contrast, the apoptotic or partially apoptotic viruses M11, M15, M16, n504R, and n406R produced high levels of ICP4 and ICP22, reduced amounts of TK, VP16, and gD, but very little if any gC. It is noteworthy that these findings indicate that the protein reductions observed with apoptotic viruses are not simply due to loss of material as a result of cell death since wild-type levels of some viral proteins can be detected. Overall, there was a very good correlation between apoptosis prevention and the accumulation of early/leaky-late gene products TK, VP16, and gD. However, this conclusion remains a correlation and it must be emphasized that it is still possible that ICP27 is acting directly as well. These results suggest that in order for HSV-1 to prevent apoptosis, it must efficiently transit from the IE to E phase of replication.

(iv) ICP27 contains regions which are not required for prevention of apoptosis. The series of ICP27 mutants helps us to define the regions of ICP27 needed for apoptosis prevention as well as those regions which play minor or no roles. Our results indicate that a large region in ICP27 corresponding to amino acids 64 to 200 does not play a significant role in ICP27's ability to block apoptosis. This region is defined by recombinant viruses d2-3, d3-4, d4-5, d5-6, and d6-7, which were all found to be nonapoptotic. Of this group, only d4-5 has a significant growth defect in Vero cells. The mutation in d3-4 affects the ICP27 NLS (Fig. 1) and, consequently, increased amounts of ICP27 can be detected in the cytoplasm (36). However, significant ICP27 enters the nucleus in d3-4-infected cells due to nuclear localization signals present in the carboxy-terminal half of the protein (36), and the virus replicates in Vero cells (Table 2). It is noteworthy that the sequences deleted in d3-4 and d4-5 make up ICP27's NuLS (Fig. 1). We can thus conclude that nucleolar localization is not required for apoptosis prevention. The functions of the regions mutated in d2-3, d5-6, and d6-7 remain unknown. Interestingly, while d2-3 and d3-4 produced wild-type levels of gC, ICP22 accumulations were reduced with these viruses. This supports our previous observation that while ICP22 may be involved in apoptosis prevention, it does not play a dominant role (2).

(v) The ICP27 RGG box is required for gC production but not for apoptosis prevention. It is interesting that the nonapoptotic virus d4-5 has a deletion of the sequences encoding the RGG box region, which was shown to be (i) involved in RNA binding in vitro (38) and in vivo (56) and (ii) required for efficient viral growth in Vero cells (Table 2 [36]). Therefore, these results suggest that ICP27's direct interaction with RNA is not necessary for apoptosis prevention. However, we cannot exclude the possibility that additional potential RNA binding sites in ICP27 such as K homology-like domains (61, 62) might compensate for the loss of the RNA binding function of the RGG box. We also found that the RGG box region is not required for the efficient synthesis of ICP4, ICP22, TK, VP16, or gD. However, it is required for the efficient production of the true late gC protein, suggesting that ICP27's RNA binding activity is required for the induction of true late genes (48, 53). Since d4-5 prevents apoptosis but cannot synthesize gC, the results obtained using this virus functionally separate these two activities of ICP27.

(vi) ICP27's ability to prevent apoptosis requires functional regions in both the amino- and carboxy-terminal halves of the protein. Vero cells have the distinction of being unable to undergo apoptosis following ICP27-null virus infection (1). Thus, virus growth in Vero cells is an ideal assay to assess the consequences of ICP27 mutations on viral replication since infection occurs in a nonapoptotic cellular environment. For example, previous studies by one of us using Vero cells demonstrated that the M11, M15, M16, and n504R viruses replicate viral DNA (Table 1) but do not exhibit gC gene expression (48, 49). We have now compared the growth of various ICP27 mutants in Vero cells with their apoptotic potential in HEp-2 cells. With one exception, ICP27 mutants which are defective for plaque formation in Vero cells are also unable to mediate the activity which prevents apoptosis. The exception is d4-5, discussed above (sections iv and v). The apoptotic mutants included those with alterations in both the amino-terminal (e.g., d1-2 and d1-5) and carboxy-terminal (e.g., M15 and n504R) parts of the protein . The mutations in d1-5 and d1-2 affect the ICP27 NES, leading to defects in shuttling, and the acidic regulation domain (50). Recent evidence indicates that this region also contains an export control sequence which appears to regulate the function of the NES (62). It is noteworthy that d1-5 has a more pronounced apoptotic phenotype than d1-2, suggesting that residues 63 to 152 (Table 1) play a role in apoptosis prevention under some circumstances. All of the mutations which affect the carboxy-terminal portion of ICP27 resulted in a loss of the ability to prevent apoptosis. This is perhaps not surprising, as many previous data suggest that important functional regions lie in this domain. Interestingly, M11, M15, and M16 are deficient for ICP27 shuttling between the nucleus and cytoplasm of infected cells (37). ICP27 shuttling seems to be an event which occurs late in infection and plays a role in regulating late genes by facilitating export of mRNAs (43, 56, 60). However, since n504R still allows ICP27 to shuttle (37) and does not block apoptosis, the role of ICP27 shuttling in apoptosis prevention remains unclear.

(vii) Definition of apoptosis during HSV-1 infection and proposal of a model for apoptosis prevention by HSV-1. We found that all HEp-2 monolayers, including mock-infected ones, display some cells with distinguishing cell morphologies indicative of apoptosis. To differentiate between the phenotypes of apoptotic, partially apoptotic, and nonapoptotic mutants, it was therefore important to quantitatively determine the proportion of cells with apoptotic features. It should be noted that we use MOIs of at least 5 PFU/cell, and therefore all cells in a given monolayer are infected. Viruses which are not able to prevent apoptosis (apoptotic or partially apoptotic) trigger greater than 30% of the cell population to have condensed chromatin. Extracts made from cells infected by these viruses display a significant level of processing of cellular death factors. In contrast, nonapoptotic viruses generate less than 20% apoptotic cells. For comparison, wild-type KOS1.1 infection produces 8% apoptotic cells in this system. Viral features which do not seem to be associated with apoptosis prevention include accumulations of ICP4 and ICP22, the act of viral DNA synthesis, and the production of true late proteins.

View larger version (32K): [in this window] [in a new window]   FIG. 9.   Schematic representation of apoptosis during productive HSV-1 infection. HSV-1 replication occurs in a sequentially ordered cascade (23, 52). Following virion binding and fusion at the cell surface, tegument dissociation, capsid translocation to the nuclear pore, viral DNA release, and circularization in the nucleus,  gene expression occurs. Viral DNA synthesis begins after  gene expression. Expression of 2 genes is distinguished from that of 1 genes by its absolute dependence on viral DNA synthesis. Infection of HEp-2 cells with HSV-1 leads to the activation of caspase-3. Under conditions in which de novo viral protein synthesis is blocked (e.g., by addition of cycloheximide) or ICP27 is absent, activated caspase-3 cleaves cytoplasmic and nuclear substrates leading to the classic structural features of apoptosis, including chromatin condensation, nuclear fragmentation, nucleosomal DNA laddering, membrane blebbing, and the formation of apoptotic bodies (1). During infection with wild-type HSV-1, infected cell proteins synthesized between 3 and 6 h p.i. are capable of preventing the process from killing the cells (2). The data in this study indicate that the accumulation of viral early () and leaky-late (1) proteins correlates with the prevention activity.