ICP0 gene expression is a herpes simplex virus type 1 apoptotic trigger

Abstract

Apoptosis is a highly regulated programmed cell death process which is activated during normal development and by various stimuli, such as viral infection, which disturb cellular metabolism and physiology. That herpes simplex virus type 1 (HSV-1) induces apoptosis but then prevents its killing of infected cells is well-established. However, little is known about the viral factor/event which triggers the apoptotic process. We previously reported that infections with either (i) a temperature-sensitive virus at its nonpermissive temperature which does not inject viral DNA into nuclei or (ii) various UV-inactivated wild-type viruses do not result in the induction of apoptosis (C. M. Sanfilippo, F. N. W. Chirimuuta, and J. A. Blaho, J. Virol. 78:224-239, 2004). This indicates that virus receptor binding/attachment to cells, membrane fusion, virion disassembly/tegument dispersal, virion RNAs, and capsid translocation to nuclei are not responsible for induction and implicates viral immediate-early (IE) gene expression in the process. Here, we systematically evaluated the contribution of each IE gene to the stimulation of apoptosis. Using a series of viruses individually deleted for alpha27, alpha4, and alpha22, we determined that these genes are not required for apoptosis induction but rather that their products play roles in its prevention, likely through regulatory effects. Sole expression of alpha0 acted as an "apoptoxin" that was necessary and sufficient to trigger the cell death cascade. Importantly, results using a recombinant virus which contains a stop codon in alpha0 showed that it was not the ICP0 protein which acted as the apoptotic inducer. Based on these findings, we propose that alpha0 gene expression acts as an initial inducer of apoptosis during HSV-1 infection. This represents the first description of apoptosis induction in infected cells triggered as a result of expression of a single viral gene. Expression of apoptotic viral genes is a unique mechanism through which human pathogens may modulate interactions with their host cells.

FIG. 1.

HSV-1 IE gene expression, but not the ICP27 gene, is required for apoptosis induction. Mock-, HSV-1(KOS)-, d109-, or Δ27-infected (MOI of 5) HEp-2 cell extracts at 24 hpi, untreated (−) or treated with CHX (+), were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with antibodies to viral IE ICP4 and cellular (PARP and caspase-3) proteins as described in Materials and Methods. The anti-caspase-3 antibody used recognizes pro-caspase-3; loss of immune reactivity indicates caspase-3 activation. Under these CHX treatment conditions, no newly synthesized viral proteins were detected, with the exception of the previously described three slow-migrating IE ICP27 (triplet) forms (58). CHX treatment of uninfected cells activates caspase-3, but PARP remains predominantly uncleaved (4); thus, PARP processing during virus infection above that observed with mock infection plus CHX indicates apoptosis. “116” and “85” denote full-length (116,000-molecular-weight) and processed (85,000-molecular-weight) PARP, respectively. Locations of prestained molecular size markers (in kDa) are indicated on the right.

FIG. 2.

The HSV-1 IE ICP4 gene is not required for apoptosis induction. Mock-, HSV-1(17)-, or CgalΔ3-infected (MOI of 5) HEp-2 cells, untreated (−) or treated with CHX (+), at 24 hpi were extracted, separated in denaturing gels, transferred to nitrocellulose, and reacted with antibodies to viral (IE ICP4, true-late gC, and IE ICP27) (A) and cellular (PARP) (B) proteins. Locations of prestained molecular size markers (in kDa) are indicated on the right. “116” and “85” denote full-length (116,000-molecular-weight) and processed (85,000-molecular-weight) PARP, respectively. Prior to extraction, cells were stained with Hoechst DNA dye and visualized by phase-contrast (Phase) and fluorescence (Hoechst) microscopy (C) and the percentages of cells with condensed chromatin were determined as described in Materials and Methods. Corresponding Phase and Hoechst panels were overlaid to produce merged images (Overlay). Original magnification, ×40.

FIG. 3.

The HSV-1 IE ICP22 gene is not required for apoptosis induction. Mock-, HSV-1(F)-, or R7802-infected (MOI of 5) HEp-2 cells, untreated (−) or treated with CHX (+), at 24 hpi were extracted, separated in denaturing gels, transferred to nitrocellulose, and reacted with antibodies to viral (IE ICP4, true-late gC, IE ICP22, and leaky-late VP22) (A) and cellular (PARP) (B) proteins. Locations of prestained molecular size markers (in kDa) are indicated on the right. “116” and “85” denote full-length (116,000-molecular-weight) and processed (85,000-molecular-weight) PARP, respectively. Prior to extraction, cells were stained with Hoechst DNA dye and visualized by phase-contrast (Phase) and fluorescence (Hoechst) microscopy (C) and the percentages of cells with condensed chromatin were determined as described in Materials and Methods. Corresponding Phase and Hoechst panels were overlaid to produce merged images (Overlay). Original magnification, ×40.

FIG. 4.

Expression of the IE ICP0 gene is necessary for the induction of apoptosis during infection by HSV-1. (A) Cell extracts at 24 hpi from mock-, HSV-1(KOS)-, 7134-, or Δ27-infected HEp-2 cells, untreated (−) or treated with CHX (+), were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with antibodies to viral (IE ICP4, IE ICP0, IE ICP27, and leaky-late VP22) proteins. Slight amounts of incoming virion VP22 were detected in the presence of CHX. (B) Mock-, HSV-1(KOS)-, 7134-, or Δ27-infected cell proteins were immunoblotted with antibodies to cellular (PARP and pro-caspase-3) proteins. Locations of prestained molecular size markers (in kDa) are indicated on the right. “116” and “85” denote full-length (116,000-molecular-weight) and processed (85,000-molecular-weight) PARP, respectively. (C) The cells used to prepare the extracts shown in panels A and B were also examined by phase-contrast microscopy (Phase) to observe morphological changes. Staining of nuclear chromatin with Hoechst 33258 was visualized by fluorescence microscopy (Hoechst). Corresponding Phase and Hoechst panels were overlaid to produce merged images (Overlay). The exact numbers of cells with condensed chromatin and fragmented nuclei (2) were determined as described in Materials and Methods. Original magnification, ×40.

FIG. 5.

Sole expression of the IE ICP0 gene during HSV-1 infection is sufficient to induce apoptosis. (A) Cell extracts at 24 hpi from mock-, HSV-1(KOS)-, d109-, d106-, or Δ27-infected HEp-2 cells, untreated (−) or treated with CHX (+), were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with antibodies to viral (IE ICP4, IE ICP0, and leaky-late VP22) proteins. (B) Mock-, HSV-1(KOS)-, d109-, d106-, or Δ27-infected cell extracts were reacted with antibodies to cellular (PARP and pro-caspase-3) proteins. Locations of prestained molecular size markers (in kDa) are indicated on the right. “116” and “85” denote full-length (116,000-molecular-weight) and processed (85,000-molecular-weight) PARP, respectively. (C) Expression of GFP in the infected cells (Without CHX) used to prepare the extracts shown in panels A and B was visualized by fluorescence microscopy. Since both d109 and d106 synthesize GFP, but only d106 triggers apoptosis, GFP expression does not trigger apoptosis. (D) The cells used to prepare the extracts shown in panels A and B were examined by phase-contrast microscopy (Phase) to observe morphological changes. Staining of nuclear chromatin with Hoechst 33258 was visualized by fluorescence microscopy (Hoechst). The exact numbers of cells with condensed chromatin and fragmented nuclei (2) were determined as described in Materials and Methods. Original magnification, ×40.

FIG. 6.

Apoptosis in ICP0-transfected, d109-infected HEp-2 cells. HEp-2 cells were transfected with 2.5 μg of pUC (empty vector) or pSH (ICP0) plasmid and mock infected or infected with HSV-1(d109) at 24 h posttransfection. Whole-cell extracts were prepared at 24 hpi, separated in a denaturing gel, transferred to nitrocellulose, and probed with anti-ICP0, -actin, and -PARP 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.

FIG. 7.

Apoptosis in ICP0-transfected HEp-2 cells. (A) Immune reactivities of transfected cell proteins and (B) cell morphologies in HEp-2 cells transfected with 0.625, 1.25, 2.5, 5.0, or 7.5 μg of pSH (ICP0) or 7.5 μg of pUC (empty vector) plasmid. Whole-cell extracts were prepared at 24 h posttransfection, separated in a denaturing gel, transferred to nitrocellulose, and probed with an anti-ICP0 antibody (A) as described in Materials and Methods. (B) Phase-contrast (Phase), corresponding fluorescence (Hoechst), and merged (Overlay) images of pSH- and pUC-transfected cells were visualized at 24 h posttransfection as described in Materials and Methods. These cells were used to prepare the whole-cell extracts shown in panel A. The percentages of cells showing apoptotic condensed chromatin are shown. hpt, hours posttransfection. Original magnification, ×40.

FIG. 8.

Expression of the IE ICP0 gene, not the ICP0 protein, is necessary for the induction of apoptosis during infection by HSV-1. (A) Cell extracts at 24 hpi from mock-, HSV-1(KOS)-, 7134-, or n212-infected HEp-2 cells, untreated (−) or treated with CHX (+), were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with antibodies to viral (IE ICP4 and IE ICP0) and cellular (PARP) (B) proteins. Locations of prestained molecular size markers (in kDa) are indicated on the right. “116” and “85” denote full-length (116,000-molecular-weight) and processed (85,000-molecular-weight) PARP, respectively. (C) The cells used to prepare the extracts shown in panels A and B were also examined by phase-contrast microscopy (Phase) to observe morphological changes. Staining of nuclear chromatin with Hoechst 33258 was visualized by fluorescence microscopy (Hoechst). Corresponding Phase and Hoechst panels were overlaid to produce merged images (Overlay). The exact numbers of cells with condensed chromatin and fragmented nuclei (2) were determined as described in Materials and Methods. Original magnification, ×40.