The herpes simplex virus type 1 regulatory protein ICP27 is required for the prevention of apoptosis in infected human cells

Abstract

The herpes simplex virus type 1 (HSV-1) ICP27 protein is an immediate-early or alpha protein which is essential for the optimal expression of late genes as well as the synthesis of viral DNA in cultures of Vero cells. Our specific goal was to characterize the replication of a virus incapable of synthesizing ICP27 in cultured human cells. We found that infection with an HSV-1 ICP27 deletion virus of at least three separate strains of human cells did not produce immediate-early or late proteins at the levels observed following wild-type virus infections. Cell morphology, chromatin condensation, and genomic DNA fragmentation measurements demonstrated that the human cells died by apoptosis after infection with the ICP27 deletion virus. These features of the apoptosis were identical to those which occur during wild-type infections of human cells when total protein synthesis has been inhibited. Vero cells infected with the ICP27 deletion virus did not exhibit any of the features of apoptosis. Based on these results, we conclude that while HSV-1 infection likely induced apoptosis in all cells, viral evasion of the response differed among the cells tested in this study.

FIG. 1

Morphologies of infected nonhuman (A to H) Vero 2.2 and Vero cells and human (I to T) 143tk⁻, HEp-2, and HeLa cell lines. Cells infected with vBSΔ27 (A, B, and I to K), KOS1.1 (C, D, and L to N), or vBSΔ27R (E, F, and O to Q) and mock-infected cells (G, H, and R to T) were observed at 24 h p.i. by phase-contrast light microscopy (magnification, ×20) as described in Materials and Methods.

FIG. 2

Accumulation of an IE protein (ICP22) and an L protein (VP16) in infected nonhuman (A and B) and human (C to E) cell lines. Total cell extracts (50 μg) prepared at 24 h p.i. from mock- (M), vBSΔ27 (Δ27)-, vBSΔ27R (Δ27R)-, and KOS1.1-infected nonhuman Vero, Vero 2.2, and human 143tk⁻, HEp-2, and HeLa cells were used for immunoblot analyses with the polyclonal anti-ICP22 antibody RGST22 and the monoclonal anti-VP16 antibody as described in Materials and Methods. The bars indicate the multiple electrophoretic forms of ICP22.

FIG. 3

Accumulation of IE and L proteins in infected cells at various infection times. Total cell extracts (50 μg) prepared at 6, 10, 12, and 24 h p.i. from vBSΔ27- and KOS1.1-infected HEp-2 (A) and Vero (B) cells were used for immunoblot analyses with the polyclonal anti-ICP22 antibody (RGST22), monoclonal anti-ICP4 (1114), anti-ICP27 (1113), anti-ICP0 (1112) antibodies (IE proteins), and the monoclonal anti-VP16 antibody (L protein). IE and L protein locations are shown in the right margins (arrows), and the bars indicate the multiple electrophoretic forms of ICP22.

FIG. 4

Morphologic changes during the course of infection in HEp-2 cells. Phase-contrast images of HEp-2 cells infected with vBSΔ27 (A to E) and KOS1.1 (F to J) at 6, 9, 12, 15, and 24 h p.i. were shown. Images of mock-infected cells (K and L) are shown at 6 and 24 h p.i. only. The arrows mark cells possessing the small, irregular phenotypes described in the text. Magnification, ×19.4.

FIG. 5

Agarose gel electrophoresis of low-molecular-weight DNA extracted from infected HEp-2 cells. The DNAs were separated in a 1.5% agarose gel and stained with ethidium bromide after extraction at 6, 9, 12, 15, and 24 h p.i. from vBSΔ27 (Δ)- or KOS1.1 (K)-infected HEp-2 cells and at 24 h p.i. from mock (M)-infected HEp-2 cells as described in Materials and Methods. The locations of 1.2- and 0.7-kb markers are shown in the left margin.

FIG. 6

Fluorescent visualization of infected HEp-2 cell DNA. Fluorescent images (Hoechst) and corresponding phase-contrast images (Phase) of HEp-2 cells at 10 or 24 h after infection with vBSΔ27 or KOS1.1 and 24 h after mock infection. The infected cells were stained with the Hoechst H33258 DNA dye as described in Materials and Methods. Yellow arrows, condensed chromatin; red arrow, marginal chromatin. Fluorescent and phase-contrast microscopy magnification, ×20.

FIG. 7

Morphologies of HEp-2 and Vero cells at 24 h p.i. in the absence (−) or presence (+) of the protein synthesis inhibitor CHX. Phase-contrast images of HEp-2 (A to H) and Vero (I to P) cells mock infected or infected with KOS1.1, vBSΔ27, and vBSΔ27R are shown. Magnification, ×20. The CHX concentrations were 10 μg/ml for HEp-2 and 100 μg/ml for Vero cells.

FIG. 8

Agarose gel electrophoresis of low-molecular-weight DNA (A and B) and morphologies (C to H) of HEp-2 cells infected in the presence of CHX. The DNAs were extracted at 6 and 15 h p.i. from vBSΔ27 (Δ27)-, KOS1.1-, or mock (M)-infected HEp-2 cells in the presence of 10 μg of CHX/ml and separated in 1.5% agarose gels. Phase-contrast images of the corresponding infected HEp-2 cells were taken prior to the DNA extractions. Magnification, ×20.