Herpes simplex virus type 1 renders infected cells resistant to cytotoxic T-lymphocyte-induced apoptosis

Abstract

Many viruses interfere with apoptosis of infected cells, presumably preventing cellular apoptosis as a direct response to viral infection. Since cytotoxic T lymphocytes (CTL) induce apoptosis of infected cells as part of the "lethal hit," inhibition of apoptosis could represent an effective immune evasion strategy. We report here herpes simplex virus type 1 (HSV-1) interference with CTL-induced apoptosis of infected cells and show that HSV-1 inhibits the nuclear manifestations of apoptosis but not the membrane changes. The HL-60 cell line (human promyelocytic leukemia) undergoes apoptosis in response to many stimuli, including incubation with ethanol. After HSV-1 infection (strains E115 and 17+), ethanol-treated cells did not produce oligonucleosomal DNA fragments characteristic of apoptosis, as assayed by gel electrophoresis and enzyme-linked immunosorbent assay. Inhibition was detected 2 h after infection and increased over time. Importantly, HSV-1-infected cells were resistant to apoptosis induced by antigen-specific CD4+ CTL, despite the fact that CTL recognition and degranulation in response to infected targets remained intact. Unlike HSV-1, HSV-2 (strains 333 and HG52) did not inhibit DNA fragmentation. In contrast to the inhibition of DNA fragmentation by HSV-1, none of the HSV-1 or -2 strains interfered with the ethanol-induced exposure of surface phosphatidylserine characteristic of apoptosis, as determined by annexin V binding. These results demonstrate that genes of HSV-1 inhibit the nuclear manifestations of apoptosis but not the membrane manifestations, suggesting that these may be mediated via separate pathways. They also suggest that HSV-1 inhibition of CTL-induced apoptosis may be an important mechanism of immune evasion.

FIG. 1

HSV-1 inhibits oligonucleosomal DNA fragmentation. HSV-infected or uninfected HL-60 cells were treated with the indicated concentrations of ethanol and assayed for oligonucleosomal DNA fragmentation by agarose gel electrophoresis. M, DNA molecular size marker.

FIG. 2

Inhibition of oligonucleosomal DNA fragmentation by HSV-1 strains E115 and 17+ and lack of inhibition by HSV-2 strain 333. HL-60 cells were infected with HSV-1 strain E115 for 13 h (A), HSV-1 strain E115 for 2 h (B), HSV-1 strain E115 for 15 min (C), HSV-1 strain 17+ for 5 h (D), HSV-2 strain 333 for 5 h (E) prior to apoptosis induction with ethanol (EtOH). Squares, uninfected control cells; diamonds, HSV-infected cells. O.D. 405/490; ratio of optical density at 405 nm to that at 490 nm.

FIG. 3

Neither HSV-1 nor HSV-2 inhibits the membrane PS exposure associated with apoptosis. HL-60 cells were mock infected or infected with HSV-1 strain E115 or HSV-2 strain 333 for 5 h and then mock treated or treated with 5% ethanol. Annexin V-FITC (x axis)-propidium iodide (y axis) labeling, followed by flow cytometry, was performed as described in the text. Quadrants: 2, necrotic cells; 3, nonnecrotic, PS-negative cells; 4, viable, PS-positive cells. The percentage of nonnecrotic, PS-positive cells is shown in quadrant 4. The small decrease in quadrant 4 cells in HSV-1-infected cells seen in this representative experiment was within the range of error of the assay and was not reproducible in multiple repeat experiments.

FIG. 4

Effects of HSV infection on DNA fragmentation induced by CTL clones ESL3.326 and 1A.B.25. A, clone ESL3.326 versus targets infected with HSV-1 strain E115 at 33 PFU/cell; B, clone ESL3.326 versus targets infected with HSV-1 strain E115 at 10 PFU/cell; C, clone ESL3.326 versus targets infected with HSV-2 strain 333 at 10 PFU/cell; D, clone 1A.B.25 versus targets infected with HSV-1 strain E115 at 10 PFU/cell; E, clone 1A.B.25 versus targets infected with UV-inactivated HSV-1 strain E115 at 10 PFU/cell; F, clone 1A.B.25 versus targets infected with HSV-2 strain 333 at 10 PFU/cell; G, clone 1A.B.25 versus targets infected with HSV-2 strain 333 at 33 PFU/cell. Squares, uninfected control cells; diamonds, HSV-infected cells. E:T ratio, effector-to-target cell ratio.

FIG. 5

HSV infection of targets does not interfere with recognition by CTL. Mock- or HSV-infected, peptide-pulsed LCL targets were incubated with CTL clone 3.326 at a 20:1 effector-to-target cell ratio. Supernatant was collected 5 h later, and CTL degranulation was measured by BLT esterase assay. O.D., optical density.

FIG. 6

Lack of inhibition of DNA fragmentation induced in target cell line YAR by CTL clone ESL4.34. Neither HSV-1 strain E115 (10 PFU/cell) (A) nor HSV-2 strain 333 (10 PFU/cell) (B) inhibited fragmentation induced by this clone. HSV-1 strain E115 (10 PFU/cell) (C) inhibited UV-induced DNA fragmentation in YAR cells. Cells were UV irradiated (30-W source at 10 cm) for the indicated times. Squares, uninfected control cells; diamonds, HSV-1 infected cells. E:T ratio, effector-to-target cell ratio.