Herpes simplex virus 1 blocks caspase-3-independent and caspase-dependent pathways to cell death - PubMed (original) (raw)

Herpes simplex virus 1 blocks caspase-3-independent and caspase-dependent pathways to cell death

V Galvan et al. J Virol. 1999 Apr.

Abstract

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type HSV blocks the execution of the cell death program triggered by viral gene products, by the effectors of the immune system such as the Fas and tumor necrosis factor pathways, or by nonspecific stress agents such as either osmotic shock induced by sorbitol or thermal shock. A report from this laboratory showed that caspase inhibitors do not block DNA fragmentation induced by infection with the HSV-1 d120 mutant. To identify the events in programmed cell death induced and blocked by HSV-1, we examined cells infected with wild-type virus or the d120 mutant or cells infected and exposed to sorbitol. We report that: (i) the HSV-1 d120 mutant induced apoptosis by a caspase-3-independent pathway inasmuch as caspase 3 was not activated and DNA fragmentation was not blocked by caspase inhibitors even though the virus caused cytochrome c release and depolarization of the inner mitochondrial membrane. (ii) Cells infected with wild-type HSV-1 exhibited none of the manifestations associated with programmed cell death assayed in these studies. (iii) Uninfected cells exposed to osmotic shock succumbed to caspase-dependent apoptosis inasmuch as cytochrome c was released, the inner mitochondrial potential was lost, caspase-3 was activated, and chromosomal DNA was fragmented. (iv) Although caspase-3 was activated in cells infected with wild-type HSV-1 and exposed to sorbitol, cytochrome c outflow, depolarization of the inner mitochondrial membrane, and DNA fragmentation were blocked. We conclude that although d120 induces apoptosis by a caspase-3-independent pathway, the wild-type virus blocks apoptosis induced by this pathway and also blocks the caspase-dependent pathway induced by osmotic shock. The block in the caspase-dependent pathway may occur downstream of caspase-3 activation.

PubMed Disclaimer

Figures

FIG. 1

DEVDase activity in cells exposed to various concentrations of sorbitol. SK-N-SH cells were exposed to various concentrations of sorbitol as shown for 2 h and then incubated for 5 h in DMEM containing 1% fetal bovine serum. Cell extracts were assayed for DEVDase activity as described in Materials and Methods. The colorimetric reaction was monitored by measuring the absorbance at 405 nm. DNA fragmentation was observed only in cells treated with the two highest concentrations tested (data not shown).

FIG. 2

Photograph of agarose gel containing electrophoretically separated low-molecular-weight DNA fragments. SK-N-SH cells were mock infected, exposed to sorbitol, infected with HSV-1(F) or d120 mutant, or infected with HSV-1(F) and exposed to sorbitol as described in the text. Cultures were mock infected or infected with 10 PFU of HSV-1(F) or the _d_120 mutant per cell and collected 6 h after infection. At 1 h after mock infection or infection with 10 PFU of HSV-1(F) per cell, cells were exposed to 1 M sorbitol for 1 h and incubated in DMEM containing 1% fetal bovine serum for 5 h. Cell lysates were assayed for the presence of oligonucleosomal DNA fragments as described in Materials and Methods.

FIG. 3

Time course of DEVDase activity in cell extracts after the addition of substrate. Extracts from infected or uninfected SK-N-SH cells or from SK-N-SH cells exposed to sorbitol were assayed for DEVDase activity at the indicated times after the addition of the colorimetric substrate as described in Materials and Methods. The colorimetric reaction was monitored by measuring the absorbance at 405 nm.

FIG. 4

Time course of DEVDase activity after exposure to sorbitol, wild-type virus, or _d_120 mutant. SK-N-SH cells, either mock infected or infected with HSV-1(F) or the _d_120 mutant or exposed to sorbitol in the presence or absence of the caspase inhibitor Ac-DEVD-CHO were harvested at the times indicated in the figure and assayed for DEVDase activity as described in Materials and Methods. The colorimetric reaction was monitored by measuring the absorbance at 405 nm. Endpoint values of absorbance are shown.

FIG. 5

Photograph of electrophoretically separated cell lysates reacted with an antibody to poly(ADP-ribose) polymerase. Lysates from infected or uninfected HEp-2 cells either left untreated or exposed to sorbitol were electrophoretically separated in a polyacrylamide gel, transferred to a nitrocellulose sheet, and reacted with antibody against poly(ADP-ribose) polymerase (PARP). (A) Cells were treated with sorbitol at 1 h after infection and collected at 2 or 5 h after the addition of sorbitol. (B) Cells were treated at the indicated times after infection and collected at 5 h after the exposure to sorbitol.

FIG. 6

Immunoblot showing cytochrome c distribution in mock-infected, infected, and sorbitol-treated HEp-2 cells. (A) HEp-2 cells were harvested at 16.5 h after mock infection or after infection with viruses as indicated. Cells were exposed to sorbitol for 1.5 h. (B) HEp-2 cells were harvested at 4 h after mock infection or after infection with viruses as indicated. Cells were exposed to sorbitol for 1.5 h. The procedures were as described in Materials and Methods. M, mitochondrial fraction; C, cytosol.

FIG. 7

Distribution of cells according to their Rh-123 fluorescence intensities. (A) Rh-123 fluorescence profiles of mock-infected or HSV-1(F)-infected cells either left untreated or exposed to sorbitol. (B) Rh-123 fluorescence profiles of HSV-1(F)-infected or _d_120-infected HEp-2 cells at 12 and 24 h after infection. The thicker line corresponds to the profile of uninfected cells and is shown as a reference.

FIG. 8

Schematic representation of the manifestations of the programmed cell death assayed in these studies. “Yes” and “no” refer to detection of or failure to detect, respectively, manifestations of intracellular events associated with programmed cell death.

References

1. Adams J, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science. 1998;281:1322–1326. - PubMed
1. Batterson W, Furlong D, Roizman B. Molecular genetics of herpes simplex virus. VIII. Further characterization of a temperature-sensitive mutant defective in release of viral DNA and in other stages of the viral reproductive cycle. J Virol. 1985;54:397–407. - PMC - PubMed
1. Batterson W, Roizman B. Characterization of the herpes simplex virion-associated factor responsible for the induction of α genes. J Virol. 1983;46:371–377. - PMC - PubMed
1. Cory S. Cell death throes. Proc Natl Acad Sci USA. 1998;95:12077–12079. - PMC - PubMed
1. Cryns V, Yuan J. Proteases to die for. Genes Dev. 1998;12:1551–1570. - PubMed

Herpes simplex virus 1 blocks caspase-3-independent and caspase-dependent pathways to cell death - PubMed (original) (raw)