p53 and hTERT determine sensitivity to viral apoptosis.

Apoptosis is a potent host defense against microbes. Most viruses have adapted strategies to counteract this response. Herpes simplex virus (HSV) produces a balance between pro- and antiapoptotic processes during infection. When antiapoptotic signals become limiting, infected cells die through HSV-dependent apoptosis (HDAP). Oncogenic pathways were previously implicated in HDAP susceptibility. Here, we exploited our ability to selectively express all, one, or no oncogenes in the well-defined HeLa cell system to dissect the requirements for HDAP. Human papillomavirus E6 and E7 oncogene expression was inhibited by the E2 viral repressor. Sole expression of E6 mediated HDAP sensitization. Next, two known cellular targets of E6 were independently modulated. This demonstrated that E6 sensitizes HeLa cells to HDAP through hTERT and p53. Given the universality of the apoptotic antiviral response, p53 and telomerase regulation will likely be important for counteracting host defenses in many other viral infections.

Caspase 3 activation during herpes simplex virus 1 infection.

During herpes simplex virus 1 (HSV-1) infection, apoptosis is initiated by immediate early gene transcription and is later modulated by proteins synthesized in infected cells. We have previously shown that procaspase 3 levels are reduced during HSV-1 replication. We now demonstrate that a replication-defective HSV-1 recombinant virus which is incapable of packaging viral DNA into capsids activated caspase 3 but retained the ability to prevent the apoptotic process from killing the infected cells. This implies that HSV-1-dependent apoptosis is not merely a response to abortive infection. Maximum accumulation of the active form of caspase 3 accompanied complete HSV-1-dependent apoptosis. Additionally, caspase 7 was found to be activated during HSV-1-dependent apoptosis. Infected MCF-7 cells which ectopically express caspase 3 underwent more efficient apoptosis than their caspase 3-null parental counterparts, confirming that caspase 3 contributes to HSV-1-dependent apoptosis. However, caspase 3 reconstitution did not make the MCF-7 cells as sensitive as HEp-2 cells to HSV-1-dependent apoptosis, suggesting that other cellular factors may be involved in conferring resistance to this process. These results indicate that caspase 3 activation is a consequence of HSV-1 infection and have important implications in our understanding of the interactions of the virus with host cells.

Susceptibility of cancer cells to herpes simplex virus-dependent apoptosis.

Apoptosis has recently been associated with herpes simplex virus 1 (HSV-1) latency and disease severity. There is an intricate balance between pro- and anti-apoptotic processes during HSV-1 infection. When anti-apoptotic pathways are suppressed, this balance is upset and the cells die by apoptosis, referred to here as HSV-1-dependent apoptosis (HDAP). It has been observed previously that HeLa cancer cells exhibit an enhanced sensitivity to HDAP. Here, a series of specific patient-derived cancer cells was utilized to investigate the cell-type specificity of HDAP. The results showed that a human mammary tumour cell line was sensitive to HDAP, whilst syngeneic normal cells were resistant. Furthermore, low-passage-number primary human mammary epithelial cells were resistant to HDAP. When the susceptibility of human colon, brain, breast and cervical cancer cells was assessed, the only cells insensitive to HDAP were those resistant to all environmental stimuli tested. This implies that the HDAP resistance was probably due to mutations in the cellular apoptotic machinery. Thus, the susceptibility of cancer cells to HDAP requires that they possess a functional ability to undergo programmed cell death.

African green monkey kidney Vero cells require de novo protein synthesis for efficient herpes simplex virus 1-dependent apoptosis.

During HSV-1 infection, IE gene expression triggers apoptosis, but subsequent synthesis of infected cell proteins blocks apoptotic death from ensuing. This "HSV-1-dependent" apoptosis was identified in HEp-2/HeLa cells infected with wild-type HSV-1 in the presence of an inhibitor of protein synthesis or a virus lacking ICP27 {HSV-1(vBSDelta27)}. Unlike HEp-2/HeLa cells, vBSDelta27-infected Vero cells fail to exhibit dramatic apoptotic morphologies at times prior to 24 hpi. Here, we examined the basis of these different apoptotic responses to HSV-1. We found that infected Vero cells take substantially longer than HEp-2/HeLa cells to display membrane blebbing, chromatin condensation, DNA laddering, and PARP cleavage. Vero, but not HEp-2/HeLa, cells required de novo protein synthesis to exhibit efficient HSV-1-dependent apoptosis, which included changes in mitochondrial membrane potential, and these factors were produced prior to 3 hpi. Vero cells infected with recombinant viruses devoid of the ICP27 and ICP4 proteins alone or both the ICP27 and ICP22 proteins were apoptotic. These results indicate a requirement for cellular or other viral protein synthesis in Vero cells and provide insight into cell type differences in HSV-1-dependent apoptosis.