Jl Parish

The Human Papillomavirus (HPV) 16 E2 Protein Induces Apoptosis in the Absence of Other HPV Proteins and via a p53-dependent Pathway

The human papillomavirus (HPV) E2 protein regulates viral gene expression and is also required for viral replication. HPV-transformed cells often contain chromosomally integrated copies of the HPV genome in which the viral E2 gene is disrupted. We have shown previously that re-expression of the HPV 16 E2 protein in HPV 16-transformed cells results in cell death via apoptosis. Here we show that the HPV 16 E2 protein can induce apoptosis in both HPV-transformed and non-HPV-transformed cell lines. E2-induced apoptosis is abrogated by a trans-dominant negative mutant of p53 or by overexpression of the HPV 16 E6 protein, but is increased by overexpression of wild-type p53. We show that mutations that block the DNA binding activity of E2 do not impair the ability of this protein to induce apoptosis. In contrast, removal of both N-terminal domains from the E2 dimer completely blocks E2-induced cell death. Heterodimers formed between wild-type E2 and N-terminally deleted E2 proteins also fail to induce cell death. Our data suggest that neither the DNA binding activity of E2 nor other HPV proteins are required for the induction of apoptosis by E2 and that E2-induced cell death occurs via a p53-dependent pathway.

Herpes simplex virus VP22–human papillomavirus E2 fusion proteins produced in mammalian or bacterial cells enter mammalian cells and induce apoptotic cell death

Infection by high‐risk HPV (human papillomavirus) is supposed to be the primary cause of cervical cancer. The HPV E2 protein (E2) is a DNA‐binding protein that regulates viral gene expression and is required for efficient viral replication. Overexpression of the E2 protein in cervical cancer cells can induce growth arrest and/or apoptotic cell death, suggesting that E2 might be useful in the treatment of this disease. In the present study, we show that VP22 (herpes simplex virus VP22 protein) can be used to deliver E2 to target cells. VP22–E2 fusion proteins induce apoptosis in transiently transfected HPV‐transformed cervical carcinoma cell lines. However, VP22–E2 fusion proteins do not kill COS‐7 cells, probably because these cells constitutively express the simian‐virus‐40 T antigen and this protein sequesters the tumour suppressor protein p53. When COS‐7 cells producing VP22–E2 are seeded into cultures of HPV‐transformed cells, VP22–E2 enters the non‐producing cells and induces apoptosis. VP22–E2 proteins produced in bacterial cells can also enter cervical cancer cells and induce apoptosis in a dose‐dependent manner. Our results suggest that local delivery of VP22–E2 fusion proteins could be used to treat cervical cancer and other HPV‐associated diseases.

Human T cell responses to HPV 16 E2 generated with monocyte-derived dendritic cells.

Persistent infection with human papillomavirus (HPV) type 16 has been implicated in the etiology of cervical cancer. The E2 protein is required early in viral infection and therefore may serve as a useful immune target for a vaccine aimed at prevention or therapy of premalignant lesions. Dendritic cells (DC) prepared from monocytes and pulsed with bacterially produced HPV 16 E2 C‐terminus protein were used to stimulate autologous T cells over several rounds of stimulation. T cells were tested for γ‐interferon release by ELISPOT and for cytotoxic activity by 51chromium release assays. To generate E2‐expressing target cells for cytotoxicity assays, we constructed a recombinant vaccinia virus encoding HPV 16 E2, which was used to infect autologous Epstein‐Barr virus‐transformed lymphoblastoid cell lines (LCL). The results show that DC pulsed with E2 C‐terminus protein induce γ‐interferon‐releasing T cells as demonstrated by ELISPOT. Furthermore, we demonstrate E2‐specific lysis of vaccinia‐E2 infected autologous LCL by CD8+ cytotoxic T lymphocytes (CTL). E2‐specific CTL did not lyse untreated autologous LCL or LCL infected with wild‐type vaccinia and showed low levels of cytotoxicity against natural killer cell‐sensitive K562 cells. In addition, T cells stimulated with DC in the absence of E2 failed to demonstrate lysis of vaccinia‐E2‐labeled targets. Phenotypically, CTL populations were CD3+/CD8+. These results will facilitate the study of naturally occurring T‐cell responses to HPV E2 in patients with cervical intraepithelial neoplasia and the development of immunotherapeutic strategies designed to treat this and other HPV‐associated diseases.