MECHANISTIC APPROACHES TO HSV-2 INDUCED TRANSFORMATION

点击翻译按钮获取中文摘要

Herpes simplex virus type 2 (HSV-2) is a large DNA virus which infects humans and is spread primarily through sexual contact. Cervical carcinoma occurs most frequently in promiscuous women or in women with promiscuous partners. Furthermore, HSV-2 antigens and nucleic acids are detected more frequently in women with cervical cancer than in those without. Thus, HSV-2 may be either the causative agent of cervical cancer or one of several cofactors required to manifest the transformed state. The BamHI-E fragment of HSV-2 induces the neoplastic transformation of established rodent cells and the minimal transforming region of BamHI-E (mtrIII) maps to a 486 base pair fragment. Transformation is mediated by DNA sequence motifs in mtrIII which resemble elements controlling recombination and transcription and not the expression of a viral protein. The questions asked in this proposal are intended to delineate the mechanism of HSV-2 induced transformation. Do cellular proto- oncogenes play a role in HSV-2 mediated transformation? To answer this question, the steady state levels of RNA in transformed cells will be compared to that in normal cells. Do the transcriptional regulatory sequences in mtrIII play a key role in HSV-2 mediated transformation and gene expression? A reporter gene, the bacterial chloramphenicol acetyltransferase gene, will be utilized to correlate the transcriptional enhancer activity of mtrIII with transformation and the expression of HSV-2 genes. Do potential stem-loop structures and DNA conformation in mtrIII mediate neoplastic transformation? Site-specific mutagenesis of a stem-loop structure in mtrIII and the effects of such mutations on transformation will be analyzed. The regions of mtrIII that have altered DNA conformation will be mapped using specific chemical probes and correlated with transformation. Do cells transformed by HSV-2 retain mtrIII DNA sequences? Transformed cells will be analyzed for integrated HSV-2 sequences by Southern blotting experiments and episomal elements which contain HSV-2 DNA will be identified and analyzed in transformed cells. Does HSV-2 posses transforming potential in its natural host, the human? The transforming potential of HSV-2 will be assayed in several human cell lines. In light of evidence which implicates HSV-2 as a causative agent of cervical cancer, these studies will define a structure/function relationship of a novel transforming domain and identify cellular genes that are altered during transformation.