MECHANISTIC APPROACHES TO HSV-2 INDUCED TRANSFORMATION

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.