This is an application to study control of p53 transcription using a unique new system. The applicant's laboratory has discovered that a transcription factor from a malignant rabbit myxoma virus, namely C7, regulates p53 mRNA levels and negatively affects cell proliferation, notably of lymphocytes. C7 is encoded by a gene present in malignant rabbit fibroma virus, MV. MV was first identified in the applicant's laboratory as a contaminant of Shope Fibroma Virus (SFV) stocks. MV is a recombinant of SFV and rabbit myxoma virus (MYX). MV, MYX, and SFV are rabbit viruses which do not infect human cells. In contrast to SFV infection which has a benign course, MV infection leads to rapid death in rabbits. When MV's C7 gene is transferred to SFV, replacing its counterpart D7, the resulting recombinant virus mimics MV. Thus, it is hypothesized that C7 is important in MV's more virulent pathology. It would be of great interest, therefore, to determine the function of C7. When a construct (pKC7) containing C7 under control of SV40 early promoter is transfected into normal human foreskin fibroblasts or rabbit kidney fibroblasts, it elicits a large increase in p53 transcript levels. This increase in p53 reflects new transcription. To explain underlying mechanisms, the applicant proposes two hypotheses: - the C7 transcription factor directly binds the p53 promoter to induce p53 expression, and - the increased p53 expression is functionally significant, i.e., it regulates cell proliferation and cell cycle progression. To test these hypotheses, the following specific aims are proposed. 1. Characterize the effect of C7 protein on p53 expression. In this aim the investigator will quantify the effect of C7 on p53 transcription, measure p53 protein levels after transfection with pKC7 or MV infection, determine p53 half life as a function of C7, identify, clone and sequence the rabbit p53 cDNA, and determine whether the p53 that is expressed in these cells is wildtype p53. 2. Raise antibodies to C7 and use these antibodies to localize C7 protein. The investigator will use C7 oligopeptide conjugates to elicit antiserum to C7, then use these antibodies to localize C7 in cells transfected with pKC7 or infected with MV (that contains the C7 gene), and measure C7 protein levels with time after pKC7 transfection and MV infection. 3. Assess C7 protein binding to the p53 promoter. The investigator will localize C7 binding sites within the human p53 promoter by DNAase I footprinting, then identify specific p53 promoter sequences bound by C7 using selective nucleotide mutation analysis, clone the p53 promoter into pGL2, making, to drive expression of luciferase, and then use pGL53luc and mutant derivatives of it to characterize C7 regulation of p53. 4. Determine the effect of other common transcriptional regulators on C7 induction of p53. The investigator will test glucocorticoids, cAMP, and retinoic acid effect on p53 in MV-infected cells and in C7-transfected cells. 5. Evaluate the biological significance of the ability of C7 to increase p53 expression. The investigator will examine the conformational state of the p53 protein induced by C7, assess the effect of C7 on cell proliferation and cell cycle progression, and correlate altered cell cycling with p53 conformation in rabbit cells in vitro and in vivo. It is proposed that these experiments will elucidate the mechanism by which the transcription factor, C7, induces p53 expression, contributes to viral pathology.
| Wali, A; Strayer, D S (1996) Regulation of p53 gene expression by a poxviral transcription factor. Virology 224:63-72 |
