Analysis of the DNA polymerase promoter of human herpesvirus-6
Abstract
Human herpesvirus-6 (HHV-6) is a recently described T-cell pathogen whose medical relevance and molecular biology are just beginning to be addressed. As a first look at the regulation of viral genes, control of the HHV-G DNA polymerase promoter was examined. Polymerase gene transcription in HHV-6 infected cells was found to initiate from a single site located 115 bases upstream of the translation start codon. A polymerase promoter-chloramphenicol acetyltransferase (CAT) reporter gene construct failed to be expressed in uninfected T-cells but was highly active in HHV-6 infected cells. Mutational data indicated that the polymerase promoter is TATA-less. Mutational analysis also revealed that the major upstream promoter regulatory element required for transcriptional activity in HHV-6 infected cells is a palindromic ATF/CREB transcription factor binding site. An adjacent purine box does not appear to contribute significantly to activity. The significance of the ATF/CREB site for promoter induction was further demonstrated by the fact that the polymerase ATF/CREB element, when appended to a heterologous basal promoter, is highly responsive to HHV-6 infection. Two protein complexes were found to bind in a specific manner to the ATF/CREB motif in both uninfected and HHV-6 infected T-cell nuclear extracts. One of these complexes was shown to contain ATF-2 or an ATF-2 like protein. Site specific mutation of the ATF/CREB site resulted in loss of protein binding as well as loss of promoter activity in HHV-6 infected cells, suggesting a requirement for protein binding for promoter function. Multiple specific protein complexes were found to bind to the purine box motif in both uninfected and infected cells. A cDNA encoding a 44 kd protein which binds to the purine box was cloned.
Subject Area
Molecular biology|Immunology
Recommended Citation
Agulnick, Alan David, "Analysis of the DNA polymerase promoter of human herpesvirus-6" (1994). Dissertations available from ProQuest. AAI9427488.
https://repository.upenn.edu/dissertations/AAI9427488