Transcriptional regulation and neoplastic transforming potential of the large subunit of ribonucleotide reductase from herpes simplex virus type 2

Abstract

Herpes simplex virus (HSV) genes are regulated in a cascade of immediate early (IE), delayed early (DE), late (L). The large subunit of ribonucleotide reductase (RR) from HSV-2, designated ICP10, has been grouped with DE proteins. The amino-terminal domain of ICP10 has protein kinase (PK) activity and properties similar to growth factor receptor kinases that can be activated to transforming potential. The studies described in this dissertation sought to develop a better understanding of regulatory aspects of ICP10 regulation as well as the role of ICP10 expression in neoplastic transformation. Regulation of expression of the ICP10 gene was studied by immunofluorescence with the intact ICP10 gene or by chloramphenicol acetyltransferase (CAT) analysis with hybrid ICP10 promoter constructions containing the wild type ICP10 promoter or site-directed mutants deficit in specific cis-response motifs. Co-transfection of these constructions with DNA encoding an HSV nonspecific transactivator (IE110) or an IE gene-specific transactivator (Vmw65), enhanced expression at least 10-fold, regardless of the assay system. In contrast, expression was minimally enhanced by DNA encoding a DE gene transactivator (IE175) at low doses and slightly reduced at high doses. Sequence analysis of the ICP10 promoter revealed the presence of both herpesvirus IE gene-specific (TAATGARAT, GA-rich, and {dollar}\alpha{dollar}H2-{dollar}\alpha{dollar}H3 motifs), as well as cellular cis-response motifs (potential SP-1, consensus AP-1, and octamer transcription factor-1 (OTF-1) binding elements). Factors that bind to the ICP10 promoter were identified by gel retardation analysis with mixtures of uninfected cell nuclear extracts and virion lysates or in vitro synthesized OTF-1 and Vmw65. The Oct-1 motif (ATGCAAAT) was necessary for optimal Vmw65 binding to, but not for transactivation of the ICP10 promoter as evidenced by competition experiments with oligonucleotides overlapping the consensus IE110 promoter virion response element and by site-directed mutagenesis of the motif. The 3{dollar}\sp\prime{dollar} portion of the TAATGARAT motif (GARAT) was dispensible for binding but necessary for activation. These data suggest that ICP10 behaves as an IE gene and could therefore affect host gene regulation independent of lytic infection. ICP10-PK has neoplastic transforming potential in vitro. Anchorage independent growth was observed in cells transfected with the vectors that express the entire ICP10 protein or just the PK domain, but not a frameshift (not expressing ICP10) mutant or a carboxy-terminus (RR domain) expression vector.