Analysis of a herpes simplex virus 2 fragment from the open reading frame of the large subunit of ribonucleotide reductase with transcriptional regulatory activity.
Expression of herpes simplex virus 2 (HSV-2)-encoded ribonucleotide reductase (RR) is required for growth in nondividing cells. The functional enzyme is composed of a large and a small subunit. In virus-infected cells, RR is expressed temporally as a delayed early protein. However, the promoter regulatory region of the large subunit can function as an immediate early promoter in transient transfection assays, suggesting that expression may be quite complex. In this study, a 95-bp fragment derived from the open reading frame of the large subunit of RR (RR-A) functioned as a silencer when placed adjacent to a heterologous promoter. If the fragment was placed distal to the promoter, repression was relieved and in human keratinocytes promoter activity was consistently higher than control constructs. Exonuclease III protection assays revealed that nuclear factors from human keratinocytes as well as other primate cells specifically bind to this fragment. A 30-bp motif containing a consensus SP-1 binding site and an alternating Pu/Py element was protected in all cell lines. These results suggest that a 95-bp fragment in the open reading frame of HSV-2 RR-A plays a role in regulating viral gene expression.
Jones
C
C
Department of Veterinary Science, University of Nebraska, Lincoln 68583-0905.
Zhu
F
F
Dhanwada
K R
KR
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D002467:Cell Nucleus; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins; D005090:Exodeoxyribonucleases; D005347:Fibroblasts; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D006801:Humans; D015603:Keratinocytes; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid / Q000502:physiology*; D012097:Repressor Proteins / Q000235:genetics; D012264:Ribonucleotide Reductases / Q000235:genetics*; D018139:Simplexvirus / Q000235:genetics*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012264:Ribonucleotide Reductases; D015500:Chloramphenicol O-Acetyltransferase; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III