Direct interactions of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 ORF50/Rta protein with the cellular protein octamer-1 and DNA are critical for specifying transactivation of a delayed-early promoter and stimulating viral reactivation.
The Kaposi's sarcoma-associated herpesvirus (KSHV) delayed-early K-bZIP promoter contains an ORF50/Rta binding site whose sequence is conserved with the ORF57 promoter. Mutation of the site in the full-length K-bZIP promoter reduced Rta-mediated transactivation by greater than 80%. The K-bZIP element contains an octamer (Oct) binding site that overlaps the Rta site and is well conserved with Oct elements found in the immediate-early promoters of herpes simplex virus type 1(HSV-1). The cellular protein Oct-1, but not Oct-2, binds to the K-bZIP element in a sequence-specific fashion in vitro and in vivo and stimulates Rta binding to the promoter DNA. The coexpression of Oct-1 enhances Rta-mediated transactivation of the wild type but not the mutant K-bZIP promoter, and Oct-1 and Rta proteins bind to each other directly in vitro. Mutations of Rta within an amino acid sequence conserved with HSV-1 virion protein 16 eliminate Rta's interactions with Oct-1 and K-bZIP promoter DNA but not RBP-Jk. The binding of Rta to both Oct-1 and DNA contributes to the transactivation of the K-bZIP promoter and viral reactivation, and Rta mutants deficient for both interactions are completely debilitated. Our data suggest that the Rta/Oct-1 interaction is essential for optimal KSHV reactivation. Transfections of mouse embryo fibroblasts and an endothelial cell line suggest cell-specific differences in the requirement for Oct-1 or RBP-Jk in Rta-mediated transactivation of the K-bZIP promoter. We propose a model in which Rta transactivation of the promoter is specified by the combination of DNA binding and interactions with several cellular DNA binding proteins including Oct-1.
Carroll
Kyla Driscoll
KD
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey/New Jersey Medical School, 225 Warren Street, Newark, NJ 07103, USA.
Khadim
Farah
F
Spadavecchia
Sophia
S
Palmeri
Diana
D
Lukac
David M
DM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics*; D001665:Binding Sites; D002460:Cell Line; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D017874:Immediate-Early Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D050811:Octamer Transcription Factor-1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D025941:Protein Interaction Mapping; D012097:Repressor Proteins / Q000235:genetics*; D020218:Response Elements; D015534:Trans-Activators / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D014775:Virus Activation / Q000235:genetics*
D050976:Basic-Leucine Zipper Transcription Factors; D017874:Immediate-Early Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; C118265:K8 protein, Human herpesvirus 8; D009687:Nuclear Proteins; D050811:Octamer Transcription Factor-1; D012097:Repressor Proteins; C417604:Rta protein, Human herpesvirus 8; D015534:Trans-Activators; D014764:Viral Proteins; D004247:DNA