The Herpesvirus Saimiri Open Reading Frame (ORF) 50 (Rta) Protein Contains an AT Hook Required for Binding to the ORF 50 Response Element in Delayed-Early Promoters

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American Society for Microbiology

RESUMO

The herpesvirus saimiri open reading frame (ORF) 50 encodes two proteins, which activate transcription directly, following interactions with delayed-early (DE) promoters containing a specific motif. In this report, we demonstrate that ORF 50 contains a DNA binding domain that has homology to an AT hook DNA binding motif. Deletion analysis of this domain reduces ORF 50-mediated transactivation of the DE ORF 6 and ORF 57 promoters by 100 and 90%, respectively. Furthermore, gel retardation experiments demonstrated that the AT hook motif is required for binding the ORF 50 response element in the promoters of DE genes. Single site-directed mutagenesis of the AT hook revealed that mutation of the glycine residue at position 408 to an alanine reduces ORF 50 transactivation of the ORF 57 promoter by 40%. Moreover, the mutation of multiple basic residues in conjunction with the glycine residue within the core element of the AT hook abolishes ORF 50-mediated transactivation. In addition, p50GFPΔAT-hook is capable of functioning as a trans-dominant mutant, leading to a reduction in virus production of approximately 50% compared to that for wild-type ORF 50.

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