Interdigitated residues within a small region of VP16 interact with Oct-1, HCF, and DNA.
Upon infection, the herpes simplex virus (HSV) activator of immediate-early (IE) gene transcription VP16 forms a multiprotein-DNA complex with two cellular proteins, Oct-1 and HCF. First, VP16 associates with HCF independently of DNA, and this association stimulates subsequent association with Oct-1 on the DNA target of VP16 activation, the TAATGARAT motif found in HSV IE promoters. We have analyzed the involvement of VP16 residues lying near the carboxy-terminal transcriptional activation domain of VP16 in associating with HCF, Oct-1, and DNA. To assay VP16 association with HCF, we developed an electrophoretic mobility retardation assay in which HCF is used to retard the mobility of a hybrid VP16-GAL4 DNA-binding domain fusion protein bound to a GAL4 DNA-binding site. Analysis of an extensive set of individual and combined alanine substitutions over a 61-amino-acid region of VP16 shows that, even within a region as small as 13 amino acids, there are separate residues involved in association with either HCF, DNA, or Oct-1 bound to DNA; indeed, of two immediately adjacent amino acids in VP16, one is important for DNA binding and the other is important for HCF binding. These results suggest that a small region in VP16 is important for linking in close juxtaposition the four components of the VP16-induced complex and support the hypothesis that the structure of the Oct-1-VP16 interaction in this complex is similar to that formed by the yeast transcriptional regulatory proteins MATa1 and MAT alpha2. We propose that HCF stabilizes this Oct-1-VP16 interaction.
Lai
J S
JS
Cold Spring Harbor Laboratory, New York 11724, USA.
Herr
W
W
D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D017405:Herpes Simplex Virus Protein Vmw65 / Q000737:chemistry* / Q000378:metabolism; D051863:Host Cell Factor C1; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D050811:Octamer Transcription Factor-1; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D017405:Herpes Simplex Virus Protein Vmw65; D051863:Host Cell Factor C1; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; D011506:Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D004247:DNA