Structural requirements in the herpes simplex virus type 1 transactivator Vmw65 for interaction with the cellular octamer-binding protein and target TAATGARAT sequences.
Herpes simplex virus type 1 virion protein Vmw65 forms a complex (TRF.C) with TAATGARAT sequences and the cellular transcription factor oct-1, which has been implicated as an intermediate in the activation of gene expression by Vmw65. To examine structural requirements within Vmw65 for this interaction, we analyzed extracts of transfected cells that express mutant Vmw65 proteins by gel retardation assay and identified two regions in the primary sequence of Vmw65 which are necessary for in vitro assembly of TRF.C. The amino-terminal boundary for complex assembly and trans activation mapped between residues 49 and 75. At the carboxyl terminus, deletion as far as residue 388 did not affect in vitro TRF.C assembly, although trans-activating activity was abolished. Deletion beyond residue 388 rapidly impaired the ability of the protein to participate in the TRF.C complex, such that a truncated mutant of 380 residues was completely inactive. These requirements towards the carboxyl terminus overlap a region of strong local sequence similarity between Vmw65 and terminal protein p3 of bacteriophage phi 29. Although substitution of corresponding p3 residues into Vmw65 failed to produce a functional chimera, site-directed mutagenesis within the region of similarity identified a number of single-point mutant proteins which were completely deficient for TRF.C formation. These mutant proteins were also unable to trans activate expression from immediate-early promoters, despite the integrity of the acidic carboxyl terminus. The extreme sensitivity of both TRF.C formation and trans activation to single-residue substitutions within this region of Vmw65 suggests that it is directly involved in the protein-protein or protein-DNA interactions required for assembly of a transcriptional complex containing oct-1.
Greaves
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RF
Marie Curie Research Institute, Oxted, Surrey, United Kingdom.
O'Hare
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002872:Chromosome Deletion; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics*; D051863:Host Cell Factor C1; D008969:Molecular Sequence Data; D009154:Mutation; D050811:Octamer Transcription Factor-1; D015345:Oligonucleotide Probes; D010957:Plasmids; D015183:Restriction Mapping; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; D015345:Oligonucleotide Probes; D015534:Trans-Activators; D014157:Transcription Factors