Characterization of a cellular factor which interacts functionally with Oct-1 in the assembly of a multicomponent transcription complex

Abstract

Induction of transcription of the immediate-early (IE) genes of herpes simplex virus involves the assembly of a DNA-binding complex containing the viral protein Vmw65 and the cellular transcription factor Oct-1. We show that Oct-1 is not sufficient for complex formation and that another cellular factor(s) which is absolutely required for complex formation can be separated from Oct-1 under native conditions. We have purified this factor by approximately 100-fold using DNA-cellulose, ion-exchange and size-exclusion chromatographies. The assay used throughout the purification procedure follows the ability of the cellular factor to form a complex when added to purified Oct-1, Vmw65 and an IE specific DNA probe. The complex forming factor (CFF) had a sedimentation coefficient of about 4.4 S (i.e. molecular mass of about 70K, under non-denaturing conditions) and the polypeptide profile of highly purified CFF demonstrated two major species with molecular masses of 80K and 70K. Unequivocal association of either of these two species with CFF activity could not presently be demonstrated due to the sensitivity of CFF to denaturation. CFF, when tested on its own or in the presence of Vmw65, did not bind to the IE-specific consensus motif. We have also used deletion mutants of Oct-1 to show that the POU domain of this protein was sufficient for CFF-dependent complex formation with Vmw65. Deletions of the POU specific region of Oct-1 significantly reduced the complex forming ability, although detectable levels of complex were reconstituted using Vmw65, CFF and just the homeodomain of Oct-1.

Broad binding‐site specificity and affinity properties of octamer 1 and brain octamer‐binding proteins

“…Surprisingly, a TAATGAATT sequence that does not appear in the context of an octamer binds Oct-1 better than some sites with almost perfect octamers. In HSV-infected cells Oct-1 forms a high-affinity complex on the TAATGARAT motif in concert with a virally encoded protein and at least one other cellular factor (Gerster and Roeder, 1988;Kristie et al, 1989;Katan et al, 1990). It is thought that such a cellular factor could cooperate with Oct-1 in uninfected cells to alter its binding specificity and direct formation of a TAATGARAT-bound complex (Goding and O'Hare, 1989).…”

Structure and VP16 binding of the Mediator Med25 activator interaction domain

“…Activators have been classified as acidic, glutamine-rich, proline-rich and serine/threonine-rich, depending on the preponderance of amino acids in their TAD 3 . An archetypal acidic activator is the herpes simplex virus protein 16 (VP16), which activates the expression of immediate early viral genes during infection [4][5][6][7][8] . VP16 exerts its activating function through a C-terminal TAD that includes residues 413-490 (refs.…”

The HCF repeat is an unusual proteolytic cleavage signal.

“…HCF [for herpes simplex virus host cell factor, but also referred to as C1 (Kristie et al 1989)) VCAF (Xiao and Capone 1990), and CFF (Katan et al 1990)], is a cellular activity required for transcriptional activation of herpes simplex virus (HSV) immediate early (IE) promoters by the viral transcriptional activator protein VPl6 (Gerster and Roeder 1988;Kristie et al 1989; for review, see Thompson and McKnight 1992;O'Hare 1993). VPl6 (also known as Vmw65 or a-TIF) is a HSV virion protein that is released into the cell upon infection and forms a heterodimeric protein complex with HCF.…”