The transcriptional transactivator of simian foamy virus 1 binds to a DNA target element in the viral internal promoter.

Abstract

The transcriptional transactivator (Tas) of simian foamy virus type 1 strongly augments gene expression directed by both the promoter in the viral long terminal repeat and the newly discovered internal promoter located within the env gene. A region of 121 bp, located immediately 5' to the TATA box in the internal promoter, is required for transactivation by Tas. The present study aimed to identify the precise Tas-responsive target(s) in this region and to determine the role of Tas in transcriptional regulation. By analysis of both clustered-site mutations and hybrid promoters in transient expression assays in murine and simian cells, two separate sequence elements within this 121-bp region were shown to be Tas-dependent transcriptional enhancers. These targets, each <30 bp in length and displaying no apparant sequence homology one to the other, are designated the promoterproximal and promoter-distal elements. By means of the gel electrophoresis mobility-shift assay, using purified glutathione S-transferase-Tas fusion protein expressed in Escherichia coli, the target proximal to the TATA box exhibited strong binding to glutathione S-transferase-Tas, whereas the distal element appears not to bind. In addition, footprint analysis revealed that 26 bp in the promoter proximal element was protected by glutathione S-transferase-Tas from DNase I. We propose a model for transactivation of the simian foamy virus type 1 internal promoter in which Tas interacts directly with the proximal target element positioned immediately 5' to the TATA box. In this model, Tas attached to this element is presumed to interact with a component(s) of the cellular RNA polymerase II initiation complex and thereby enhance transcription directed by the viral internal promoter.Foamy viruses, members of the spumavirus genus of retroviruses, have complex genomes encoding virion structural genes, a transcriptional transactivator, and one or two additional open reading frames (ORFs) of undefined function (1-3). Human foamy virus (HFV) and the simian foamy viruses (SFVs) contain two promoters for initiation of viral transcription. One promoter is located in the 5' long terminal repeat (LTR) and produces the full-length viral transcript, which is the precursor to spliced subgenomic transcripts (4). Another promoter is embedded in sequences within the 3' portion of the envelope gene and thus directs transcription of the viral transactivator and the ORF(s) (5-7). The transactivator has been designated taf for SFV (8, 9) and bel-i for HFV (10, 11).At the First International Foamy Virus Meeting (November 1994, London), it was recommended that taf and bel-i be designated tas (transactivator of spumavirus).Transient expression assays in several cell types have revealed that SFV and HFV transcriptional transactivator (Tas) transactivates both the homologous viral LTR and the internal promoter. Transactivation is mediated through target elements in the U3 domain of the LTR (9, 12-15) and sequences immediately 5' to the TATA box in the internal promote...

Induction of Cellular Genes Is Mediated by the Bel1 Transactivator in Foamy Virus-Infected Human Cells

“…It is well known that Bel1 activates both HFV promoters by direct binding to short defined but promiscuous DNA target sequences of the FV promoters (9,10,33) and that for full transactivation levels, interactions of Bel1 with unknown cellular proteins seem to be required (10). Since the potent Bel1 transactivator has the capacity to bind different DNA target sequences, it is likely that Bel1-mediated activation of cellular genes proceeds by a similar mechanism.…”

Bel1-mediated Transactivation of the Spumaretroviral Internal Promoter Is Repressed by Nuclear Factor I

“…To gain insight into the mechanism of repression, we focused on the core IP.BRE region from Ϫ166 to Ϫ116 that allowed us to define the roles of NFI and Bel1 proteins in DNA binding by carrying out EMSAs, supershifts, and competition EMSAs. Several groups have reported that Bel1 binds its target sequence directly (10,16,23). The minimal IP.BRE of 27 bp seems to be the one with a high DNA binding affinity compared with the corresponding LTR BRE (10).…”

“…Expression of Bel1 is initiated at the IP until threshold levels of the Bel1 transactivator are reached which subsequently turn on the 5Ј-LTR promoter to direct synthesis of virus structural proteins (13)(14)(15). The transactivator Tas of simian foamy virus type 1 binds to corresponding proximal and distal DNA target sites directly that are not homologous to the Bel1 response element (BRE) of the IP (16). Bell 1 was shown to be a nuclear protein (17,18) that contains at least two domains, a centrally located DNA binding region and a C-terminal activation domain (19).…”

Development of Foamy Virus Vectors for Gene Therapy for Neurological Disorders and Other Diseases

“…FVs transactivators of different genera have been named differently; the simian FVs transactivator, for example, was termed taf. However the First International Foamy Virus Meeting determined that these transactivators, including Taf and Bel1, should be designated Tas (Zou et al, 1996). The activity of the functional IP and P of FVs depends on this transactivator.…”