Targeting of the visna virus tat protein to AP-1 sites: interactions with the bZIP domains of fos and jun in vitro and in vivo

Abstract

The visna virus Tat protein is required for efficient viral transcription from the visna virus long terminal repeat (LTR). AP-1 sites within the visna virus LTR, which can be bound by the cellular transcription factors Fos and Jun, are also necessary for Tat-mediated transcriptional activation. A potential mechanism by which the visna virus Tat protein could target the viral promoter is by protein-protein interactions with Fos and/or Jun bound to AP-1 sites in the visna virus LTR. Once targeted to the visna virus promoter, the Tat protein could then interact with basal transcription factors to activate transcription. To examine protein-protein interactions with cellular proteins at the visna virus promoter, we used an in vitro protein affinity chromatography assay and electrophoretic mobility shift assay, in addition to an in vivo two-hybrid assay, to show that the visna virus Tat protein specifically interacts with the cellular transcription factors Fos and Jun and the basal transcription factor TBP (TATA binding protein). The Tat domain responsible for interactions with Fos and Jun was localized to an alpha-helical domain within amino acids 34 to 69 of the protein. The TBP binding domain was localized to amino acids 1 to 38 of Tat, a region previously described by our laboratory as the visna virus Tat activation domain. The bZIP domains of Fos and Jun were found to be important for the interactions with Tat. Mutations within the basic domains of Fos and Jun abrogated binding to Tat in the in vitro assays. The visna virus Tat protein was also able to interact with covalently cross-linked Fos and Jun dimers. Thus, the visna virus Tat protein appears to target AP-1 sites in the viral promoter in a mechanism similar to the interaction of human T-cell leukemia virus type 1 Tax with the cellular transcription factor CREB, by binding the basic domains of an intact bZIP dimer. The association between Tat, Fos, and Jun would position Tat proximal to the viral TATA box, where the visna virus Tat activation domain could contact TBP to activate viral transcription.

FIG. 1

GST-visna virus Tat fusion protein constructs used in in vitro affinity chromatography experiments. Fusion proteins were expressed in E. coli JM109, purified, and used for in vitro affinity chromatography as described in Materials and Methods. Shaded areas refer to regions of the visna virus Tat protein.

FIG. 2

Interactions of visna virus Tat protein with Fos, Jun, and TBP. GST alone (lanes 1 to 3), GST-Tat (lanes 4 to 6), and GST-VP16 (lanes 7 to 9) were immobilized on glutathione-Sepharose beads and incubated with in vitro-translated, [³⁵S]methionine-labeled Fos, Jun, and TBP for 2 h at 4°C. Following incubation, bound proteins were washed extensively, eluted, and analyzed by SDS-PAGE on a 10% gel followed by autoradiography.

FIG. 3

Interactions of different domains of the visna virus Tat protein with Fos, Jun, and TBP. GST-Tat 1-38 (lanes 1 to 3), GST-Tat 34-69 (lanes 4 to 6), and GST-Tat 60-94 (lanes 7 to 9) were immobilized on glutathione-Sepharose beads and incubated with in vitro-translated, [³⁵S]methionine-labeled Fos, Jun, and TBP, and bound proteins were analyzed as described for Fig. 2.

FIG. 4

Fos and Jun proteins used in in vitro affinity chromatography experiments. Plasmids containing Fos and Jun wild-type and mutated sequences were transcribed, translated, and labeled in vitro with [³⁵S]methionine as described in Materials and Methods. Leucine zipper regions of the Fos and Jun bZIP domains of the proteins are marked by darkly shaded areas; basic domains of the bZIP domains are represented by lightly shaded regions. The point mutations in Fos ΔL5P and JunΔL1V,ΔL2V are in boldface type.

FIG. 5

Interactions of GST-Tat with Fos and Jun. Fos and Jun proteins were translated in vitro with [³⁵S]methionine, and GST-Tat was incubated with equivalent counts per minute of each Fos and Jun protein. (A) In vitro-translated Fos proteins were incubated with GST-Tat as described for Fig. 2 (lanes 1 to 6). In vitro-translated Fos proteins (IVT Protein) were loaded directly onto the gel, without interaction with GST-Tat, in lanes 7 to 12 to show that equal counts were loaded. Lanes 7 to 12 were exposed to radiography roughly 1/10 as long as lanes 1 to 6. (B) In vitro-translated Jun proteins were incubated with GST-Tat as described for Fig. 2 (lanes 1 to 3). In lanes 4 to 6, a 1/10 dilution of the amount of each Jun protein incubated GST-Tat was loaded directly on the gel to show that equal counts were loaded.

FIG. 6

Mammalian two-hybrid analysis. (A) The Fos and Jun bZIP domains were fused to the activation domain of VP16. (B) Portions of visna virus Tat were fused to the DNA binding domain of Gal4. (C) Schematic showing that the interaction of visna virus Tat with the bZIP domains of Fos or Jun will target the VP16 activation domain to a reporter construct containing Gal4 binding sites and drive transcription of the CAT gene when cotransfected into SCP cells.

FIG. 7

Visna virus Tat interaction with the Fos and Jun bZIP domains in vivo by mammalian two-hybrid analysis. SCP cells were transfected with the E1b5CAT plasmid (lanes 1 to 9) and cotransfected with Gal4 DNA binding domain-Tat fusion constructs and with VP16 activation domain-bZIP constructs (shown in Fig. 6) as described in Materials and Methods. The activity of the E1b5CAT vector alone was assigned an activity of 1, and the CAT activities of the other samples are plotted relative to this background activity. This result is representative of at least three independent experiments, and the standard error is shown.

FIG. 8

Visna virus Tat interaction with Fos and Jun dimers. In vitro-translated Fos and Jun were cross-linked and then tested for the ability to bind GST by in vitro affinity chromatography as described in Materials and Methods. Chemically cross-linked in vitro-translated Fos and Jun proteins were tested for the ability to bind GST alone (lanes 1 to 3) and GST-Tat (lanes 4 to 6) as described for Fig. 2. The samples were analyzed by electrophoresis on an SDS–4 to 20% gradient polyacrylamide gel and autoradiography.

FIG. 9

(A) EMSA using a fragment of the visna virus LTR containing the TATA proximal AP-1 site (−65 to +56) and bacterially expressed and purified Jun bZIP (aa 199 to 334), Fos bZIP (aa 199 to 334), and full-length Tat proteins. The indicated proteins (100 ng of each) were incubated with the ³²P-labeled visna virus LTR fragment as indicated in Materials and Methods. (B) EMSA using a ³²P-labeled oligonucleotide probe containing a consensus AP-1 site. Bacterially expressed and purified Jun bZIP, Fos bZIP, and Tat proteins were incubated with the ³²P-labeled oligonucleotide by the procedure used for panel A. (C) Antibody (Ab) specific to the visna virus Tat N-terminal region (lane 2) or a nonspecific antibody (antibody to SIV Nef; lane 4) was preincubated with the Tat protein on ice for 1 h and then run in an EMSA with the Jun protein and labeled AP-1 probe as for panel B. The antibody to visna virus Tat and the control antibody were also included in the reactions with Jun alone (lanes 1 and 3).