Functional differences between human and bovine immunodeficiency virus Tat transcription factors

Abstract

Transcriptional transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) promoter element by the essential viral Tat protein requires recruitment of positive transcription elongation factor b (P-TEFb) to the viral TAR RNA target. The recruitment of P-TEFb, which has been proposed to be necessary and sufficient for activation of viral gene expression, is mediated by the highly cooperative interaction of Tat and cyclin T1, an essential component of P-TEFb, with the HIV-1 TAR element. Species, such as rodents, that encode cyclin T1 variants that are unable to support TAR binding by the Tat-cyclin T1 heterodimer are also unable to support HIV-1 Tat function. In contrast, we here demonstrate that the bovine immunodeficiency virus (BIV) Tat protein is fully able to bind to BIV TAR both in vivo and in vitro in the absence of any cellular cofactor. Nevertheless, BIV Tat can specifically recruit cyclin T1 to the BIV TAR element, and this recruitment is as essential for BIV Tat function as it is for HIV-1 Tat activity. However, because the cyclin T1 protein does not contribute to TAR binding, BIV Tat is able to function effectively in cells from several species that do not support HIV-1 Tat function. Thus, BIV Tat, while apparently dependent on the same cellular cofactor as the Tat proteins encoded by other lentiviruses, is nevertheless unique in terms of the mechanism used to recruit the BIV Tat-cyclin T1 complex to the viral LTR promoter.

FIG. 1

Comparison of hTat and bTat function in cells from three distinct species. Human 293T cells, murine L cells, and avian QCl-3 cells were transfected with indicator constructs consisting of the wild-type HIV-1 LTR, or an HIV-1 LTR in which the hTAR element had been substituted with bTAR, linked to the cat gene. These plasmids were cotransfected with pBC12/CMV-based expression constructs expressing full-length hTat or bTat as well as a pBC12/CMV-based internal control plasmid encoding β-Gal. Plasmid pBC12/CMV also served as a negative control. Cultures were harvested at ∼48 h after transfection, and CAT and β-Gal activities were determined as described elsewhere (–5). The indicated data are corrected for minor differences in transfection efficiency, as measured by the β-Gal internal control, and are representative of three independent transfection experiments.

FIG. 2

Interaction of bTat with CycT1. (A) Mutation of the bTat cysteine motif blocks bTat function. Human 293T cells were cotransfected with either the pHIV/bTAR/CAT or pHIV/SLIIB/CAT indicator construct together with an effector plasmid encoding bTat or the Rev-bTat or Rev-C38S fusion protein. Data were derived as described in the legend to Fig. 1. (B) The bTat protein binds CycT1 specifically. The S. cerevisiae two-hybrid indicator strain Y190 was transformed with plasmids expressing the GAL4 DNA binding domain fused to wild-type or mutant bTat and with plasmids expressing the VP16 transcription activation domain in an unfused form (Neg.) or fused to wild-type hCycT1 or mCycT1. Induced β-Gal activities were determined as previously described (–5) after selection for transformants. (C) Western analyses of GAL4 fusion protein expression levels in yeast (lanes 1 to 4) and Rev fusion expression in 293T cells (lanes 5 to 7) were performed using a commercial GAL4 DNA binding domain antibody or a rabbit polyclonal anti-Rev antiserum as previously described (3). Neg., control cells not expressing a GAL4 (lane 1) or Rev (lane 5) fusion protein. OD 595 (here and in Fig. 3 and 4), optical density at 595 nm.

FIG. 3

The bTat and hTat proteins differ in the ability to bind their cognate TAR elements. In these three-hybrid protein-RNA interaction assays, S. cerevisiae L40uraMS2 cells were transformed with an expression plasmid encoding the MS2 operator linked to full-length hTAR (A) or bTAR (B). In addition, these cells were transformed with a plasmid encoding the VP16 activation domain fused to hTat, bTat, or the C38S bTat mutant and finally with a plasmid encoding the nonfused form of hCycT1 or mCycT1. The appropriate empty vectors served as negative controls. Induced β-Gal activity was measured in pooled transformants as previously described (–5).

FIG. 4

The bTat protein can recruit CycT1 to bTAR. This yeast three-hybrid assay was performed essentially as described for Fig. 5 except that the CycT1 proteins were expressed as VP16 fusions whereas bTat and the C38S bTat mutant were expressed in a nonfused form.

FIG. 5

The bTat protein binds bTAR in vitro. In this RNA gel shift experiment, a labeled bTAR RNA probe was incubated with the indicated recombinant GST fusion proteins, and the resultant RNA-protein complexes were then resolved by nondenaturing gel electrophoresis. C1 indicates the position of the proposed bTAR-bTAT complex, while C2 indicates the position of the proposed bTAR-bTat-CycT1 ternary complex.