Highly divergent lentiviral Tat proteins activate viral gene expression by a common mechanism

Abstract

The human immunodeficiency virus type 1 (HIV-1) Tat protein (hTat) activates transcription initiated at the viral long terminal repeat (LTR) promoter by a unique mechanism requiring recruitment of the human cyclin T1 (hCycT1) cofactor to the viral TAR RNA target element. While activation of equine infectious anemia virus (EIAV) gene expression by the EIAV Tat (eTat) protein appears similar in that the target element is a promoter proximal RNA, eTat shows little sequence homology to hTat, does not activate the HIV-1 LTR, and is not active in human cells that effectively support hTat function. To address whether eTat and hTat utilize similar or distinct mechanisms of action, we have cloned the equine homolog of hCycT1 (eCycT1) and examined whether it is required to mediate eTat function. Here, we report that expression of eCycT1 in human cells fully rescues eTat function and that eCycT1 and eTat form a protein complex that specifically binds to the EIAV, but not the HIV-1, TAR element. While hCycT1 is also shown to interact with eTat, the lack of eTat function in human cells is explained by the failure of the resultant protein complex to bind to EIAV TAR. Critical sequences in eCycT1 required to support eTat function are located very close to the amino terminus, i.e., distal to the HIV-1 Tat-TAR interaction motif previously identified in the hCycT1 protein. Together, these data provide a molecular explanation for the species tropism displayed by eTat and demonstrate that highly divergent lentiviral Tat proteins activate transcription from their cognate LTR promoters by essentially identical mechanisms.

FIG. 1

Comparison of the Tat proteins and TAR elements of HIV-1 and EIAV. (A) This alignment of the hTat and eTat proteins shows reasonable conservation of the basic domain required for TAR binding. In contrast, the cysteine-rich and core motifs, essential for CycT1 binding by hTat, are poorly conserved. Alignment of the amino- and carboxy-terminal regions of these Tat proteins is essentially arbitrary. (B) The eTAR element lacks both the U-rich bulge, required for Tat binding, and the terminal hexanucleotide loop, required for CycT1 recruitment, that are critical for hTAR function.

FIG. 1

Comparison of the Tat proteins and TAR elements of HIV-1 and EIAV. (A) This alignment of the hTat and eTat proteins shows reasonable conservation of the basic domain required for TAR binding. In contrast, the cysteine-rich and core motifs, essential for CycT1 binding by hTat, are poorly conserved. Alignment of the amino- and carboxy-terminal regions of these Tat proteins is essentially arbitrary. (B) The eTAR element lacks both the U-rich bulge, required for Tat binding, and the terminal hexanucleotide loop, required for CycT1 recruitment, that are critical for hTAR function.

FIG. 2

The species specificity of eTat transactivation is determined by eTAR and can be overcome by heterologous recruitment. Canine D17 (A) and human 293T (B) cells were transfected with the indicated reporter plasmids, pBC12/CMV/lacZ, and hTat or eTat either unfused or fused to the SLIIB RNA binding protein, Rev. Forty-eight hours after transfection, levels of CAT enzyme activity in cell lysates were determined and normalized according to the level of β-Gal internal control measured in parallel.

FIG. 3

Predicted amino acid sequence of eCycT1. The sequences of three independent eCycT1 clones were determined, and the consensus (with correction for a small number of Taq-induced errors) was aligned with sequences of hCycT1 and mCycT1. The critical cysteine residue in hCycT1 is indicated by an asterisk.

FIG. 4

Expression of eCycT1 in human cells rescues the defect in eTat transactivation via eTAR. Human 293T cell were transfected with pEIAV/eTAR/CAT (A) or pHIV/eTAR/CAT (B), pBC12/CMV/lacZ, peTat, and either pBC12/CMV/eCycT1 or pBC12/CMV/hCycT1. In control transfections, the parental vector pBC12/CMV replaced either pBC12/CMV/CycT1 or peTat, or both. Normalized CAT enzyme activities were determined as for Fig. 2.

FIG. 5

The N-terminal 300 amino acids of eCycT1 are sufficient to rescue eTat activity in human cells. (A) Schematic representation of chimeric CycT1 proteins that were generated by exchange of restriction fragments. (B) 293T cells were transfected as for Fig. 2, using the pEIAV/eTAR/CAT reporter and chimeric CycT1 expression plasmids. (C) Murine LmTK-cells were transfected with pHIV/hTAR/CAT, pBC12/CMV/lacZ, chimeric CycT1 expression plasmids, and either pcTat or the parental control vector. Normalized CAT enzyme levels were determined as for Fig. 2.

FIG. 6

Interactions between CycT1, Tat proteins, and TAR RNA elements in yeast cells. (A) Two-hybrid interactions between Tat proteins and CycT1 proteins. Yeast cells were transformed with plasmids expressing the indicated Tat proteins fused to the GAL4 DNA binding domain and CycT1 proteins fused to herpes simplex virus VP16 activation domain. (B) Yeast cells were transformed with plasmids expressing the indicated MS2-TAR fusion RNAs, VP16-CycT1 fusion proteins, and the indicated, unfused Tat proteins. After growth on selective media, β-Gal activities induced in lysed yeast cells were determined as described previously (2). The approximate limit of detection of this assay is 10 milli-optical density units at 595 nm (mOD 595).

FIG. 7

In vitro interaction between different TAR elements and Tat proteins in the presence and absence of CycT1 proteins. (A) A ³²P-labeled eTAR RNA probe was incubated in the presence of the indicated GST fusion proteins, and RNA-protein complex formation was visualized by nondenaturing polyacrylamide gel electrophoresis followed by autoradiography. (B) As for panel A except that an hTAR RNA probe was used.