Visualization of in Vivo Direct Interaction between HIV-1 TAT and Human Cyclin T1 in Specific Subcellular Compartments by Fluorescence Resonance Energy Transfer

Abstract

Human cyclin T1, a component of the P-TEFb kinase complex, was originally identified through its biochemical interaction with the Tat transactivator protein of human immunodeficiency virus type 1 (HIV-1). Current understanding suggests that binding of Tat to P-TEFb is required to promote efficient transcriptional elongation of viral RNAs. However, the dynamics and the subnuclear localization of this process are still largely unexplored in vivo. Here we exploit high resolution fluorescence resonance energy transfer (FRET) to visualize and quantitatively analyze the direct interaction between Tat and cyclin T1 inside the cells. We observed that cyclin T1 resides in specific subnuclear foci which are in close contact with nuclear speckles and that Tat determines its redistribution outside of these compartments. Consistent with this observation, strong FRET was observed between the two proteins both in the cytoplasm and in regions of the nucleus outside of cyclin T1 foci and overlapping with Tat localization. These results are consistent with a model by which Tat recruits cyclin T1 outside of the nuclear compartments where the protein resides to promote transcriptional activation.The human immunodeficiency virus type 1 (HIV-1) 1 transactivator protein Tat is a small polypeptide (86 -101 amino acids, according to the viral strains) essential for efficient transcription of viral genes. The protein is a highly unusual transcription factor since, at the HIV LTR promoter, it interacts with a cis-acting RNA element (trans-activation-responsive region, TAR) present at the 5Ј-end of each viral transcript (1).Through this interaction, Tat activates HIV-1 transcription by promoting the assembly of transcriptionally active complexes at the LTR by multiple protein-protein interactions (for a recent review, see Ref. 2). Over the last few years a number of cellular proteins have been reported to interact with Tat and to mediate or modulate its activity. These include general transcription factors, among which TBP, TAFII250, TFIIB, TFIIH (3-7); RNA polymerase II (8); transcription factor Sp1 (9); the transcriptional co-activators and histone acetyltransferases p300/CBP and P/CAF (10 -12); and the cyclin subunit of the positive transcription elongation factor complex (P-TEFb), cyclin T1 (13-16).The finding that Tat biochemically and functionally interacts with several cellular proteins raises some fundamental questions. Does Tat directly interact with its partners inside live cells? Which is the subcellular compartment of these interactions? Are they occurring simultaneously or consecutively? Some of these questions can be successfully addressed by taking advantage of fluorescence resonance energy transfer (FRET) measurements (17), allowing investigation of direct interaction of proteins labeled with optically matched fluorophores. FRET exploits radiationless energy transfer driven by dipole-dipole interaction occurring from a fluorophore (the donor) in the excited state to another fluorophore (the acceptor) when in close proximity...

The Growth Factor Granulin Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription

“…5A, panels 1 to 3), whereas RFP-cyclin T1 was predominantly nuclear (panels 4 and 5). In agreement with previous observations made with either endogenous or fluorescently tagged cyclin T1 (17,38,42,45), RFP-cyclin T1 was present in the nonnucleolar nucleoplasm, giving a dotted pattern. Coexpression of untagged GEP led to a dramatic shift in the distribution of RFP-cyclin T1 into the cytoplasm (panel 6), consistent with a biochemical interaction in this compartment.…”

The Human I-mfa Domain-Containing Protein, HIC, Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription

“…COS cells were transfected singly with EGFPtagged HIC or its C-terminally deleted derivatives and with red fluorescent protein (RFP)-tagged cyclin T1⌬PEST or Tat. As expected from previous reports (8,15,27,33), RFP-cyclin T1 was distributed in a speckled pattern in the nucleus but was excluded from the nucleolus, and RFP-Tat localized in the nucleus concentrated in the nucleolus (Fig. 3A).…”

Subnuclear distribution of the largest subunit of the human origin recognition complex during the cell cycle

“…FRET experiments were performed by transfection of human HeLa cells with plasmids expressing HP1α and Orc1p fused at their N-terminus with EGFP and BFP respectively. We have already observed that the BFP:EGFP fluorescent protein pair has excitation and emission properties favorable for FRET and we have exploited these properties to study the in vivo interactions of other nuclear proteins in the same experimental conditions (Marcello et al, 2001;Marcello et al, 2003). FRET image analysis of individual transfected cells are shown in Fig.…”

“…Cells were fixed in 2% paraformaldehyde after 48 hours and mounted directly in 70% glycerol for FRET analysis. FRET measurements were carried out with an epifluorescence Axioskop 2 Zeiss microscope mounting a 103 W HBO lamp, a 100×/NA=1.3, oilimmersion Plan-Neofluar objective and Nomarsky optics, performed as described in detail elsewhere (Marcello et al, 2001;Marcello et al, 2003). Briefly, FRET experiments consisted of collecting two EGFP emission signals by exciting the EGFP with two different excitation wavelengths, 480 and 350 nm, the former being optimal for EGFP and the latter for BFP.…”