Visualization of in vivo direct interaction between HIV-1 TAT and human cyclin T1 in specific subcellular compartments by fluorescence resonance energy transfer.
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.
Marcello
A
A
Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy.
Cinelli
R A
RA
Ferrari
A
A
Signorelli
A
A
Tyagi
M
M
Pellegrini
V
V
Beltram
F
F
Giacca
M
M
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D016466:CHO Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D056906:Cyclin T; D016213:Cyclins / Q000378:metabolism*; D015696:Gene Products, tat / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
C503684:CCNT1 protein, human; D056906:Cyclin T; D016213:Cyclins; D015696:Gene Products, tat