title: Functional characterization of human cyclin T1 N-terminal region for human immunodeficiency virus-1 Tat transcriptional activation.
abstract: Transcription of the human immunodeficiency virus type 1 (HIV-1) requires the interaction of the cyclin T1 (CycT1) subunit of a host cellular factor, positive transcription elongation factor b, with the viral Tat protein at the transactivation response (TAR) element of nascent viral transcripts. The involvement of the interaction between Tat and CycT1 is known to be through the Tat-TAR recognition motif (TRM) on CycT1. Here, we have further characterized this molecular interaction and clarified the role of the CycT1 N-terminal region in Tat action. We found crucial and distinctive roles of Q46, Q50 and F176 of human CycT1 protein in Tat-mediated transcription by creating various Ala substitution mutants of CycT1 based on its three-dimensional structure. We confirmed the involvement of these amino acid residues in binding to Tat with Q46 and Q50, and to a lesser extent with F176, by in vitro pull-down assay. Relative transactivation activities of wild-type CycT1 chimeras and mutant derivatives on the HIV-1 long terminal repeat were determined by luciferase reporter assays. Whereas CycT1 Q46A alone had impaired transcriptional activity, the CycT1(Q46A)-Tat chimeric protein retained almost full activity of the wild-type CycT1. However, CycT1 mutants (C261Y, Q50A or F176A) or their chimeric counterparts had lost the transactivation capacity. Moreover, a triple-mutant chimera containing Q46A, Q50A and F176A mutations completely abolished the transcriptional activity, indicating that these amino acid residues are involved through distinct mechanisms. These findings provide new insights for the development of anti-HIV drugs.
authors.affiliation: Department of Molecular and Cellular Biology, Nagoya City University Graduate School for Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Asamitsu
authors.forename: Yurina
authors.initials: Y
authors.lastname: Hibi
authors.forename: Kenichi
authors.initials: K
authors.lastname: Imai
authors.forename: Ann Florence B
authors.initials: AF
authors.lastname: Victoriano
authors.forename: Eiji
authors.initials: E
authors.lastname: Kurimoto
authors.forename: Koichi
authors.initials: K
authors.lastname: Kato
authors.forename: Takashi
authors.initials: T
authors.lastname: Okamoto
mesh_terms: D056906:Cyclin T / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: C503684:CCNT1 protein, human; D056906:Cyclin T; D050505:Mutant Proteins; D011994:Recombinant Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus