A minimal chimera of human cyclin T1 and tat binds TAR and activates human immunodeficiency virus transcription in murine cells.
The transcriptional elongation of human immunodeficiency virus type 1 (HIV-1) is mediated by the virally encoded transactivator Tat and its cellular cofactor, positive transcription elongation factor b (P-TEFb). The human cyclin T1 (hCycT1) subunit of P-TEFb forms a stable complex with Tat and the transactivation response element (TAR) RNA located at the 5' end of all viral transcripts. Previous studies have demonstrated that hCycT1 binds Tat in a Zn(2+)-dependent manner via the cysteine at position 261, which is a tyrosine in murine cyclin T1. In the present study, we mutated all other cysteines and histidines that could be involved in this Zn(2+)-dependent interaction. Because all of these mutant proteins except hCycT1(C261Y) activated viral transcription in murine cells, no other cysteine or histidine in hCycT1 is responsible for this interaction. Next, we fused the N-terminal 280 residues in hCycT1 with Tat. Not only the full-length chimera but also the mutant hCycT1 with an N-terminal deletion to position 249, which retained the Tat-TAR recognition motif, activated HIV-1 transcription in murine cells. This minimal hybrid mutant hCycT1-Tat protein bound TAR RNA as well as human and murine P-TEFb in vitro. We conclude that this minimal chimera not only reproduces the high-affinity binding among P-TEFb, Tat, and TAR but also will be invaluable for determining the three-dimensional structure of this RNA-protein complex.
Fujinaga
Koh
K
Departments of Medicine, Microbiology and Immunology, UCSF-Mt. Zion Cancer Center, University of California at San Francisco, 2340 Sutter Street, San Francisco, CA 94143-0703, USA.
Irwin
Dan
D
Taube
Ran
R
Zhang
Fan
F
Geyer
Matthias
M
Peterlin
B Matija
BM
D016475:3T3 Cells; D000818:Animals; D056906:Cyclin T; D016213:Cyclins / Q000378:metabolism*; D015696:Gene Products, tat / Q000378:metabolism*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D051379:Mice; D012367:RNA, Viral / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014158:Transcription, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus
C503684:CCNT1 protein, human; C503685:Ccnt1 protein, mouse; D056906:Cyclin T; D016213:Cyclins; D015696:Gene Products, tat; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus