A Minimal Chimera of Human Cyclin T1 and Tat Binds TAR and Activates Human Immunodeficiency Virus Transcription in Murine Cells

Abstract

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 threedimensional structure of this RNA-protein complex.

Transcriptional Profiles of Latent Human Immunodeficiency Virus in Infected Individuals: Effects of Tat on the Host and Reservoir

“…This statement is supported by direct binding data and heterologous RNA tethering of Tat, e.g., via the regulator of virion gene expression Rev and its Rev response element, where the Rev.Tat chimera functioned equally well on the modified HIV LTR in cells from both species (6,24,42). Rather, the cysteine at position 261 in the human CycT1, which is a tyrosine in the murine CycT1, coordinates the Zn 2ϩ -dependent interaction between the two proteins and brings arginine-rich motifs (ARM) in CycT1 and Tat into close proximity so that they bind the 5Ј bulge and central loop in TAR (26).…”

“…Importantly, although the abundant expression of many proteins in ␤ cells of the pancreas from the insulin promoter leads to diabetes mellitus (4,48), the sTat.GFP chimera was innocuous. Because Tat can interact productively with the murine P-TEFb for all other purposes other than HIV transcription (6,24,42) and can enter murine and human cells equivalently (20,27,51), we conclude that, at these levels, Tat is highly specific for the HIV LTR.…”

Functional Characterization of Human Cyclin T1 N-Terminal Region for Human Immunodeficiency Virus-1 Tat Transcriptional Activation

“…Fujinaga et al 21 previously demonstrated that the CycT1 N-terminal region (amino acids 1-59) was crucial to support Tat-mediated transactivation using CycT1-Tat chimeric proteins in NIH3T3 cells. Apart from introducing single point mutations of those amino acids that were found earlier to exhibit defective phenotype (Figs.…”

Identification of a Cyclin T-Binding Domain in Hexim1 and Biochemical Analysis of Its Binding Competition with HIV-1 Tat

“…Site-directed mutagenesis was performed using the megaprimer method with both sense and antisense oligonucleotides. The plasmid coding for the CycT1-Tat fusion protein was described previously (20).…”