Tat competes with HEXIM1 to increase the active pool of P-TEFb for HIV-1 transcription

Human immunodeficiency virus type 1 (HIV-1) transcriptional transactivator (Tat) recruits the positive transcription elongation factor b (P-TEFb) to the viral promoter. Consisting of cyclin dependent kinase 9 (Cdk9) and cyclin T1, P-TEFb phosphorylates RNA polymerase II and the negative transcription elongation factor to stimulate the elongation of HIV-1 genes. A major fraction of nuclear P-TEFb is sequestered into a transcriptionally inactive 7SK small nuclear ribonucleoprotein (snRNP) by the coordinated actions of the 7SK small nuclear RNA (snRNA) and hexamethylene bisacetamide (HMBA) induced protein 1 (HEXIM1). In this study, we demonstrate that Tat prevents the formation of and also releases P-TEFb from the 7SK snRNP in vitro and in vivo. This ability of Tat depends on the integrity of its N-terminal activation domain and stems from the high affinity interaction between Tat and cyclin T1, which allows Tat to directly displace HEXIM1 from cyclin T1. Furthermore, we find that in contrast to the Tat-independent activation of the HIV-1 promoter, Tat-dependent HIV-1 transcription is largely insensitive to the inhibition by HEXIM1. Finally, primary blood lymphocytes display a reduced amount of the endogenous 7SK snRNP upon HIV-1 infection. All these data are consistent with the model that Tat not only recruits but also increases the active pool of P-TEFb for efficient HIV-1 transcription

Mechanistic insights into the translocation of full length HIV-1 Tat across lipid membranes

AbstractThe mechanism of how full length Tat (aa 1–86) crosses artificial lipid membranes was elucidated by means of fluorescence spectroscopy and fluorescence microscopy. It was shown that full length Tat (aa 1–86) neither forms pores in large unilamellar vesicles (LUVs) nor in giant unilamellar vesicles (GUVs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). In contrast, an N-terminally truncated Tat protein (aa 35–86) that lacks the structurally defined proline- and cysteine-rich region as well as the highly conserved tryptophan residue at position 11 generates pores in artificial POPC-membranes, through which a water-soluble dye up to a size of 10kDa can pass. By means of fluorescence microscopy, the transfer of fluorescently labeled full length Tat across POPC-bilayers was unambiguously visualized with a concomitant accumulation of the protein in the membrane interface. However, if the dye was attached to the protein, also pore formation was induced. The size of the pores was, however smaller than the protein size, i.e. the labeled protein with a mass of 11.6kDa passed the membrane, while a fluorescent dye with a mass of 10kDa was excluded from the vesicles' interior. The results demonstrate that pore formation is not the prime mechanism by which full length Tat crosses a membrane

Crystal Structure of the Human tRNA m(1)A58 Methyltransferase-tRNA3(Lys) Complex: Refolding of Substrate tRNA Allows Access to the Methylation Target

Human tRNA(3) (Lys) is the primer for reverse transcription of HIV; the 3′ end is complementary to the primer-binding site on HIV RNA. The complementarity ends at the 18th base, A58, which in tRNA(3) (Lys) is modified to remove Watson–Crick pairing. Motivated to test the role of the modification in terminating the primer-binding sequence and thus limiting run-on transcription, we asked how the modification of RNA could be accomplished. tRNA m(1)A58 methyltransferase (m(1)A58 MTase) methylates N1 of A58, which is buried in the TψC-loop of tRNA, from cofactor S-adenosyl-L-methionine. This conserved tRNA modification is essential for stability of initiator tRNA in Saccharomyces cerevisiae. Reported here, three structures of human tRNA m(1)A58 MTase in complex with human tRNA(3) (Lys) and the product S-adenosyl-L-homocysteine show a dimer of heterodimers in which each heterodimer comprises a catalytic chain, Trm61, and a homologous but noncatalytic chain, Trm6, repurposed as a tRNA-binding subunit that acts in trans; tRNAs bind across the dimer interface such that Trm6 from the opposing heterodimer brings A58 in to the active site of Trm61. T-loop and D-loop are splayed apart showing how A58, normally buried in tRNA, becomes accessible for modification. This result has broad impact on our understanding of the mechanisms of modifying internal sites in folded tRNA. The structures serve as templates for design of inhibitors that could be used to test tRNA m(1)A58 MTase's impact on retroviral priming and transcription