A discrete element 3' of human immunodeficiency virus 1 (HIV-1) and HIV-2 mRNA initiation sites mediates transcriptional activation by an HIV trans activator.

Replication of HIV requires the Tat protein, which activates elongation of RNA polymerase II transcription at the HIV-1 promoter by interacting with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex b (P-TEFb). The transactivation domain of Tat binds directly to the CycT1 subunit of P-TEFb and induces loop sequence-specific binding of P-TEFb onto nascent HIV-1 trans-activation responsive region (TAR) RNA. We used systematic RNA-protein photocross-linking, Western blot analysis, and protein footprinting to show that residues 252-260 of CycT1 interact with one side of the TAR RNA loop and enhance interaction of Tat residue K50 to the other side of the loop. Our results show that TAR RNA provides a scaffold for two protein partners to bind and assemble a regulatory switch in HIV replication. RNA-mediated assembly of RNA-protein complexes could be a general mechanism for stable ribonucleoprotein complex formation and a key step in regulating other cellular processes and viral replication.H IV-1 encodes a transcriptional activator protein, Tat, which is expressed early in the viral life cycle and is essential for viral gene expression, replication, and pathogenesis (1-3). Tat enhances processivity of RNA polymerase II (pol II) elongation complexes that initiate in the HIV long terminal repeat region. In nuclear extracts, HIV-1 Tat associates tightly with the CDK9-containing positive transcription elongation factor complex b, P-TEFb (4-6). Recent studies indicate that Tat binds directly through its transactivation domain to the cyclin T1 (CycT1) subunit of the P-TEFb complex and induces loop sequence-specific binding of the P-TEFb complex to trans-activation responsive region (TAR) RNA (7-9).Recruitment of P-TEFb to TAR has been proposed to be both necessary and sufficient for activating transcription elongation from the HIV-1 long terminal repeat promoter (10). Neither CycT1 nor the P-TEFb complex bind TAR RNA in the absence of Tat; thus TAR binding is highly cooperative for both 9). In the C-terminal boundary of the CycT1 cyclin domain, Tat appears to contact residues that are not critical for CycT1 binding to CDK9 (8,(11)(12)(13)(14)(15). Mutagenesis studies showed that the CycT1 sequence containing amino acids 1-303 was sufficient to form complexes with Tat-TAR and CDK9 (8,(11)(12)(13)(14)(15). Recent fluorescence resonance energy-transfer studies using fluorescein-labeled TAR RNA and a rhodamine-labeled Tat protein showed that CycT1 remodels the structure of Tat to enhance its affinity for TAR RNA, and that TAR RNA further enhances interaction between Tat and CycT1 (16).The mechanism by which CycT1 induces loop sequence-specific binding of the P-TEFb complex onto nascent HIV-1 TAR RNA is not understood presently. Does CycT1 interact directly with the TAR loop or merely reorganize Tat structure to bind the loop residues? Does Tat bind TAR loop in the presence of CycT1? What regions of CycT1 and Tat interact directly with the TAR loop sequence? Does phosphorylation of P-TEFb chan...

Section: Methodsmentioning

confidence: 99%

TAR RNA loop: A scaffold for the assembly of a regulatory switch in HIV replication

Richter

Ping²,

Rana

2002

“…Upon binding the regulatory protein Tat and the host cellular proteins cyclin T and CDK9, the TAR hairpin stabilizes RNA polymerase II to allow viral transcription (1)(2)(3)(4)(5)(6). Only the apical portion of the stem loop from positions ϩ19 to ϩ42 is required for transcriptional activation in vivo (7). Extensive mutagenesis studies have shown that the region of TAR important for binding Tat involves a set of nucleotides that surround a characteristic UCU nucleotide bulge; important residues include U23, the first bulged nucleotide, and the two base pairs that immediately precede and follow the bulge (8)(9)(10)(11)(12).…”

mentioning

confidence: 99%

A 1.3-Å resolution crystal structure of the HIV-1 trans-activation response region RNA stem reveals a metal ion-dependent bulge conformation

Ippolito

Steitz

1998

144

147

The crystal structure of an HIV-1 transactivation response region (TAR) RNA fragment containing the binding site for the trans-activation protein Tat has been determined to 1.3-Å resolution. In this crystal structure, the characteristic UCU bulge of TAR adopts a conformation that is stabilized by three divalent calcium ions and differs from those determined previously by solution NMR. One metal ion, crucial to the loop conformation, binds directly to three phosphates in the loop region. The structure emphasizes the inf luence of metal ion binding on RNA structure and, given the abundance of divalent metal ion in the cell, raises the question of whether metal ions play a role in the conformation of TAR RNA and the interaction of TAR with Tat and cyclin T in vivo.

“…The Tat/TAR interaction may have functional consequences at both the transcriptional (8,9) and posttranscriptional levels (10,11). At the transcriptional level, Tat has been shown to enhance or stabilize binding of transcription complexes required for efficient initiation and elongation of viral RNAs (12,13).…”

mentioning

confidence: 99%

Tat-dependent adenosine-to-inosine modification of wild-type transactivation response RNA.

Sharmeen

Bass

Sonenberg

et al. 1991

Tat is a potent activator of gene expression in human immunodeficiency virus type 1 (HIV-1). (dsRNA)-modifying activity, which converts adenosines to inosines. This activity is located in the nuclei of Xenopus oocytes. The specificity and extent of modification of adenosines in TAR is dependent on Tat. We propose that the dsRNA-modifying activity may be one of the cellular proteins that interacts with TAR in the nucleus. The possible role of TAR RNA modification in the expression ofHIV-1 is discussed.