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

Abstract

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...

Genetic Analysis of the Structure and Function of 7SK Small Nuclear Ribonucleoprotein (snRNP) in Cells

“…This finding and the previous data on residues from positions 178 -220 of HEXIM1, which contribute to its binding specificity (Fig. 3), support the model where RNA-binding residues from positions 150 -177 and those from positions 178 -220 of HEXIM1 form a tripartite HEXIM1-CycT1-7SK complex that resembles the Tat-CycT1-TAR complex (21,47,48).…”

“…In addition, there is another bulge (from positions 75-77) containing a uridine residue that could interact with HEXIM1. Finally, 7SK contains a central loop (from positions 50 -59) that resembles the central loop of TAR where CycT1 binds to form a stable complex between Tat, P-TEFb, and TAR (21,47,48). Therefore, we constructed a series of mutant M3 plasmid targets to identify other residues critical for these interactions.…”

First Snapshots of the HIV-1 RNA Structure in Infected Cells and in Virions

“…The in vitro binding of a Tat-derived peptide to this three-nucleotide bulge strongly protected this residue against Me 2 SO 4 modification (35). Thus, our results suggested that Tat and the P-TEFb complex (positive transcription elongation factor complex b) that binds the TAR apical loop during transcription trans-activation (36) do not remain associated with the genomic RNA. To note, this stem is involved in the packaging of the genomic RNA (37,38).…”

Crystal structure of HIV-1 Tat complexed with human P-TEFb and AFF4

“…On the other hand, the photocrosslinking and protein footprinting studies by Rana and colleagues revealed that the TRM interacts with the loop in TAR RNA. 40 This provides a plausible mechanism for the modulation of TAR RNA recognition through stabilization of the TRM in Cyclin T1 by interaction of acetylated Lys28 of Tat with Asn257 in the TRM.…”