The Apical Loop of the HIV-1 TAR RNA Hairpin Is Stabilized by a Cross-loop Base Pair

Tadeusz Kulinski,Mikolaj Olejniczak,Hendrik Huthoff,Lukasz Bielecki,Katarzyna Pachulska-Wieczorek,Atze T Das,Ben Berkhout,Ryszard W Adamiak

doi:10.1074/jbc.m301939200

Abstract

The TAR hairpin of the HIV-1 RNA genome is indispensable for trans-activation of the viral promoter and virus replication. The TAR structure has been studied extensively, but most attention has been directed at the three-nucleotide bulge that constitutes the binding site of the viral Tat protein. In contrast, the conformational properties of the apical loop have remained elusive. We performed biochemical studies and molecular dynamics simulations, which indicate that the TAR loop is structured and stabilized by a cross-loop base pair between residues C30 and G34. Mutational disruption of the cross-loop base pair results in reduced Tat response of the LTR promoter, which can be rescued by compensatory mutations that restore the base pair. Thus, Tat-mediated transcriptional activation depends on the structure of the TAR apical loop. The C30-G34 cross-loop base pair classes TAR in a growing family of hairpins with a structured loop that was recently identified in ribosomal RNA, tRNA, and several viral and cellular mRNAs.

Highlights

The trans-activation response element (TAR) hairpin of the HIV-1 RNA genome is indispensable for trans-activation of the viral promoter and virus replication
Thermal Denaturation of HIV-1 RNA with a Mutated TAR Loop—To test if the TAR loop is structured, we performed a UV-melting experiment with transcripts corresponding to the 5Ј-terminal 466 nucleotides of the wild-type HIV-1 RNA and a mutant in which the apical loop residues 32–34 were changed from GGG to AAA (Fig. 1)
The high-temperature transition Tm3 has previously been shown to correspond to the TAR hairpin, and the intense Tm2 transition reflects melting of the extended LDI stem in the downstream leader domain [50]

Summary

EXPERIMENTAL PROCEDURES

Oligoribonucleotide Synthesis—The synthesis of 29-mer HIV-1 TAR RNA and its 2-aminopurine riboside modified analogues AP34 and AP35 was performed using solid-support aided phoshoramidite chemistry and applying the 2Ј-O-tBDMSi protection [43]. The RNA was renatured by incubation at 85 °C for 3 min, followed by slow cooling to room temperature. The RNA was refolded by heating at 80 °C for 30 s, followed by slow cooling to room temperature in a buffer containing 10 mM Tris-HCl, pH 7.2, 40 mM NaCl, 10 mM MgCl2, and 8 ␮M carrier tRNA. Prepared Pb(OAc) was added to final concentrations of 0.5, 1, and 2 mM This reaction mixture was incubated for 15 min at room temperature. Plasmid Constructs—The pBlue3ЈLTR-luc plasmid contains an XhoIHindIII fragment from the HIV-1 LAI proviral DNA, encoding the complete U3 region, the transcription start site (ϩ1) and R sequences up to position ϩ82, coupled to the Firefly luciferase reporter gene [51]. Isolates with a complete TAR sequence were considered in the analysis, yielding a total of 654 sequences from most of the prevailing HIV-1 subtypes

RESULTS

TAR RNA

DISCUSSION