Potential Intra- and Intermolecular Interactions Involving the Unique-5′ Region of the HIV-1 5′-UTR

Abstract

The 5′-untranslated region (5′-UTR) of the human immunodeficiency virus type-1 (HIV-1) genome regulates multiple RNA-dependent functions during viral replication and has been proposed to adopt multiple secondary structures. Recent phylogenetic studies identified base pair complementarity between residues of the unique 5′ element and those near the gag start codon (gagAUG) that is conserved among evolutionarily distant retroviruses, suggesting a potential long-range RNA-RNA interaction. However, nucleotide accessibility studies led to conflicting conclusions about the presence of such interactions in virions and in infected cells. Here we show that an 11-nucleotide oligo-RNA spanning residues 105-115 of the 5′-UTR (U5) readily binds to oligoribonucleotides containing the gag start codon (AUG), disrupting a pre-existing stem loop and forming a heteroduplex stabilized by 11 Watson Crick base pairs (Kd = 0.47 ± 0.16 μM). Addition of the HIV-1 nucleocapsid protein (NC), the transacting viral factor required for genome packaging, disrupts the heteroduplex by binding tightly to U5 (Kd = 122 ± 10 nM). The structure of the NC:U5 complex, determined by NMR, exhibits features similar to those observed in NC complexes with HIV-1 stem loop RNAs, including the insertion of guanosine nucleobases to hydrophobic clefts on the surface of the zinc fingers and a 3′-to-5′ orientation of the RNA relative to protein. Our findings indicate that the previously proposed long-range U5-gagAUG interaction is feasible, and suggest a potential NC-dependent mechanism for modulating the structure of the 5′-UTR.

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

“…5B,C ;Vuilleumier et al 1999;Bourbigot et al 2008;Bazzi et al 2011;Darlix et al 2011). In the three RNA-NC complexes for which high-resolution structures have been obtained (De Guzman et al 1998;Amarasinghe et al 2000;Spriggs et al 2008), the protein is oriented anti-parallel to the 5 ′ -3 ′ direction of the nucleic acid chain. As a result, the N-terminal zinc finger (ZF1) binds a residue 3 ′ to the residue binding to the C-terminal zinc finger (ZF2) (Fig.…”

Properties and functions of the nucleocapsid protein in virus assembly

“…This hydrophobic plateau appears to be essential in the interactions between NC and small oligonucleotides (ODN) since the structures of all ODN/NCp7 complexes resolved today show that this hydrophobic plateau pilots the binding of NCp7 to nucleic acids (NA) via multiple contacts with the nucleotide bases and the phosphate backbone. [27][28][29][30][31][32] In addition, amino acid Trp37 of the second ZnF always stacks with a guanine base, which represents a major contribution to the NCp7/NA binding energy. The nucleoprotein complex is further stabilized by means of numerous electrostatic interactions with the NC basic amino acids present in the N-terminal disordered sequence and the ZnF linker ( Fig.…”

Biophysical studies of the nucleic acid chaperone properties of the HIV-1 nucleocapsid protein

“…82,83 Major changes in NMR spectroscopy constitutes another biophysical approach to investigate the chaperone properties of NCp7 at the molecular level. Beside its crucial role in solving the structure of both free NCp7, [2][3][4]74 and NCp7 complexed to various ODNs, [6][7][8][9]75 NMR spectroscopy was used to determine how NCp7 destabilizes NAs. Although technically challenging, NMR analysis of NA-protein complexes is probably one of the few techniques, together with fluorescence spectroscopy, which can provide useful dynamic data and thus, contribute to a deeper understanding of the molecular mechanisms of the NCp7 chaperone functions.…”

“…This plateau is composed of the residues Val13, Phe16, Thr24 and Ala25 of the proximal ZF and the residues Trp37, Gln45 and Met46 of the distal ZF. [2][3][4] This hydrophobic plateau directs the nucleic acid (NA) binding properties of NCp7, [5][6][7][8][9] with notably a strong contribution in the stabilization of the complexes due to the stacking of Trp37 with the guanine bases. 10 The complexes are further stabilized by electrostatic interactions with the basic amino acids of the protein.…”