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 (gag(AUG)) 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 (K(d) = 0.47 +/- 0.16 microM). Addition of the HIV-1 nucleocapsid protein (NC), the trans-acting viral factor required for genome packaging, disrupts the heteroduplex by binding tightly to U5 (K(d) = 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-gag(AUG) interaction is feasible and suggest a potential NC-dependent mechanism for modulating the structure of the 5'-UTR.

Figure 1

(a) HIV-1 genome showing the location of the 5′- Untranslated Region (UTR). (b) One of the predicted secondary structures of the HIV-1 5′- UTR showing proposed U5-gag^AUG long-range interactions (12, 33). The U5 sequence is shown in blue and the AUG sequence is shown in green, with the gag start codon in red. (c) Alternate secondary structure showing the hairpin structure of AUG (also called SL4) that was predicted on the basis of chemical modification and mutagenesis experiments (7, 20, 25, 29).

Figure 2

U5 and AUG form a stable duplex. (a) U5 and AUG RNA constructs used for this study. (b) Native PAGE results for AUG RNA upon titration with U5 RNA showing that U5 and AUG form a stable heterodimer at 1:1 U5:AUG molar ratios. No additional bands were observed at U5:AUG ratios greater than 1:1. (c) Representative ITC data obtained upon titration of AUG with U5 (K_d = 0.47 ± 0.16 μM). (d) Downfield region of the 2D ¹H-¹H NOESY spectrum obtained for the U5:AUG duplex showing sequential connectivities between base-paired Watson-Crick imino protons. A weak cross peak between the U337 and G338 imino protons is observed at lower contour levels (dashed circle).

Figure 3

(a) Native PAGE results obtained upon titration of U5 (left), a 19-residue form of AUG that spans residues A334-C352 (AUG¹⁹; center) and U5:AUG¹⁹ (right) with NC (NC/RNA ratios are shown at the top). NC binds to both U5 and AUG¹⁹, disrupting the U5:AUG¹⁹ complex. NC:U5 migrates slowly due to the low overall negative charge. Weak band intensities observed at NC:U5:AUG¹⁹ ratios of 1:1:1 are due to significant smearing that appears to be related to exchange between the different equilibrium species. (b) ITC data obtained upon titration of U5 with NC at varying NaCl concentrations. Upper Panel: the raw data showing the time-dependent heat profiles obtained upon NC injection. Bottom panel: Calculated binding isotherms obtained after subtraction of blank (heat of dilution) data. (c) Plot of the log (K_d) versus -log [NaCl].

Figure 4

Portions of the 800 MHz 2D NOESY data obtained for the unlabeled NC:U5^GUU complex. Selected intermolecular cross peaks are shown for the aromatic protons of Phe 16 (N-terminal zinc knuckle) and Trp 37 (C-terminal knuckle) are labeled. Selected intermolecular NOEs involving the Ile24-δCH₃, Ile24-γCH₃ and Ala25-CH₃ protons are shown in the upper panels.

Figure 5

Stereoviews of the 20 lowest-energy structures calculated for the NC:U5^GGU complex. (a) Best-fit superposition of the backbone atoms of the N-terminal zinc knuckle. The figure displays the backbone atoms of residues Val 13-Cys 49 (black), heavy atoms of the N-terminal zinc knuckle (Cys and His in yellow and blue, respectively), and RNA residues U107 (orange), G106 and G108 (green). The backbone atoms of residues that link the two zinc knuckles are shown in red. (b) Same as in (a), except that the backbone atoms of the C-terminal zinc knuckle are superposed.

Figure 6

Structure of the lowest-energy NC:U5^GGU structure. (a) Stereoview showing the relative positions of the two zinc knuckles and the RNA residues that interact with NC. (b) Electrostatic surface potential representation (positive and negative potential shown in blue and red, respectively) of residues Val 13 - Cys 49 in the NC:U5^GGU complex. Residues G106-C109 are shown as sticks. Phosphate groups and basic residues of NC that are poised for favorable electrostatic interactions are labeled. (c, d) Stick figure representations showing H-bonding interactions between G108 and G106 and the N- and C-terminal zinc knuckles, respectively (Cys = yellow, His = blue, Zn = brown).