NMR detection of structures in the HIV-1 5'-leader RNA that regulate genome packaging

Abstract

The 5'-leader of the HIV-1 genome regulates multiple functions during viral replication via mechanisms that have yet to be established. We developed a nuclear magnetic resonance approach that enabled direct detection of structural elements within the intact leader (712-nucleotide dimer) that are critical for genome packaging. Residues spanning the gag start codon (AUG) form a hairpin in the monomeric leader and base pair with residues of the unique-5' region (U5) in the dimer. U5:AUG formation promotes dimerization by displacing and exposing a dimer-promoting hairpin and enhances binding by the nucleocapsid (NC) protein, which is the cognate domain of the viral Gag polyprotein that directs packaging. Our findings support a packaging mechanism in which translation, dimerization, NC binding, and packaging are regulated by a common RNA structural switch.

Fig. 1. Structure of the HIV-1 5´-leader

(A–C), Secondary structure predictions showing AUG (green) in hairpin (B) and U5:AUG (C) conformations. (D) 5´-L forms a monomer at low ionic strength (LI buffer = 10 mM Tris-HCl, 10 mM NaCl, pH 7.0) ([RNA] = 60 µM). (E) 2D ¹H-¹³C HMQC NMR spectrum obtained for 5´-L containing ¹³C-labeled AUG (5´-L^AUG*; LI buffer). (F) Overlay of spectra obtained for 5´-L^AUG* (gray) and oligo-AUG* hairpin (green). (G) 5´-L exists as a reversible monomer-dimer equilibrium in PI buffer ([5´-L] = 30 µM in left panel). The equilibrium shifts to the monomer upon subsequent dilution (100-fold) in PI buffer. (H) ¹H-¹³C HMQC NMR data obtained for 5´-L^AUG* in LI buffer (black) and immediately upon dissolution in PI buffer (red). Spectral changes correlate with an equilibrium shift toward the dimer. Residues 349, 351, 352 and 355 are unstructured and detectable, and residues 337 and 341 are rotationally restricted and undetectable, in the [5´-L]₂ dimer. (I) Similar spectral changes were observed upon titration of an isolated AUG* hairpin (black) with an unlabeled U5 oligonucleotide, except that all U5:AUG signals were detectable (red).

Fig. 2. AUG base pairs with U5 in the dimer

(A) lr-AID mutations (red) designed to probe for predicted U5:AUG base pairing. Black arrows denote ¹H-¹H NOEs. (B) Portions of 1D ¹H NMR spectra showing (top): the TAR A46-H2 signal of native [5´-L]₂; (middle): A46G substitution ([5´-L^A46G]₂) eliminates the TAR A46-H2 signal; (bottom): A338-H2 signal observed for lr-AID substituted [5´-L^A46G]₂. (C,D) Similarities in NOE spectra observed for A46-H2 of an isolated TAR oligonucleotide (C) and native [5´-L]₂ (D). (E,F) Similar A338-H2 NOEs observed for an isolated lr-AID U5:AUG oligonucleotide (E) and lr-AID substituted [5´-L^A46G]₂ (F).

Fig. 3. U5:AUG formation promotes dimerization

(A) 5´-L^ΔAUG (1 µM) forms a stable monomer. (B) Addition of AUG-17 (10-fold molar excess) promotes dimerization of 5´-L^ΔAUG. (C,D) Substitutions that enhance U5:DIS interactions favor the monomer. (E) Proposed U5:DIS base pairing and lr-AID sequences used for structural probing. (F) The A259-H2 signal is only observed when U5 and DIS both contain lr-AID substitutions. Top: 5´-L^{ΔAUG, A46G}. Middle: 5´-L^ΔAUG,A46G containing the lr-AID substitution in the DIS loop only. Bottom: 5´-L^ΔAUG,A46G containing lr-AID substitution in both DIS and U5. (G) 2D NOE data for A259-H2 in lr-AID substituted 5´-L^ΔAUG,A46G, assigned from the corresponding U5:DIS oligonucleotide control spectrum.

Fig. 4. U5:AUG formation promotes NC binding and packaging

(A) 5´-L^AUG-HP and 5´-L^U5:AUG form predominantly monomers and dimers, respectively, and native 5´-L adopts a 70:30 monomer:dimer equilibrium (PI buffer; [RNA] = 0.8 µM). MIX = 70:30 mixture of 5´-L^AUG-HP and 5´-L^U5:AUG. (B) ITC NC titration data for samples shown in (A). (C) Packaging of native HIV-1_NL4-3 5´-L, 5´-L^U5:AUG and 5´-L^AUG-HP RNAs under competition conditions (assayed by RNase protection; see Supplementary Methods). Lanes 2–8: 2: undigested probe; 3: RNA size standards; 4: native HIV-1_NL4-3 helper versus native HIV-1_NL4-3 test vector RNAs; 5: native HIV-1_NL4-3 helper versus 5´-L^U5:AUG test vector; 6: native HIV-1_NL4-3 helper versus 5´-L^AUG-HP test vector; 7: HIV-1_NL4-3 helper expressed without test RNA; 8: mock transfected-cells. Bands corresponding to native HIV-1_NL4-3 helper RNA (helper) and co-packaged test RNAs (test) are labeled. (D) Nucleotide displacement mechanism regulates dimeric genome packaging.