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. 2012 Mar 30;417(3):224-39.
doi: 10.1016/j.jmb.2012.01.033. Epub 2012 Jan 27.

Identification of a minimal region of the HIV-1 5'-leader required for RNA dimerization, NC binding, and packaging

Xiao Heng  1 , Siarhei Kharytonchyk, Eric L Garcia, Kun Lu, Sai Sachin Divakaruni, Courtney LaCotti, Kedy Edme, Alice Telesnitsky, Michael F Summers
Affiliations

Identification of a minimal region of the HIV-1 5'-leader required for RNA dimerization, NC binding, and packaging

Xiao Heng et al. J Mol Biol. .

Abstract

Assembly of human immunodeficiency virus type 1 (HIV-1) particles is initiated in the cytoplasm by the formation of a ribonucleoprotein complex comprising the dimeric RNA genome and a small number of viral Gag polyproteins. Genomes are recognized by the nucleocapsid (NC) domains of Gag, which interact with packaging elements believed to be located primarily within the 5'-leader (5'-L) of the viral RNA. Recent studies revealed that the native 5'-L exists as an equilibrium of two conformers, one in which dimer-promoting residues and NC binding sites are sequestered and packaging is attenuated, and one in which these sites are exposed and packaging is promoted. To identify the elements within the dimeric 5'-L that are important for packaging, we generated HIV-1 5'-L RNAs containing mutations and deletions designed to eliminate substructures without perturbing the overall structure of the leader and examined effects of the mutations on RNA dimerization, NC binding, and packaging. Our findings identify a 159-residue RNA packaging signal that possesses dimerization and NC binding properties similar to those of the intact 5'-L and contains elements required for efficient RNA packaging.

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Figures

Fig. 1
Fig. 1
(a) Representation of HIV-1 viral genome showing relative positions of splice sites and coding versus non-coding regions. (b) Secondary structure of the 5´-L conformer that promotes dimerization, NC binding and genome packaging. Long range base pairing interactions between U5 (blue) and AUG (green) help expose the dimer-promoting GC-rich loop of the DIS hairpin (cyan). Residues in pairing patterns that give rise to resolved, upfield-shifted adenosine C2-H signals are highlighted (colored boxes). The 3´-most residues of AUG (light green) are deleted in the 5´-L344 construct. (c) Mutations in AUG that affect base pairing with U5 influence the monomer-dimer equilibrium. Lane 1: native 5´-L exists as a mixture of monomer and dimer when incubating within PI buffer. Lane 2: Substitution of 331-GAGAGAUGGGUGCGAGAGCGUCGGU-355 by 331-AACUAUACAAUCGGAGACGAUUGCA-355, which has been reported in our previous study known as 5´-LHP-AUG), favors the AUG hairpin structure and exists mainly as a monomer. This mutation has been shown to inhibit packaging. Lane 3: Substitution of 333-UGGGUGC-339 by 333-CUCUAGA-339, which favors the AUG hairpin structure, mainly exists as a monomer, and has been reported with packaging deficiency. Lane 4: Substitution of 337-UGGGUGCGAGAGCGUC-353 by 337-CGGGCACAAGAAAAAA-353 (5´-LU5:AUG) favors the formation of U5:AUG base pairs and promotes dimerization and packaging. Lane 5: 5´-L344, in which residue 345–356 was truncated from 5´-L. The AUG hairpin cannot form in 5´-L344, but the residues involved in U5:AUG base pairs are maintained. This truncation stabilizes the RNA as a dimer. (d) 5´-L344 readily forms a dimer upon incubation in PI buffer. (e) Portion of the 2D NOESY spectrum obtained for a dimeric 5´-L344 RNA sample containing protons on the non-exchangeable adenosine C-2 and ribose (A2,R), the guanosine ribose (GR), and the cytosine ribose (CR) carbons and deuterons at all other non-exchangeable aromatic and ribose carbons: A2,R,GR,CR-[5´-L344]2. NMR signal assignments were made based on comparisons with isolated oligo-RNAs corresponding to the TAR, lower-PBS, DIS, and Ψ-RNA hairpins (Lu, Heng and Summers, in preparation).
Figure 2
Figure 2
(a) RNA constructs used in the present studies: (1) 5´-L, (2) 5´-L344, (3) 5´-L344-ΔTAR, (4) 5´-L344-ΔPBS, (5) 5´-L344-ΔTAR-ΔPBS, (6) 5´-L344-ΔTAR-ΔPolyA-ΔPBS. (b) Denaturing PAGE gels show sample purity and their relative electrophoretic migration. (c) Portions of 2D NOESY spectra showing the A220 C2-H peaks observe for (from left to right) A2,R,GR,CR- labeled [5´-L344]2, 5´-L344-ΔPBS, 5´-L344-ΔTAR-ΔPolyA-ΔPBS, and for a fully protonated oligoribonucleotide corresponding to the lower PBS hairpin (5´-ggCUCUGGUgagaGCCAGAGcc; lower case = non-native), showing that the lower PBS stem structure is maintained in these 5´-L constructs. (d) Chemical shifts of the C2-H signals for the PBS lower stem, Ψ, and DIS hairpins are unaffected by removal of the TAR, Poly(A) and PBS upper loop (gray and black spectra correspond to RNAs labeled 2 and 6 in panel (a), respectively.
Figure 3
Figure 3. Effects of TAR, PolyA and PBS deletions on NC binding
(a) Native gel electrophoresis data (2% high-gelling-temperature agarose gel containing TRIS-borate buffer and ethidium bromide for RNA visualization) showing that 5´-L344-ΔTAR, 5´-L344-ΔPBS, 5´-L344-ΔTAR-ΔPBS and 5´-L344-ΔTAR-ΔPolyA-ΔPBS (labeled 3–6, respectively, as in Fig. 2) form dimers after incubation in PI buffer (140 mM KCl, 10 mM NaCl, 1 mM MgCl2, pH 7.0), indicating that deletions of TAR, PolyA and PBS do not affect RNA dimerization (b) ITC data obtained upon titration of NC into 5´-L344. The raw data are shown in the top panel, and the integrated heat changes are shown in the bottom panel. First two calorimetric data points were deleted before non-linear least squares isotherm fitting using “Two-Set of Sites” mode (MicroCal Origin 5.0). The first set of binding parameters were utilized to characterize the high-affinity interactions between 5´-L344 and NC (Table 1). (c) ITC NC titration data obtained for 5´-L344-ΔTAR, 5´-L344-ΔPBS, 5´-L344-ΔTAR-ΔPBS, showing that these deletions have modest effects on high affinity NC binding. (d) ITC NC titration data obtained for 5´-L344-ΔTAR-ΔPolyA-ΔPBS. The post-high affinity endothermic contributions to NC binding observed for 5´-L344 (and at diminished levels in the other 5´-L344 mutants) are nearly undetectable.
Figure 4
Figure 4. TAR, Poly(A) and PBS deletions do not affect RNA packaging
RNA packaging efficiency of 5´-L variants were monitored by RNase protection assay. (a) RNA samples obtained from the co-expression of a series of packaging test vectors with CMVΔR8.2, an HIV-1 Ψ- helper construct. Lanes 1–5: products of RNA samples harvested from media of cells transfected with the indicated constructs plus pCMVΔR8-2 helper; lane 6: from cells transfected with pCMVΔR8-2 alone; lane 7: from mock-transfected cells. Lanes 8–14 are RNase protection assay products of cellular samples from the same transfections used in lanes 1–7. P, undigested riboprobe; ΔpA = Δpoly(A). The mobilities of size standards are indicated to the left; the mobilities of riboprobe fragments protected by transcripts from each of the test vectors and from the Ψ- helper are indicated at right. (b) RNase protection assay of viral samples using two different riboprobes specific to 7SL RNA or HIV-1 vectors RNA (Ps = the two undigested riboprobes). Lanes 1–5: products of RNA samples harvested from media of cells transfected with the indicated constructs plus CMVΔR8-2 helper; lane 6: from cells transfected with CMVΔR8-2 alone; lane 7: from mock-transfected cells. The mobilities of riboprobe fragments protected by transcripts from each of the test vectors and the Ψ- helper are indicated at right as HIV RNA; the mobilities of riboprobe products protected by 7SL RNA indicated as 7SL RNA
Figure 5
Figure 5. Effect of gag deletion on RNA packaging
RNA samples obtained from the co-expression of a series of native and 3´ truncated packaging test vectors with CMVΔR8.2. Lanes 1–2: RNase protection assay products of RNA samples harvested from media of cells transfected with the indicated constructs plus pCMVΔR8-2 helper; lane 3: from cells transfected with pCMVΔR8-2 alone; lane 4: from mock-transfected cells. Lanes 5–8 are RNase protection assay products of cellular samples from the same transfections used in lanes 1–4.
Figure 6
Figure 6
(a) Non-denaturing gel electrophoresis (2% agarose) results showing that 5´-LΔAUG exists predominantly as a monomer, as expected, and that truncation of TAR (5´-LΔAUG-ΔTAR) strongly promotes dimerization. (b) [5´-L344]2 (green) versus 5´-LΔAUG (black). (c) 5´-LΔAUG (black) versus 5´-LΔAUG-ΔTAR (red); The star and arrow symbols highlight the expected loss of the TAR signals (stars) and unexpected loss of the DIS A268-C2-H signal (arrow) that were observed upon deletion of TAR. These data indicate that removal of TAR leads to misfolding of DIS hairpin and induces non-native dimerization of the monomeric form of the 5´-leader.
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