5'-Cap sequestration is an essential determinant of HIV-1 genome packaging

Abstract

HIV-1 selectively packages two copies of its 5'-capped RNA genome (gRNA) during virus assembly, a process mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein and encapsidation signals located within the dimeric 5' leader of the viral RNA. Although residues within the leader that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency remain unknown. Here, we show that a previously characterized 159-nt region of the leader that possesses all elements required for RNA dimerization, high-affinity NC binding, and packaging in a noncompetitive RNA packaging assay (Ψ^CES) is unexpectedly poorly packaged when assayed in competition with the intact 5' leader. Ψ^CES lacks a 5'-tandem hairpin element that sequesters the 5' cap, suggesting that cap sequestration may be important for packaging. Consistent with this hypothesis, mutations within the intact leader that expose the cap without disrupting RNA structure or NC binding abrogated RNA packaging, and genetic addition of a 5' ribozyme to Ψ^CES to enable cotranscriptional shedding of the 5' cap promoted Ψ^CES-mediated RNA packaging to wild-type levels. Additional mutations that either block dimerization or eliminate subsets of NC binding sites substantially attenuated competitive packaging. Our studies indicate that packaging is achieved by a bipartite mechanism that requires both sequestration of the 5' cap and exposure of NC binding sites that reside fully within the Ψ^CES region of the dimeric leader. We speculate that cap sequestration prevents irreversible capture by the cellular RNA processing and translation machinery, a mechanism likely employed by other viruses that package 5'-capped RNA genomes.

Keywords: 5′ cap; HIV-1; RNA; genome; packaging.

Fig. 1.

Secondary structure of the HIV-1_NL4-3 gRNA 5′-L and constructs used to probe RNA packaging. (A) Native 5′-L. The PBS region is shaded in gray; nucleotides of TAR, polyA, U5, DIS, SD (splice donor site), Ψ^HP, and AUG are color-coded. The two three-way junctions are labeled as Ψ^3WJ-1 and Ψ^3WJ-2. (B) Schematic representation of the 5′-L^ΔPBS RNA (PBS substituted by a GAGA tetraloop). (C) Schematic representation of Ψ^CES (5′-L^{ΔTAR-ΔPolyA-ΔPBS}). Native residues U106 and G345 are substituted by G and C, respectively.

Fig. 2.

RNA packaging signal in the HIV-1 5′-L. (A, Top) The proviral DNA of HIV-1_NL4-3. (A, Middle) The Ψ⁺-helper vector is a replication-defective version of the HIV-1_NL4-3 proviral DNA. (A, Bottom) The test vector’s 5′-L includes the 5′ untranslated region plus 353 nt from the gag-coding region. In the test vectors, the puromycin cassette (Puro*) has an extra CMV promoter region, for which the riboprobe is targeted. LTR, long terminal repeat. (B) Packaging of test RNAs containing 5′-L, 5′-L^ΔPBS, and Ψ^CES in competition with Ψ⁺, assayed by ribonuclease protection. M, RNA size marker; P, undigested probe. (C) Native gel electrophoresis showing that eIF4E binds the dimeric ^CapΨ^CES but not capped RNAs containing TAR–polyA (^Cap5′-L, ^Cap5′-L^ΔPBS, and ^CapTAR–polyA). (D) Portion of 2D NOESY spectra overlay showing cap chemical shifts and NOEs indicative of a sequestered 5′ cap in ^CapTAR–polyA (black) and exposed 5′ cap in ^CapΨ^CES (green); ppm, parts per million. Corresponding spectra for 7mG (orange) and ^CapTPUA^MAL [TAR, polyA, and U5:AUG regions of the HIV-1_MAL leader (23)] (blue) are also shown. The chemical shifts and NOEs observed for the cap-CH₃ and -H8 protons of ^CapTPUA^MAL were similar to those of the HIV-1_MAL 5′-L (23). Residue G105 of HIV-1_NL4-3 gRNA corresponds to G103 in the HIV-1_MAL leader (SI Appendix, Fig. S1).

Fig. 3.

Cap sequestration is required for competitive gRNA packaging. (A) The AAGG sequence was inserted after the transcription start site. (B) The nonnative AAGG residues extend the cap away from the TAR hairpin. (C and D) The inserted AAGG residues do not affect dimerization (C) and NC binding (D) (D, dimer; M, monomer). (E) Portion of 2D NOESY spectra overlay showing cap chemical shifts and NOEs indicative of the exposed 5′ cap for ^CapΨ^CES (green) and ^Cap5′-L^AAGG (purple). NOEs for 7mG are shown in orange. ppm, parts per million. (F) eIF4E binds the ^Cap5′-L^AAGG dimer. (G) Packaging of test leader RNA with an AAGG insertion under competitive conditions as measured by ribonuclease protection.

Fig. 4.

Cap-dependent modulation of Ψ^CES packaging. (A) Construct design of δ-Ψ^CES and δ^mut-Ψ^CES. (B) The wild-type HDV ribozyme cotranscriptionally cleaves the capped 5′ fragment in vitro but the C-to-U mutant does not. (C) δ-Ψ^CES and δ^mut-Ψ^CES exhibit the same dimerization properties as the parent Ψ^CES RNA. M, monomer; D, dimer; W, water; PI, PI buffer. (D) Packaging of test RNAs containing δ-Ψ^CES and δ^mut-Ψ^CES in competition with Ψ⁺ as measured by ribonuclease protection.

Fig. 5.

Dimeric Ψ^CES is required for authentic packaging fidelity and efficiency. (A) TP-Ψ^3WJ-1 and TP-Ψ^HP contain the proximal three-way junction and the extended Ψ-hairpin, respectively. TP, cap-sequestering TAR–polyA cassette. (B) Test RNAs containing TP-Ψ^3WJ-1 and TP-Ψ^HP were not detectably packaged in competition with Ψ⁺. (C–E) Substitution of the native DIS loop by GAGA (C) prevents dimerization (D) and attenuates packaging of test RNAs containing 5′-L^DISm and 5′-L^DPBS-DISm under competitive conditions (E). (F) Working model for HIV-1 RNA packaging. Cap-dependent capture by the RNA processing or translation machinery precludes packaging of host and viral mRNAs. gRNAs are selected by a bipartite mechanism that involves structural sequestration of the 5′ cap and exposure of a cluster of high-affinity Gag binding sites. CBP, cap-binding protein; ORF, open reading frame.