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 by mechanisms that have yet to be established. We developed an NMR 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 protein (NC), 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.

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

“…Thus, packaging signals may act as ‘structural switches’ that favor either translation or dimerization/packaging of the gRNA, and also help the virus differentiate between spliced and unspliced viral RNAs. Several riboswitch models have been proposed for HIV-1 [50–53], and in the case of FIV, the existence of dual structures has been validated by SHAPE and RNA dimerization assays [54]. Flexibility in retroviral gRNA is created via long-range interactions that have now been observed in several retroviruses, from HIV-1, FIV, MLV, to MPMV and MMTV [6,25,26,33,55–59].…”

Specific recognition of the HIV-1 genomic RNA by the Gag precursor

“…7), and substitution of the SL4 apical loop, which destroys the U5-AUG interaction, had little effect on Pr55 Gag binding (Figs 4 and 5), indicating that the U5-AUG interaction does not regulate the initial Pr55 Gag binding to the gRNA. We suggest that the U5-AUG riboswitch controls later stages of viral particle assembly, as numerous NCp7 proteins are still able to bind the 5 0 -region of HIV-1 RNA when this interaction is disrupted 40 . Thus, the putative long-range interaction exposing the lower part of SL1 for Pr55 Gag binding remains to be identified.…”

“…The most probable explanation is that a long-distance interaction between the regions 5 0 to SL1 and 3 0 to SL4 exposes the lower part SL1 for Pr55 Gag binding. The so-called U5-AUG long-distance interaction was recently proposed to act as a structural switch regulating RNA packaging 40 . The sequences involved in the U5-AUG interaction are all present in RNA N1-SL4WT that weakly bound Pr55 Gag (Fig.…”

“…3), several studies also indicated that the regions upstream and downstream of the Psi region play a role in RNA packaging 24,34,35,37,51 . In addition, tertiary interactions present in the gRNA might be absent in the isolated Psi region 26,39,40 . Therefore, we introduced the mutations described above in the context of RNAs M1-615 and N1-600, and tested their effect on Pr55 Gag binding using filter-binding assays (Fig.…”

“…1b). Long-distance interactions that can only take place in the gRNA 26,38 , and especially the so-called U5-AUG interaction, may also contribute to the specificity of gRNA packaging 39,40 , while an RNA structural element overlapping the gag-pol frameshift signal may enhance it 41 .…”