RNA Packaging in HIV

Abstract

Successful replication of the AIDS retrovirus, HIV, requires that its genomic RNA be packaged in assembling virus particles with high fidelity. However, cellular mRNAs can also be packaged under some conditions. Viral RNA (vRNA) contains a 'packaging signal' (ψ) and is packaged as a dimer, with two vRNA monomers joined by a limited number of base pairs. It has two conformers, only one of which is capable of dimerization and packaging. Recent years have seen important progress on the 3D structure of dimeric ψ. Gag, the protein that assembles into the virus particle, interacts specifically with ψ, but this is obscured under physiological conditions by its high nonspecific affinity for any RNA. New results suggest that vRNA is selected for packaging because ψ nucleates assembly more efficiently than other RNAs.

Keywords: HIV; RNA; retroviruses; virus assembly.

Figure 1.

HIV Gag Protein and its Nucleocapsid Domain. Gag is the building block of HIV virus particles. After the virus is released from the cell, Gag is cleaved by the viral protease into six fragments, which are all present in the mature particle. These are (from N- to C-terminus) Matrix (MA), Capsid (CA), “Spacer 1”, Nucleocapsid (NC), “Spacer 2”, and p6. The interactions of Gag with RNA are mediated principally by its NC domain. The lower portion of the figure shows the sequence of the NC protein of the BH10 isolate of HIV-1. The two zinc fingers in NC are highlighted in the sequence.

Figure 2.

The Packaging Signal within HIV RNA. (A) The 5’ end of HIV RNA (nucleotides ^~1-345) can be folded into two alternative structures. In one of these, indicated here as “DIS-sequestered” but resembling the earlier proposal of “Long Distance Interaction” (LDI) [21, 22], the AUG codon for initiation of Gag translation is exposed, while the palindromic Dimer Initiation Site (DIS) is base-paired to the “U5” region of the molecule. In contrast, in the “DIS-exposed” structure (resembling the Branched Multiple Hairpin or BMH model [21, 22]), the Gag AUG is occluded by base-pairing and the DIS is exposed. Exposure enables the DIS to engage in intermolecular base-pairing with the DIS of another viral RNA molecule; the resulting RNA dimer is a substrate for selective packaging into assembling virus particles. (B) NMR-based model of 3-dimensional structure of the DIS-exposed conformer, representing nucleotides 105-344 of HIV RNA. It contains two consecutive 3-way junctions: one between SL3, the U5:AUG helix, and the connecting helix H1, and the other between H1, the primer-binding site (PBS) stem-loop, and the DIS stem-loop. Although nucleotides 282-300 can theoretically be folded into a small stem-loop, indicated as SL2 in (A), in the DIS-exposed structure they actually pair with bases immediately 3’ of “U5” to form H1. (Adapted from [25, 35]).

Figure 3.

ψ-containing RNA Supports Particle Assembly More Efficiently than Control RNAs. Recombinant HIV-1 Gag protein was titrated into solutions of 401-base fluorophore-tagged RNAs. One of these, representing nucleotides 200-600 of HIV RNA, contained ψ, while the other two did not. The incorporation of the RNAs into virus-like particles was monitored by sedimenting them through sucrose gradients. The results show that the ψ-containing RNA supports particle formation more efficiently than the control RNAs. (Adapted from [32]).

Figure 4.

Model Explaining Selective Packaging of ψ-Containing RNA. Either viral genomic RNA, which contains ψ, or cellular mRNAs, which lack ψ, can be packaged into assembling virus particles. We propose that assembly proceeds in two steps, nucleation and lattice formation. We suggest that nucleation is the rate-limiting step in assembly and that it occurs more rapidly on ψ than on other RNAs. In the Figure, Gag molecules are represented as blue ovals, while the RNAs are shown as larger colored blobs.