A riboswitch regulates RNA dimerization and packaging in human immunodeficiency virus type 1 virions

Abstract

The genome of retroviruses, including human immunodeficiency virus type 1 (HIV-1), consists of two identical RNA strands that are packaged as noncovalently linked dimers. The core packaging and dimerization signals are located in the downstream part of the untranslated leader of HIV-1 RNA-the Psi and the dimerization initiation site (DIS) hairpins. The HIV-1 leader can adopt two alternative conformations that differ in the presentation of the DIS hairpin and consequently in their ability to dimerize in vitro. The branched multiple-hairpin (BMH) structure folds the poly(A) and DIS hairpins, but these domains are base paired in a long distance interaction (LDI) in the most stable LDI conformation. This LDI-BMH riboswitch regulates RNA dimerization in vitro. It was recently shown that the Psi hairpin structure is also presented differently in the LDI and BMH structures. Several detailed in vivo studies have indicated that sequences throughout the leader RNA contribute to RNA packaging, but how these diverse mutations affect the packaging mechanism is not known. We reasoned that these effects may be due to a change in the LDI-BMH equilibrium, and we therefore reanalyzed the structural effects of a large set of leader RNA mutations that were presented in three previous studies (J. L. Clever, D. Mirandar, Jr., and T. G. Parslow, J. Virol. 76:12381-12387, 2002; C. Helga-Maria, M. L. Hammarskjold, and D. Rekosh, J. Virol. 73:4127-4135, 1999; R. S. Russell, J. Hu, V. Beriault, A. J. Mouland, M. Laughrea, L. Kleiman, M. A. Wainberg, and C. Liang, J. Virol. 77:84-96, 2003). This analysis revealed a strict correlation between the status of the LDI-BMH equilibrium and RNA packaging. Furthermore, a correlation is apparent between RNA dimerization and RNA packaging, and these processes may be coordinated by the same LDI-BMH riboswitch mechanism.

FIG. 1.

Alternative foldings of the HIV-1 leader RNA. (A) Schematic of the 9.2-kb HIV-1 genome. The 5′ and 3′ long terminal repeats (LTR) and all nine open reading frames are indicated. The untranslated leader RNA consists of several regulatory domains. poly A, polyadenylation domain with the AAUAAA sequence; PAS, primer activation signal; SD, major splice donor; ψ, core packaging signal; AUG, Gag start codon. (B) The HIV-1 leader RNA can adopt two conformations. Detailed structure models of the LDI and BMH structures have been presented elsewhere. The poly(A) (orange) and DIS (pink) sequences are base paired to form the LDI structure. The same sequences form the poly(A) and DIS hairpins in the BMH structure. The riboswitch model for regulated dimerization argues that the ground-state LDI structure must first be rearranged into the BMH conformation to expose the DIS hairpin, which mediates subsequent RNA dimerization.

FIG. 2.

HIV-1 leader mutants. The RNA packaging and dimerization values for 38 HIV-1 leader RNA mutants were taken from previous studies. Black blocks indicate the positions of the mutations in the leader RNA; several regulatory domains within the leader are indicated at the top. The packaging efficiencies were measured as the ratio of genomic RNA to spliced RNA within virions in the studies of Clever et al. (9) and Russell et al. (35). In the study of Helga-Maria et al. (20), packaging was determined as the ratio of virion-associated RNA to CA-p24 protein. All RNA packaging values are relative to that of the corresponding wild-type (wt) construct, which was set at 100%. To obtain these values, we converted the data of Clever et al. into relative values (in parentheses). The dimerization values in the study of Russell et al. reflect the percentages of dimeric and monomeric RNA genomes from virions, as analyzed on a nondenaturing gel. The thermodynamic stabilities (ΔG, in kilocalories per mole) of the LDI and BMH structures were calculated by using the Mfold program and were used to calculate ΔΔG values for LDI-BMH.

FIG. 3.

HIV-1 RNA packaging is controlled by the LDI-BMH equilibrium. The relative RNA packaging efficiencies of the mutant sets shown in Fig. 2 are plotted against the ΔΔG values for LDI-BMH. (A, B, and C) Data sets from the studies of Clever et al. (9), Russell et al. (35), and Helga-Maria et al. (20), respectively. The wild-type (wt) constructs are indicated by arrows.

FIG. 4.

HIV-1 RNA dimerization and RNA packaging are coupled. (A) The RNA dimerization of the mutant set of Russell et al. (35) shown in Fig. 2 is plotted against ΔΔG values for LDI-BMH. (B) The RNA packaging of the mutant set of Russell et al. is plotted against the RNA dimerization. The wild-type (wt) constructs are marked by arrows.