Structure-Based Alignment and Consensus Secondary Structures for Three HIV-Related RNA Genomes

Abstract

HIV and related primate lentiviruses possess single-stranded RNA genomes. Multiple regions of these genomes participate in critical steps in the viral replication cycle, and the functions of many RNA elements are dependent on the formation of defined structures. The structures of these elements are still not fully understood, and additional functional elements likely exist that have not been identified. In this work, we compared three full-length HIV-related viral genomes: HIV-1NL4-3, SIVcpz, and SIVmac (the latter two strains are progenitors for all HIV-1 and HIV-2 strains, respectively). Model-free RNA structure comparisons were performed using whole-genome structure information experimentally derived from nucleotide-resolution SHAPE reactivities. Consensus secondary structures were constructed for strongly correlated regions by taking into account both SHAPE probing structural data and nucleotide covariation information from structure-based alignments. In these consensus models, all known functional RNA elements were recapitulated with high accuracy. In addition, we identified multiple previously unannotated structural elements in the HIV-1 genome likely to function in translation, splicing and other replication cycle processes; these are compelling targets for future functional analyses. The structure-informed alignment strategy developed here will be broadly useful for efficient RNA motif discovery.

Fig 1. RNA structure-based comparative genome analysis.

Sequence alignments were created through SHAPE-dependent pairwise comparisons, which were then combined into multiple sequence alignments [24]. Windowed linear regression analysis of SHAPE data was then used to define regions where structural conservation is implied by correlation of SHAPE reactivities. For these regions, consensus secondary structures were modeled using SHAPE- and sequence-dependent folding [50]. Consensus secondary structures were found for both HIV-1/SIVcpz and three-genome alignments. Base pairs that did not disagree between the two consensuses and that had a pairing probability greater than 95% were used to constrain a final model for HIV-1 using SHAPE-directed folding [29].

Fig 2. SHAPE-structure dependent alignment.

(A) SHAPE-directed alignment over one 200-nt window in the RRE. Sequences are numbered relative to the HIV-1 RNA genome, with the transcription start site as +1. (B, C) Windowed linear regression statistics as a function of the HIV-1 (NL4-3) sequence, computed over 200-nt windows. Correlations of SHAPE values across the three-genome sequence alignment were evaluated by F-test (results shown as p-values; black); pairwise comparisons with HIV-1 were evaluated by t-test (results shown as p-values). The entire HIV-1 alignment is shown; RNA landmarks are given at the bottom of the figure.

Fig 3. Secondary structure models for six structurally conserved elements in the 5' half of HIV-related RNA genomes.

Nucleotides are colored by HIV-1 SHAPE reactivities. Secondary structures are shown for the final constrained HIV-1 secondary structure model. Base pairs are colored by level of structural consensus; black base pairs appear only in constrained HIV-1 predictions. Positions of predicted elements are shown on the HIV-1 (NL4-3) genome; annotations indicate statistical dependence, known RNA elements, major splice sites, and protein reading frames.

Fig 4. Secondary structure models for five structurally conserved elements in the 3' half of HIV-related RNA genomes.

Secondary structures are shown for the final constrained HIV-1 secondary structure model. Other figure annotations are described in the Fig 3 legend.

Fig 5. Secondary structure models for RNA elements with prior well-established functions.

Secondary structures correspond to the final, fully automated constrained HIV-1 prediction. Other figure annotations are described in the Fig 3 legend.

Fig 6. Novel conserved, likely functional, elements in the final HIV-1 structure model.

(A) Structural elements located near protein-protein junctions. Protein domain junctions are labeled. (B) Conserved structural elements with the potential to form long (>20 bp) helical stacks. (C) Conserved structure at the A1 splice site, described in Pollom et al., recapitulated in this work [11].

Fig 7. Consensus structures for the cPPT and PPT elements.

(A) cPPT and PPT sequences and SHAPE reactivities. The six sequences correspond to two elements from each of three genomes. Regions with consistently low or high SHAPE reactivities are highlighted by shading. (B) Structure models for cPPT- and PPT-containing elements in the final HIV-1 model.