Codon alignment and predicted pairing partners in the Gag-Pro-Pol frameshift region of SIVmac239 and HIV-1_NL4-3.

<p>(A) RNA structures at the Gag-Pro-Pol frameshift stem for SIVmac239 (left) and HIV-1 (right). The main stem is emphasized by green brackets and the poly(U) slippery sequence is emphasized by a curved line. The dotted line indicates nucleotides between the poly(U) sequence and the beginning of the frameshift stem. (B) The sequences of HIV-1, SIVagm, and SIVmac239 were aligned horizontally. The poly(U) slippery sequence is boxed. Curved lines represent base pairs between the nucleotides within HIV-1 and SIVmac239; the curved lines connecting nucleotides downstream of the poly(U) sequence correspond to the frameshift stems (green), the dotted line represents the extended frameshift stem in HIV-1. Gray boxes indicate regions of strong alignment, and spaces in the sequence indicate regions of poor alignment. The structure of the HIV-1 hairpin is modified from the previous model <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003294#ppat.1003294-Watts1" target="_blank">[17]</a> based on the updated folding parameters as described in the Supporting Material.</p

Genomic organization and SHAPE reactivity of SIVmac239 and comparison with HIV-1_NL4-3.

<p>(A) Organization of the SIVmac239 genome. Gray boxes indicate protein coding regions, dark lines indicate the boundaries of the mature viral proteins. (B) SIVmac239 median SHAPE reactivity values calculated over a 75 nucleotide sliding window (red). Regions with SHAPE reactivities below 0.3 are numbered (and listed explicity in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003294#ppat.1003294.s010" target="_blank">Table S2</a>). SHAPE reactivity values of HIV-1_NL4-3 (gray) are shown as medians calculated over a 75 nucleotide sliding window and overlayed with those of SIVmac239. Viruses were codon-aligned based on the Los Alamos Database alignment (<a href="http://www.hiv.lanl.gov" target="_blank">www.hiv.lanl.gov</a>). Green dashed line indicates SHAPE reactivity of 0.4, and the gray bars below indicate regions where median reactivity values for both viruses are below 0.4. SHAPE reactivities for SIVmac239 and HIV-1_NL4-3 are included in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003294#ppat.1003294.s006" target="_blank">Datasets S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003294#ppat.1003294.s008" target="_blank">S4</a>, respectively. (C) Percent concordance of SHAPE reactivity and pairing prediction of the SIVmac239 genome over a 76 nucleotide sliding window. A concordant nucleotide is defined as having low SHAPE reactivity (below 0.4) and predicted to be paired or high reactivity (0.4 or above) and predicted to be unpaired.</p

Profiles of HIV-1^wt and HIV-1^SLSA1m transcripts.

<p>(A) Diagram displaying reading frames (open boxes) of the HIV-1_NL4-3 genome. Solid lines indicate different classes of mRNA including unspliced, 4 kb, and 1.8 kb, with their corresponding genes labeled on the right. Gray boxes represent exons 2 (between SA1 to SD2) and 3 (between SA2 and SD3). Splice donors (SD1-4) and acceptors (SA1-7) are labeled on the top of the unspliced length of RNA The sites of NarI cleavage and primer-binding for the forward and reverse primers used to create the splicing profile are shown on the unspliced RNA. (B) SHAPE-derived structure for the SLSA1 hairpin in HIV-1_NL4-3. The first splice acceptor (SA1) is labeled. The mutated nucleotides in HIV^SLSA1m are boxed, and the mutations are identified next to the arrows. Exonic splicing enhancer sequences (ESEVif and ESEM1) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003294#ppat.1003294-Exline1" target="_blank">[52]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003294#ppat.1003294-Mandal1" target="_blank">[71]</a> are labeled. (C) Splicing profiles for HIV-1^wt and HIV-1^SLSA1m grown for three days in CEMx174 cells are shown. The cDNA from the 1.8 kb class (left) or the 4 kb class (right) of transcripts, amplified with the corresponding primers, was separated on a 6% polyacrylamide gel and labeled according to common nomenclature <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003294#ppat.1003294-Purcell1" target="_blank">[10]</a> (solid lines) or left unidentified (dotted lines). Decreased band intensity between the WT and SLSA1m transcripts is marked by thicker lines.</p

Comparison between SIVmac239 and HIV-1_NL4-3 RNA genome secondary structure models.

<p>SHAPE-directed folding used Δ<i>G</i>_SHAPE parameters of m = 1.9 and b = −0.7.</p

Codon alignment and predicted pairing partners in the stem-loop surrounding SA1 of SIVmac239 and HIV-1_NL4-3.

<p>(A) Structures for the conserved stem in SIVmac239 (left) and HIV-1_NL4-3 (right). Blue lines indicate the base pairs that are exactly conserved between the two viruses. (B) The sequences of SIVmac239 (top) and HIV-1_NL4-3 are aligned horizontally. Curved lines indicate base-pairing partners. Gray boxes indicate regions of amino acid alignment. Bold letters represent the bases that are involved in the conserved pairing interactions.</p

Model for the structure of the SIVmac239 RNA genome as determined by SHAPE probing and directed RNA structure refinement.

<p>The genome is divided into (A) 5′ and (B) 3′ halves. Colors of nucleotides indicate SHAPE reactivity on the scale shown on the left. Each sphere corresponds to a nucleotide, and side-by-side spheres indicate a base pair. Protein coding region boundaries are indicated by letters with the code shown at the bottom. Splice acceptor and donor sites <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003294#ppat.1003294-Victoria1" target="_blank">[49]</a> are labeled SA and SD, respectively. tRNA^Lys3 interaction is shown in gray. Heavy blue bars indicate base pairs in stems that are conserved between codon-aligned SIVmac239 and HIV-1_NL4-3 RNA structures (71 total pairs). Areas of structure with a median reactivity below 0.3 over a 75 nucleotide window are numbered in green and correspond to motif numbers in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003294#ppat-1003294-g002" target="_blank">Figure 2B</a>. All positions are numbered in reference to the GenBank accession number M33262 for SIVmac239. A full structure, including nucleotide identity, is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003294#ppat.1003294.s001" target="_blank">Figure S1</a>. Helix files for SIVmac239 and HIV-1_NL4-3 are included in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003294#ppat.1003294.s005" target="_blank">Datasets S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003294#ppat.1003294.s007" target="_blank">S3</a>, respectively.</p