Identification of NPHV/HCV chimeras capable of replication and infectious virus production in Huh-7.5 cells.

<p><b>(A)</b> Schematic showing the secondary structures of 5’UTR regions of HCV/NPHV chimeras that contain the NPHV entire 5’UTR (NPHV-5’UTR), IRES (NPHV-IRES), Stem Loop 1 with microRNA Binding Region (miRBR) (NPHV-SL1/miRBR), or only SL1 (NPHV-SL1). All chimeras were constructed on the HCV J6/JFH Clone 2 backbone. <b>(B,E)</b> NS5A positive cells post transfection of Huh-7.5 cells. Results represent mean±SEM from 3 independent transfections. <b>(C,F)</b> Infectious virus production quantified by limiting dilution assay on naïve Huh-7.5 cells post transfection (n = 3). <b>(D)</b> Schematic of predicted miR-122 binding modes to NPHV and HCV. The adaptive mutation of NPHV-SL1/miRBR at C83A site is indicated. Asterisks, *p < 0.05, **p < 0.01, Student’s t test.</p

Evolutionary selected NPHV/HCV variants can replicate and produce infectious particles in non-hepatic cells.

<p><b>(A)</b> Quantification of NS5A positive cells on day 2 post-transfection of 293T cells with HCV and NPHV/HCV variants. <b>(B)</b> Quantification of NS5A positive 293T cells post-transfection as in (A) but compared to a daclatasvir (DCV) treated culture. <b>(C)</b> Infectious virus production from 293T cells with or without exogenous ApoE expression. Virus titers were quantified by limiting dilution assay on naïve Huh-7.5 cells. In (A)-(C), results represent mean±SEM from 3–6 independent experiments. Asterisks, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Student’s t test.</p

Growth kinetics of wild type HCV and NPHV/HCV chimeras in ΔmiR-122 Huh-7.5 cells.

<p><b>(A)</b> NS5A positive cells post transfection in Huh-7.5 (open bars) and ΔmiR-122 Huh-7.5 cells (hatched bars). Results represent mean±SEM from 3 independent transfections. <b>(B)</b> Infectious virus production of supernatants from transfected ΔmiR-122 Huh-7.5 and Huh-7.5 cells. Virus titers were quantified by limiting dilution assay on naïve Huh-7.5 cells (n = 3).</p

AGO-CLIP provides a miRNA binding map for NPHV <i>in vivo</i>.

<p><b>(A)</b> Schematic of standard AGO-CLIP and CLEAR-CLIP. After UV-induced cross-linking of RNA-protein complexes, precipitation of the AGO complex and RNA library preparation, standard AGO-CLIP provides a map of AGO/miRNA interactions across the transcriptome. In CLEAR-CLIP, chimeras of miRNAs with cellular or viral RNAs are induced. Analysis of these allows unambiguous detection of specific miRNA interactions. <b>(B)</b> <i>In vivo</i> AGO/miRNA binding across the NPHV genome from horse liver. Binding is observed across the four miR-122 seed sites conserved among all published isolates. Non-conserved sites present in the NZP1 isolate are indicated.</p

miR-122 but not other miRNAs binds the NPHV 5’UTR.

<p><b>(A)</b> Percentage of NS5A positive ΔmiR-122 Huh-7.5 or Huh-7.5 cells post-transfection with the indicated virus constructs. Results represent mean±SEM from 3 independent transfections. <b>(B)</b> Standard AGO (left) and miRNA-specific chimera-derived (right) binding maps on the 5’ end of NPHV-SL1/miRBR (top) and NPHV-SL1/miRBR-m15 (bottom) in Huh-7.5 cells four days post-transfection. Chimera-supported specific miRNA binding across the entire viral genome is shown below for both panels. The binding site and chimera-supported interactions for miR-122 are shown in red and those of miR-15 in blue.</p

Graphical View of All Duplicated Segments

<p>The 12 chromosomes are depicted along the perimeter of a circle, not in order but slightly rearranged so as to untangle the connections between segments. Overall, we cover 65.7% of the genome.</p

Functional Classifications from GO, Focused on Plant-Specific Categories Outlined by Gramene

<p>(A) compares predicted genes from <i>Arabidopsis</i> and Beijing <i>indica</i>. (B) compares predicted genes from Beijing <i>indica</i> with nr-KOME cDNAs. We ignore categories with less than 0.1% of the genes.</p

A Region on Beijing <i>indica</i> Chromosome 2, Showing Three Gene Islands Separated by Two Intergenic Repeat Clusters of High 20-mer Copy Number

<p>Transposable elements identified by RepeatMasker are classified based on the nomenclature of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030038#st002" target="_blank">Table S2</a>. Depicted genes include both nr-KOME cDNAs and FGENESH predictions.</p

Distribution of Substitutions per Silent Site (Ks) for Homolog Pairs in Segmental, Tandem, and Background Duplications

<p>In (A), contributions from the recent segmental duplication on Chromosomes 11 and 12 are colored in red. The tandem duplication data are shown on two different scales, one to emphasize the magnitude of the zero peak (B) and another to highlight the exponential decay (C). Background duplications are shown in (D).</p

Overlapping FGENESH Predictions in All Three Rice Assemblies

<p>Two predictions are shared when 50% of their coding regions can be aligned. Because of imprecision in the predictions and overlap criteria, we get slightly different numbers for each assembly, and these are encoded through multiple color-coded numbers in the Venn diagram. EST confirmation requires 100 bp of exact match. Unlike the genes, we do not bother to show a different number for each assembly, because they are very similar.</p