Evolutionary Selection Against Short Nucleotide Sequences in Viruses and Their Related Hosts

Viruses are under constant evolutionary pressure to effectively interact with the host intracellular factors, while evading its immune system. Understanding how viruses co-evolve with their hosts is a fundamental topic in molecular evolution and may also aid in developing novel viral based applications such as vaccines, oncologic therapies, and anti-bacterial treatments. Here, based on a novel statistical framework and a large-scale genomic analysis of 2,625 viruses from all classes infecting 439 host organisms from all kingdoms of life, we identify short nucleotide sequences that are under-represented in the coding regions of viruses and their hosts. These sequences cannot be explained by the coding regions’ amino acid content, codon, and dinucleotide frequencies. We specifically show that short homooligonucleotide and palindromic sequences tend to be under-represented in many viruses probably due to their effect on gene expression regulation and the interaction with the host immune system. In addition, we show that more sequences tend to be under-represented in dsDNA viruses than in other viral groups. Finally, we demonstrate, based on in vitro and in vivo experiments, how under-represented sequences can be used to attenuated Zika virus strains

Melatonin expression patterns in <i>Nematostella vectensis</i> developmental stages.

<p>Confocal sections of whole mounts indicated that melatonin is enriched near the area of invagination in the early gastrula stage (A) and at the oral and aboral poles during the late gastrula stage of the forming planulae (B). In swimming planulae (older larvae with an actinopharynx and developing mesenteries), melatonin exhibited a definite early preference for the actinopharynx and the apical tuft (at), both of which are highly neuralized components of the developing nerve net (C). By the 4-tentacle primary polyp stage (D, E), this preference extends to include other neural areas, such as the tentacle (tn) tips (arrowheads) and mesenteries (mes, arrowhead). The concentration of melatonin in the developed mesenteries is very clear in (E). The asterisks denote the oral poles. Scale bars: A-E = 50 µm.</p

Melatonin Distribution Reveals Clues to Its Biological Significance in Basal Metazoans

<div><p>Although nearly ubiquitous in nature, the precise biological significance of endogenous melatonin is poorly understood in phylogenetically basal taxa. In the present work, we describe insights into the functional role of melatonin at the most “basal” level of metazoan evolution. Hitherto unknown morphological determinants of melatonin distribution were evaluated in <em>Nematostella vectensis</em> by detecting melatonin immunoreactivity and examining the spatial gene expression patterns of putative melatonin biosynthetic and receptor elements that are located at opposing ends of the melatonin signaling pathway. Immuno-melatonin profiling indicated an elaborate interaction with reproductive tissues, reinforcing previous conjectures of a melatonin-responsive component in anthozoan reproduction. In situ hybridization (ISH) to putative melatonin receptor elements highlighted the possibility that the bioregulatory effects of melatonin in anthozoan reproduction may be mediated by interactions with membrane receptors, as in higher vertebrates. Another intriguing finding of the present study pertains to the prevalence of melatonin in centralized nervous structures. This pattern may be of great significance given that it 1) identifies an ancestral association between melatonin and key neuronal components and 2) potentially implies that certain effects of melatonin in basal species may be spread widely by regionalized nerve centers.</p> </div

The detection of melatonin by HPLC.

<p>Overlaid chromatograms of a melatonin standard (green, 1×10⁻⁸ gr mL⁻¹) and a sea anemone tissue extract (<i>N. vectensis</i>) with (black) and without (red) melatonin enrichment (1×10⁻⁸ gr mL⁻¹). Chromatographic separation of tissue extracts was conducted based on fluorimetric detection (λex = 280 nm and λem = 345 nm). The mobile phase consisted of a mixture of 0.1% (v/v) formic acid in acetonitrile:water 17∶81 (v/v), which was delivered isocratically at a flow-rate of 1 mL min⁻¹. The detailed conditions are described in the Materials and Methods.</p

A schematic diagram of <i>Nematostella</i> illustrating the combinatorial expression of putative biosynthetic (<i>HIOMT</i>) and receptor gene elements overlaid with the distribution of melatonin immunoreactivity in different morphological features of the sea anemone.

<p>The neural architecture in <i>Nematostella</i> (as assayed by FMRFamide, which is a known neuro-marker in the anthozoan nervous system <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052266#pone.0052266-Grimmelikhuijzen2" target="_blank">[34]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052266#pone.0052266-Pernet1" target="_blank">[36]</a>), overlaps with melatonin immunoreactivity in several areas. <i>HIOMT</i> expression patterns are highly correlated with melatonin immunoreactivity in key neural areas and reproductive tissues, corresponding to abundant expression of putative melatonin receptors. Varying levels in the amount/abundance of melatonin or mRNA expression of the gene elements among different body regions are schematically represented in the diagram by varying color intensities or by the number of consecutive cells that indicate the presence of a particular element.</p

Gene expression patterns of putative melatonin receptors in <i>Nematostella vectensis</i>.

<p>Two representative melatonin receptor orthologs were evaluated using in situ hybridization (ISH) with specific probes. (A, B) A similar expression pattern between orthologs in adult polyps was observed and indicated that the mRNA for the putative receptor was predominantly expressed in the circumference of the actinopharynx (ph) and at substantially lower levels in the hypostome. (C) Higher magnification of the actinopharynx area indicated that the melatonin receptors are also expressed in the inner epithelium (arrowhead); albeit at lower levels (D) Substantial melatonin receptor expression was evident in fertile gonads (peripheral surfaces of endodermal cells), implying a reproduction-related receptor-mediated mechanism of action for melatonin. (E, F) The expression of putative melatonin receptors in the body wall was predominantly characterized by an endodermal distribution but was also sporadically observed at lower levels in both ectodermal cells and in the epithelial muscular layer (arrowheads) at the base of the endoderm. Scale bars: A = 500 µm; B = 200 µm; C = 100 µm; D, E = 50 µm; F = 20 µm.</p

The expression pattern of hydroxyindole-O-methyltransferase (<i>HIOMT</i>) mRNA in <i>Nematostella vectensis</i>.

<p>Two representative <i>HIOMT</i> orthologs were evaluated using in situ hybridization (ISH) with specific probes. (A–C) The <i>HIOMT</i> expression pattern was similar between orthologs and indicated that melatonin is predominantly produced in the circumference of the actinopharynx (ph). High <i>HIOMT</i> expression levels were also evident in the endodermal layer of the hypostome (retracted individual, B). A higher magnification image of the outer surface of the endothelium in the actinopharynx folds (C). (D, E) Substantial <i>HIOMT</i> expression in reproductive tissues suggested that the considerable level of the melatonin that is observed in these tissues (see Fig. 1E–F) is locally produced. A uniform <i>HIOMT</i> expression pattern was observed among the cells. (F, G) In the body wall, predominantly endodermal <i>HIOMT</i> expression was observed throughout the apical end of the anemone and was uniformly distributed throughout the cells. This pattern differed from the pattern of melatonin immunoreactivity (see Fig. 1B–C). Note that melatonin production occurs also in tentacles (tn), albeit at lower levels. Endoderm (en), ectoderm (ec). Scale bars: A = 200 µm; B, C = 100 µm; E, F = 50 µm; D, G = 20 µm.</p

Confocal colocalization of melatonin and the <i>Nematostella vectensis</i> neural network in adult polyps.

<p>(A) The distribution of melatonin immunoreactivity (red) generally corresponded with RFamide-expressing neurons (green). (B) Neuro-melatonin interactions were implied by specific colocalization (orange) along both the major longitudinal fasciculated neurite tracts and the minor inter-crossing neuronal pathways (arrowheads). (C) Melatonin distribution also paralleled the neural tracts in other areas of the <i>N. vectensis</i> neural net, such as the tentacles and the mesenterial endomesodermal cells that were proximate to the actinopharynx (arrowheads). (D, E) Confocal sections in the pharyngeal nerve ring area indicated neuro-melatonin associations. The asterisk denotes the oral pole. Scale bars: A = 500 µm; B, C-E = 200 µm.</p

The colocalization of melatonin and the neural network in <i>Nematostella vectensis</i> sections.

<p>(A–C) An overlay of FMRFamide (green) and melatonin (red), revealing little colocalization (orange) in the hypostome (A), extensive colocalization in the actinopharynx (ph) (B) and sporadic neuro-melatonin interactions in a subset of the fertile gonad cells (arrowheads) (C). (D–F) Pervasive colocalization of melatonin and FMRFamide was observed in several neurons in the body wall (arrowheads) (D); however, sensory cells (sc), ganglion cells (gc) and basiepithelial neurites (ne) were generally not melatonin-positive despite being located in highly melatonin-enriched surroundings (E–F). Ectoderm (ec). Scale bars: A, B = 50 µm; C, D = 20 µm; E, F = 10 µm.</p

The distribution of melatonin immunoreactivity in <i>Nematostella vectensis</i> polyps.

<p>(A) Overlaid fluorescent and differential interference contrast (DIC) micrographs of a young adult polyp that was stained with a melatonin-specific antibody (red). Melatonin accumulation is noticeable in the tentacle tips (tn), mesenteries (mes) and actinopharynx (ph). Oral ring (or). (B–C) Confocal micrographs of melatonin immunoreactivity in <i>Nematostella</i> sections. Melatonin appears to be unevenly distributed throughout the layers (arrow) of the hypostomal body wall (retracted polyp), implying layer-specific differences in melatonin sources/targets (B, C). (D) Increased magnification of extensive melatonin accumulation in the outer surfaces of the endothelium in the actinopharynx folds (arrowheads in B). (E) The punctate immunoreactive pattern lining the external surfaces of the gonads suggests a reproductive role for melatonin. (F) Uniform melatonin immunoreactivity was detected in gonadal endodermal cells. (G-I) Melatonin immunoreactivity was observed within both endodermal and ectodermal cells in the body wall; the endodermal signals were generally stronger. Occasionally, melatonin accumulated at the base of the endoderm, implying an interaction with epithelial muscular cells (arrowhead). Scale bars: B = 200 µm; A, E = 100 µm; C, D, F, G = 20 µm; I = 10 µm; H = 5 µm.</p