Endothelial FGF signaling is protective in hypoxia-induced pulmonary hypertension

Hypoxia-induced pulmonary hypertension (PH) is one of the most common and deadliest forms of PH. Fibroblast growth factor receptors 1 and 2 (FGFR1/2) are elevated in patients with PH and in mice exposed to chronic hypoxia. Endothelial FGFR1/2 signaling is important for the adaptive response to several injury types and we hypothesized that endothelial FGFR1/2 signaling would protect against hypoxia-induced PH. Mice lacking endothelial FGFR1/2, mice with activated endothelial FGFR signaling, and human pulmonary artery endothelial cells (HPAECs) were challenged with hypoxia. We assessed the effect of FGFR activation and inhibition on right ventricular pressure, vascular remodeling, and endothelial-mesenchymal transition (EndMT), a known pathologic change seen in patients with PH. Hypoxia-exposed mice lacking endothelial FGFRs developed increased PH, while mice overexpressing a constitutively active FGFR in endothelial cells did not develop PH. Mechanistically, lack of endothelial FGFRs or inhibition of FGFRs in HPAECs led to increased TGF-β signaling and increased EndMT in response to hypoxia. These phenotypes were reversed in mice with activated endothelial FGFR signaling, suggesting that FGFR signaling inhibits TGF-β pathway-mediated EndMT during chronic hypoxia. Consistent with these observations, lung tissue from patients with PH showed activation of FGFR and TGF-β signaling. Collectively, these data suggest that activation of endothelial FGFR signaling could be therapeutic for hypoxia-induced PH

Genetic Variability and Evolutionary Implications of RNA Silencing Suppressor Genes in RNA1 of Sweet Potato Chlorotic Stunt Virus Isolates Infecting Sweetpotato and Related Wild Species

Peer reviewe

Viral RNase3 Co-Localizes and Interacts with the Antiviral Defense Protein SGS3 in Plant Cells

Sweet potato chlorotic stunt virus (SPCSV; family Closteroviridae) encodes a Class 1 RNase III endoribonuclease (RNase3) that suppresses post-transcriptional RNA interference (RNAi) and eliminates antiviral defense in sweetpotato plants (Ipomoea batatas). For RNAi suppression, RNase3 cleaves double-stranded small interfering RNAs (ds-siRNA) and long dsRNA to fragments that are too short to be utilized in RNAi. However, RNase3 can suppress only RNAi induced by sense RNA. Sense-mediated RNAi involves host suppressor of gene silencing 3 (SGS3) and RNA-dependent RNA polymerase 6 (RDR6). In this study, subcellular localization and host interactions of RNase3 were studied in plant cells. RNase3 was found to interact with SGS3 of sweetpotato and Arabidopsis thaliana when expressed in leaves, and it localized to SGS3/RDR6 bodies in the cytoplasm of leaf cells and protoplasts. RNase3 was also detected in the nucleus. Co-expression of RNase3 and SGS3 in leaf tissue enhanced the suppression of RNAi, as compared with expression of RNase3 alone. These results suggest additional mechanisms needed for efficient RNase3-mediated suppression of RNAi and provide new information about the subcellular context and phase of the RNAi pathway in which RNase3 realizes RNAi suppression.Peer reviewe

Suppression of RNAi by dsRNA-Degrading RNaseIII Enzymes of Viruses in Animals and Plants

Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)–mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.Peer reviewe

Functional characterization of a novel plant viral RNAi suppressor

Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus; family Closteroviridae) consists of a bipartite single-stranded RNA (ssRNA) genome. Open reading frames in RNA1 encode a Class 1 RNase III-like protein (designated as RNase3) and a 22 kDa protein (designated as p22). RNase3 and p22 suppress RNA silencing, the basal antiviral defense mechanism in plants. RNase3 is sufficient to render sweet potato (Ipomoea batatas) virus-susceptible and leads to the development of a severe disease (sweet potato virus disease - SPVD) following infection with unrelated viruses. This study aimed to identify how the genetic variability of silencing supressor genes in different SPCSV isolates affected the ability to suppress RNAi. No differences in silencing suppression capacities were detected for p22 and RNase3 proteins of tested SPCSV isolates. Furthermore, a new RNA virus (that belongs to the genus Crinivirus) was found to encode an RNase3 homolog functional in the suppression of RNAi. --- The double-stranded RNA (dsRNA)-specific RNase3 eliminates antiviral defense in sweet potato in an endoribonuclease activity dependent manner. RNase3 cleaves long dsRNA molecules, synthetic small-interfering RNAs (siRNAs), and plant- and virus-derived siRNAs. Studies in Nicotiana benthamiana have indicated that RNase3 inhibits only sense-transcript-induced post-transcriptional gene silencing (S-PTGS). In this study, conditions for efficient expression and purification of recombinant RNase3 were determined to establish activity assays for characterization of substrate specificity in vitro. RNA-binding was dependent on the dimerization of the protein confirming the affiliation of RNase3 to functional Class1 of RNase III enzymes. RNA-processing was assessed on a range of small dsRNA molecules consisting of mature double-stranded siRNA (ds-siRNA) and microRNA (miRNA) and revealed that small RNA (sRNA) containing asymmetric bulges (as they often appear in miRNA) and methylation did not support cleavage. ------ Some viruses infecting insects, fish and poikilothermic animals encode Class 1 RNase III-like molecules among which ascovirus RNase III was recently characterized as a silencing suppressor. It is still unknown how the different viral RNase III-type molecules act in the host cells. In vivo experiments carried out in N. benthamiana, Drosophila melanogaster and Caenorhabditis elegans revealed that a putative RNase3 homolog encoded by pike-perch iridovirus (designated as PPIV RNase3; genus Ranavirus; family Iridoviridae) was a Class 1 RNase III protein and suppressed RNAi in C. elegans and plant tissues in an endoribonuclease-dependent manner. However, SPCSV RNase3 only suppressed RNAi (in detail, S-PTGS) in plants. The viral Class 1 RNase III-like enzymes appear unique as homologous proteins produced by RNA and DNA viruses and are able to suppress RNAi in plants and animals. -------- The function of SPCSV RNase3 in RNAi suppression (limited to S-PTGS in plants) was postulated to be facilitated through localization or interaction with plant-host factors rather than substrate specificity. Experiments in N. benthamiana revealed that RNase3 localized in sub-cellular structures (punctate granules), which co-localized with characteristic granules that are implicated in antiviral defense and contain SGS3 and RDR6 proteins. Bimolecular fluorescence complementation studies revealed interaction of RNase3 with SGS3. However, no interference with associated trans-acting siRNA production or transitivity was detected. Keywords: Ipomoea, RNase III, RNAi, suppression of silencing, plant virus, Ranavirus, siRNA, miRNA, protein-protein interaction, SGS3, C. elegans.available shortl

Suppression of RNA silencing by the p22 and RNase3 proteins of SPCSV isolates and the RNase3-like protein of the new virus KML33b.

<p>Upper row of leaves: “Silencing on the spot” to induce “strong silencing” of the <i>gfp</i> gene for green fuorescent protein (GFP) was achieved by co-expressing <i>gfp</i> from one <i>A. tumefaciens</i> strain and double-stranded (hairpin) RNA homologous to <i>gfp</i> from another strain in coinfiltrated leaf tissue of <i>Nicotiana benthamiana</i>, and coinfiltration of a third strain expressing p22 protein to suppress gfp silencing. The p22 proteins of isolates ARU59 (<i>I. sinensis</i>) and HOM89 (sweetpotato) were compared with the previously characterized p22 protein of isolate Ug by expressing them at the opposite sites of the midrib in the same leaf. An <i>Agrobacterium</i> strain expressing ß-glucuronidae (GUS) was included as the negative control. Leaves were illuminated with UV light and photographed from the underside with a digital camera 3 days postinfiltration. Lower row of leaves: Cosuppression of <i>gfp</i> in transgenic <i>N. benthamiana</i> plants (line 16c) constitutively expressing <i>gfp</i> (note the green fluorescence in veins). The spots were co-infiltrated with a mixture of two <i>Agrobacterium</i> strains, one expressing <i>gfp</i> to achieve cosuppression (silencing) of <i>gfp</i> and another expressing RNase3 of isolate SOR71 (<i>I. obscura</i>), Ug, or the RNase3-like protein of the new virus KML33b (<i>I. sinensis</i>). Leaves were illuminated with UV light and photographed from the underside with a digital camera 6 days postinfiltration. </p

Symptoms and virus accumulation in single or double infections with Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV) in sweetpotato plants of cv.

<p>Tanzania. (<b>A</b>) A plant infected with SPFMV (isolate RUK73) shows no obvious virus symptoms 6 weeks post-inoculation. (<b>B</b>) Chlorosis and purpling of older leaves induced by SPCSV isolate HOM76. (<b>C</b>) Typical symptoms of sweetpotato virus disease (SPVD) including retarded growth, severe leaf strapping and puckering induced by co-infection with SPCSV isolate HOM76 and SPFMV. (<b>D</b>) Chlorosis and purpling of older leaves induced by SPCSV isolate SOR71, and (<b>E</b>) similar symptoms in a plant co-infected with SOR71 and SPFMV. Young leaves develop normally in (D) SOR71-infected plants and (E) plants co-infected with SOR71 and SPFMV. In both cases the plants display only mild chlorosis typical of SPCSV infection, which indicates that SOR71 is not able to induce SPVD in co-infection with SPFMV. (<b>F</b>) SPCSV RNA detected by dot blot hybridization with a digoxigenin-labelled RNA probe specific to the <i>RNase3</i> gene in (a) plants infected with SPCSV alone or (b) plants co-infected with the SPCSV and SPFMV. The amounts of total plant RNA dotted on the membrane are indicated. The SPCSV isolates tested were 1, KTK39; 2, KTK40; 3, KTK41; 4, SOR71; 5, MAS69; 6, SET5; 7, MSK7; 8, TOR16; and 9, HOM76. Isolates 1 to 4 are lacking the p22 gene. Note that SOR71 (4a and 4b) accumulates at very low concentrations in sweetpotato leaves and is barely detectable. H, non-inoculated healthy plant of cv. Tanzania. (<b>G</b>) SPFMV RNA detected by dot blot hybridization with a digoxigenin-labelled RNA probe specific to the CP-encoding region. Samples 1 to 9 are those co-infected with SPCSV and SPFMV and tested for SPCSV (i.e., samples 1b to 9b) in (F). Three additional samples co-infected with SPCSV (10, MAS46; 11, TOR14; and 12, MPG88) and SPFMV were included. Note that SOR71 synergises SPFMV, which is detected by the enhanced SPFMV concentrations (sample 4) as compared to the samples (FM) from cv. Tanzania infected with SPFMV only. H, non-inoculated healthy plant of cv. Tanzania.</p

Phylogenetic analysis of genes coding for RNase3 of <i>Sweet</i><i>potato</i><i>chlorotic</i><i>stunt</i><i>virus</i> and the corresponding sequence of an unknown related virus (KML33b) detected in this study.

<p>The branch of KML33b is not fully depicted. Names of isolates characterized from wild plants are indicated in bold, whereas the ten SPCSV isolates lacking the p22 gene are indicated with a black triangle (▲). Numbers at branches represent bootstrap values of 1000 replicates. Only bootstrap values of ≥ 50% are shown. Scale indicates Kimura units in nucleotide substitutions per site [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081479#B55" target="_blank">55</a>].</p

Alignment of the 43 different RNase3 protein amino acid (aa) sequences of <i>Sweet</i><i>potato</i><i>chlorotic</i><i>stunt</i><i>virus</i>.

<p>Groups of isolates containing identical RNase3 aa sequences are represented each by a single isolate. Numbers on top of the alignment indicate the aa positions with reference to isolate Ug (AJ428554) whereas numbers on the right indicate the number of the last amino acid at that position for each isolate. Numbers at the bottom of the alignment indicate aa positions in the new unknown virus (KML33b) related to SPCSV and detected in this study. The class 1 RNase III signature motif at aa positions 36-44 in SPCSV isolates (aa 42-50 in KML33b) is boxed. Two aa sites predicted to be under positive selection are indicated with black shades. Names of isolates characterized from wild plants are in bold.</p

Co-localization of RNase3 and RDR6 in epidermal cells of <i>N</i>. <i>benthamiana</i> following co-expression by agroinfiltration.

<p>The red signals of RNase3-dsRED and green signals of (a) AtRDR6-GFP (<i>A</i>. <i>thaliana</i>) and (b) NtRDR6-GFP (<i>N</i>. <i>tabacum</i>) co-localized in cytoplasmic, punctate bodies detected by confocal microscopy at 2 dpi. Images in (a) and (b) illustrate optical planes in which many RDR6-containing bodies were observed. Scale bars, 10 μm.</p