<i>S. stercoralis</i> ILP promoters are active in the nervous system and other tissues.

<p>Transgenic <i>S. stercoralis</i> post-free-living larvae expressing enhanced green fluorescent protein (EGFP) under the control of three insulin-like peptide (ILP) promoters were assessed for tissue-specific expression. (A–D) Transgenic larvae carrying the <i>Ss-ilp-1</i> promoter::<i>egfp</i> reporter construct; (A,C) DIC images and (B,D) fluorescent images. The <i>Ss-ilp-1</i> promoter is active in the hypodermis/body wall and a single pair of head neurons (D, arrow). (E–H) Transgenic larvae carrying the <i>Ss-ilp-6</i> promoter::<i>egfp</i> reporter construct; (E,G) DIC images and (F,H) fluorescent images. The <i>Ss-ilp-6</i> promoter is active in the hypodermis/body wall and several head neurons. (I–L) Transgenic larvae carrying the <i>Ss-ilp-7</i> promoter::<i>egfp</i> reporter construct; (I,K) DIC images and (J,L) fluorescent images. The <i>Ss-ilp-7</i> promoter is active in the intestine and a single pair of head neurons, with a single process that extends dorsally almost to the anterior portion of the intestine (L, arrow), most consistent with the SIAV neurons in <i>C. elegans</i>.</p

Identification of <i>S. stercoralis</i> heterotrimeric G protein orthologs.

<p>Identification of <i>S. stercoralis</i> heterotrimeric G protein orthologs.</p

Location of EGFP expression in transgenic <i>S. stercoralis</i> post-free-living larvae under the control of ILP promoters.

<p>Location of EGFP expression in transgenic <i>S. stercoralis</i> post-free-living larvae under the control of ILP promoters.</p

Transcript abundance of <i>S. stercoralis</i> homologs of <i>C. elegans</i> chemosensory 7TM GPCRs.

<p>Transcript abundance of <i>S. stercoralis</i> homologs of <i>C. elegans</i> chemosensory 7TM GPCRs.</p

Regulation of <i>C. elegans</i> dauer development by cellular signaling pathways.

<p>Dauer entry in <i>C. elegans</i> is regulated by four signaling pathways: a cyclic guanosine monophosphate (cGMP) signaling pathway, an insulin/IGF-1-like signaling (IIS) pathway, a dauer transforming growth factor β (TGFβ) signaling pathway, and a DAF-12 nuclear hormone receptor (NHR) regulated by dafachronic acid (DA) steroid ligands. Unfavorable conditions stimulate dauer entry (left panel) by down-regulating cGMP production, increasing expression of antagonistic insulin-like peptides that down-regulate IIS, decreasing expression of the dauer TGFβ ligand, and inhibiting production of DAs. When dauer larvae encounter favorable conditions, these pathways are hypothesized to act in reverse. Dotted lines signify down-regulated signaling through the pathway, while black lines signify up-regulated signaling through the pathway. Colored proteins are active; grayed out proteins are inactive. Grayed out ligands are absent. Adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004235#ppat.1004235-Fielenbach1" target="_blank">[12]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004235#ppat.1004235-McGrath1" target="_blank">[32]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004235#ppat.1004235-Wollam1" target="_blank">[43]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004235#ppat.1004235-Cornils1" target="_blank">[91]</a>.</p

<i>S. stercoralis</i> L3i are activated by 8-bromo-cGMP.

<p>The membrane-permeable cGMP analog, 8-bromo-cGMP, induced resumption of feeding, a hallmark of activation, in <i>S. stercoralis</i> L3i. Feeding was assessed by ingestion of a FITC dye after incubation at 37°C and 5% CO₂ in air for 24 hours for all conditions. (A) At 200 µM, 8-bromo-cGMP dissolved in M9 buffer results in potent resumption of feeding in L3i, with 85.1% (±2.2, SD) of larvae feeding after 24 hours. In comparison, host-like cues consisting of DMEM, 10% canine serum (S), and 12.5 mM reduced glutathione (G), resulted in 43.9% (±2.6, SD) of L3i feeding, while the M9 buffer negative control resulted in 0.6% (±0.3, SD) of L3i feeding, after 24 hours. (B) Kinetics of activation were determined for both 200 µM 8-bromo-cGMP and host-like cues, consisting of DMEM, 10% canine serum, and 3.75 mM reduced glutathione, after incubation for 4, 6, 12, 18, or 24 hours. All conditions were incubated for a total of 24 hours at 37°C and 5% CO₂ in air. Error bars represent ±1 standard deviation (SD).</p

Immunofluorescence microscopy of rVSV-SFTSV particles following UGCG knockdown.

<p><b>(A-D)</b> U-2 OS cells were transfected with siRNAs targeting UGCG (siUGCG) or a non-targeting control (siNegCtrl) and plated onto glass coverslips. At 72 hours post-transfection cells were chilled to 4°C on ice and rVSV-SFTSV was bound by centrifugation (1200xg, 30’, 4°C). Following centrifugation, media was replaced with pre-warmed media (37°C) and the cells placed in a 37°C incubator for 20 or 40 minutes before fixation in 1% paraformaldehyde for 15 minutes. Cells were then immunostained for viral antigen (anti-VSV M, red), cellular markers (green), and nuclei stained with DAPI (blue). Images are representative from at least 3 independent experiments. (<b>A,B</b>) U-2 OS cells fixed after 20 minutes were co-stained for rVSV-SFTSV (red) and EEA1 (A) or TGN46 (B) (green). (<b>C,D</b>) U-2 OS cells fixed after 40 minutes and stained as above. Boxes indicate zoomed-in regions. Scale bar represents 5μm.</p

Pharmacological inhibitors of UGCG activity inhibit rVSV-SFTSV entry.

<p><b>(A)</b> A549 cells were pre-treated with the UGCG inhibitor D,L-<i>threo</i>-PDMP for 24 hours before infection with rVSV-SFTSV. Drug was kept in the media throughout the infection. Ten hours post-infection cells were harvested, immunostained for VSV M, and analyzed by flow cytometry. Mean ± S.E.M. for 3 independent experiments. <b>(B)</b> A549 cells were pre-treated with the UGCG inhibitor N-butyldeoxynojirimycin-HCl (NB-DNJ) for 24, 48, or 72 hours before infection with rVSV-SFTSV. Drug was kept in the media throughout the infection. Ten hours post-infection, cells were harvested, immunostained for VSV M, and analyzed by flow cytometry. Mean ± S.E.M. for 3 independent experiments. <b>(C,D)</b> A549 cells were pre-treated with NB-DNJ (C) or N-(n-Butyl) deoxygalactonojirimycin (NB-DGJ) (D) for 48 hours before infection with rVSV-SFTSV, VSV, SV40, or RVFV. Drug was kept in the media throughout infection. Ten hours (rVSV-SFTSV, VSV, RVFV) or 24 hours (SV40) post-infection, cells were harvested, immunostained for viral antigen, and analyzed by flow cytometry. Mean ± S.E.M. for 3 independent experiments. **** p<0.0001 using Student’s t-test with Bonferroni correction.</p

Immunofluorescence microscopy and quantification of incoming virus particles.

<p>(<b>A, B</b>) A549 cells prepared as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006316#ppat.1006316.g008" target="_blank">Fig 8</a> were fixed 40 minutes post-warming and co-stained for rVSV-SFTSV (red) and the early endosome marker EEA1 (green) (A) or TGN46 (B). Cells were treated with NB-DNJ (bottom panels) or left untreated (top panels). Boxes indicate zoomed-in regions. Scale bar represents 5μm. (<b>C, D</b>) Quantitative image analysis was performed to measure the volume of discrete VSV M-stained puncta within z-stack images in untreated and NB-DNJ treated cells at both 20 and 40 minutes post-warming. Puncta were counted for at least 6 independent z-stacks per sample for both rVSV-SFTSV (C) and VSV (D) infected cells. (**** p<0.0001 using Welch’s one-tailed t-test).</p

Mechanistic studies on UGCG’s role in SFTSV entry.

<p><b>(A)</b> Binding and Internalization Assay. U-2 OS cells were transfected with negative control or UGCG siRNAs, replated the following day into 24 wells dishes, and the assay was performed 72 hours post-transfection. Assay details provided in Materials and Methods. vRNA levels were normalized to GAPDH mRNA levels, and are expressed relative to bound vRNA for the negative control siRNA. Mean ± S.E.M. for 3 independent experiments. <b>(B)</b> RNA collected from (A) was also analyzed for UGCG mRNA expression. UGCG mRNA levels were measured by RT-qPCR, normalized to GAPDH mRNA levels, and expressed relative to the negative control siRNA (bound). <b>(C,D)</b> The binding and internalization assay was carried out essentially as in (A) with the exception that U-2 OS cells were instead pre-treated with NB-DNJ for 48 hours prior to binding with rVSV-SFTSV (C) or SV40 (D) and qPCR for SV40 genomes did not require reverse transcription. Mean ± S.E.M. for 3 independent experiments. ** p<0.01 using Student’s t-test.</p