11 research outputs found

    Dual Mechanisms Implemented by LIN-28 for Positive Regulation OF HBL-1 Are Necessary for Proper Development of Distinct Tissues in Caenorhabditis elegans

    Get PDF
    In Caenorhabditis elegans, the heterochronic pathway is comprised of a hierarchy of genes that control the proper timing of developmental events. hbl-1 (Hunchback Like-1) encodes an Ikaros family zinc-finger transcription factor that promotes the L2 stage cell fate events of the hypodermis. The downregulation ofhbl-1 is a crucial step for the transition from the L2 to the L3 stage. There are two known processes through which negative regulation of hbl-1 occurs: suppression of hbl-1 expression by 3 let-7 miRNAs through the hbl-1 3’UTR and inhibition of HBL-1 activity by LIN-46. The mechanisms by which hbl-1 is positively regulated have not yet been full defined. Currently, this positive regulation seems to be the responsibility of the conserved developmental regulator lin-28. lin-28 is purported to oppose the activities of the 3 let-7 miRNAs and the Caenorhabditis specific heterochronic gene lin-46. Here I demonstrate the removal of 3 let-7 miRNA binding sites in 3’UTR of hbl-1 does not abolish negative regulation of hbl-1 in seam cells. I find lin-28 negatively regulates lin-46 expression by direct binding of the 5’UTR of lin-46. I report a novel sterilely phenotype due to the loss of HBL-1 activity in postembryonic development. Due to the increased sensitivity of the somatic gonad to HBL-1 protein levels, I utilize the development of this tissue as an alternate means to study the genetic relationships between lin-28, lin-46 and hbl-1. My results suggest lin-28 acts through a branched pathway, partially bypassing lin-46 to positively regulate hbl-1 either through its 3’UTR or by targeting a third unknown factor

    A role for glycolipid biosynthesis in severe fever with thrombocytopenia syndrome virus entry

    Get PDF
    A novel bunyavirus was recently found to cause severe febrile illness with high mortality in agricultural regions of China, Japan, and South Korea. This virus, named severe fever with thrombocytopenia syndrome virus (SFTSV), represents a new group within the Phlebovirus genus of the Bunyaviridae. Little is known about the viral entry requirements beyond showing dependence on dynamin and endosomal acidification. A haploid forward genetic screen was performed to identify host cell requirements for SFTSV entry. The screen identified dependence on glucosylceramide synthase (ugcg), the enzyme responsible for initiating de novo glycosphingolipid biosynthesis. Genetic and pharmacological approaches confirmed that UGCG expression and enzymatic activity were required for efficient SFTSV entry. Furthermore, inhibition of UGCG affected a post-internalization stage of SFTSV entry, leading to the accumulation of virus particles in enlarged cytoplasmic structures, suggesting impaired trafficking and/or fusion of viral and host membranes. These findings specify a role for glucosylceramide in SFTSV entry and provide a novel target for antiviral therapies

    UGCG activity and expression important for wild-type SFTSV infection.

    No full text
    <p><b>(A)</b> U-2 OS cells were transfected with siRNAs to UGCG or a non-targeting control and 72 hours later were infected with SFTSV (strain HB29, MOI 0.1). Supernatants from infected cells were collected 48 hours post-infection and viral output was determined by plaque assay. Infectious output is expressed relative to the negative control siRNA. <b>(B)</b> U-2 OS cells were pre-treated with NB-DNJ for 48 hours before infection with SFTSV (MOI 5). Supernatants from infected cells were collected 12 hours post-infection and viral output was determined by plaque assay. Infectious output is expressed relative to the untreated control. *** p<0.001, **** p<0.0001 using Student’s t-test with Bonferroni correction.</p

    Immunofluorescence microscopy and quantification of incoming virus particles.

    No full text
    <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.

    No full text
    <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

    Pharmacological inhibitors of UGCG activity inhibit rVSV-SFTSV entry.

    No full text
    <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 of incoming virus particles, 20 minutes.

    No full text
    <p>(<b>A, B</b>) A549 cells were plated onto glass coverslips and the following day replaced with media containing NB-DNJ (200μM) or left untreated. Forty-eight hours later, cells were chilled to 4°C on ice, then 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 minutes before fixation in 2% paraformaldehyde for 10 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>) A549 cells co-stained for rVSV-SFTSV (red) and early endosome marker EEA1 (green). (<b>B</b>) A549 cells co-stained for rVSV-SFTSV (red) and <i>trans</i>-Golgi marker TGN46 (green). Boxes indicate zoomed-in regions. Scale bar represents 5μm.</p
    corecore