Journal: Journal of Virology

Article Title: Genome-Wide Screening Uncovers the Significance of N-Sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection

doi: 10.1128/JVI.00432-17

Figure Lengend Snippet: Golgi compartment-resident TM9SF2-dependent localization and stability of NDST1. (A) The cell surface expressions of HS, syndecan-1, and glypican-3 in wild-type HAP1 and HAP1ΔTM9SF2 cells were analyzed by flow cytometry. The numbers indicate the G-MFIs. (B) Intracellular localization of endogenous TM9SF2. Cells were methanol fixed and double stained with anti-TM9SF2, anti-GM130, or anti-Golgin97 antibodies. Green, TM9SF2; red, GM130 or Golgin97 (Golgi apparatus markers). (C) The cell surface expression of TM9SF2 in wild-type HAP1 (middle) and mutant HAP1ΔTM9SF2 cells (right) was analyzed by flow cytometry. The numbers indicate the G-MFIs. Wild-type HAP1 cells stained only with secondary antibody were used as the negative control (left). (D) Molecular sizes of glypican-3 in various HAP1-derived knockout and rescued cells. Cell lysates of these cells were subjected to immunoblotting to analyze the expression of glypican-3. (E) Analysis of N-sulfation and HS chain numbers in wild-type HAP1 (wild type+vector), TM9SF2-knockout cells (ΔTM9SF2+vector), and rescued TM9SF2-knockout cells (ΔTM9SF2+TM9SF2). Cells were incubated with or without 0.3 U of heparinase III at 37°C for 1 h and then stained with F58-10E4 (10E4) to detect HS expression and F69-3G10 (3G10) to determine HS chain numbers. Cells stained only with secondary antibody (Alexa 488-conjugated anti-mouse Ig or IgM antibody) were used as the negative control (without first antibody). Solid line, no enzyme treatment; dotted line, heparinase III treatment. (F) Quantitative analysis of the results in panel E. Relative ratios of cell surface N-sulfated HS (left columns), HS chain numbers (middle), and N-sulfates per HS chain (right) were calculated from the G-MFI values detected in panel E. The data represent the means ± the SE for three independent experiments. *, P < 0.05; **, P < 0.01. (G) Expression and intracellular localization of exogenously expressed EGFP-fused NDST1 (NDST1-EGFP) in wild-type HAP1 and HAP1ΔTM9SF2 cells. Cells stably expressing NDST1-EGFP were fixed and stained with anti-GM130 antibody. The pictures were taken under the same conditions. (H) Stability of NDST1-EGFP in HAP1ΔTM9SF2 cells. The construct shown in the upper panel, from which NDST1-EGFP and puro-T2A-BFP were cistronically transcribed, was stably integrated into wild-type HAP1, HAP1ΔNDST1, and HAP1ΔTM9SF2 cells. The stability of NDST1-EGFP was calculated as the ratio of EGFP intensity to the BFP intensity, which was measured by flow cytometry. The data represent the means ± the SD for two independent experiments. **, P < 0.01. (I) The susceptibilities of NDST1 and TM9SF2 knockout cells to CHIKV Thai#16856, SINV, JEV, and YFV were examined. The susceptibility of each cell is shown as the virus titer (CCID50/50 μl). The data represent the means ± the SD for three independent experiments. Statistical significances between wild-type HAP1 and other HAP1 mutants or between mutant cell lines (showed on the horizontal bar) are indicated (*, P < 0.05; **, P < 0.01; ns, not significant).

Article Snippet: The mouse MAb clones used here were as follows: F58-10E4 against HS (US Biologicals, Salem, MA), CS56 against chondroitin sulfate (CS; Abcam, Cambridge, United Kingdom), 1G12 against glypican-3 (Santa Cruz Biotechnology, Santa Cruz, CA), MI15 against syndecan-1 (BioLegend, San Jose, CA), and F69-3G10 against delta HS and clone 35 anti-GM130 (BD Biosciences, San Jose, CA).

Techniques: Flow Cytometry, Staining, Expressing, Mutagenesis, Negative Control, Derivative Assay, Knock-Out, Western Blot, Plasmid Preparation, Incubation, Stable Transfection, Construct, Virus