Journal: mBio

Article Title: Identification of the Cell-Surface Protease ADAM9 as an Entry Factor for Encephalomyocarditis Virus

doi: 10.1128/mBio.01780-19

Figure Lengend Snippet: A genome-wide haploid screen identifies novel host factors for EMCV. (A) Bubble plot indicating the significance of enrichment of gene trap insertions in genes ( y axis) in an EMCV-infected cell population compared to an uninfected control population. Each bubble represents a gene, with size corresponding to the number of gene trap insertions per gene. Genes were randomly distributed on the x axis. The names of the top 20 hits are indicated. (B) HAP1 cells deficient for various genes were infected with EMCV or coxsackievirus and adenovirus receptor (CAR)-binding CV-B3, followed by staining of capsid proteins (EMCV) or 3A protein (CV-B3) and nuclei (blue) at 5 h postinfection. Representative confocal micrographs are shown. The values (percentages) in the micrographs are the means ± standard errors of the means (SEM) for ≥3 technical replicates, normalized to the WT value. (C) HAP1 clones were infected with EMCV, followed by staining of capsid proteins (green) and nuclei (blue) at the indicated times postinfection. Representative confocal micrographs are shown. Percentage values denote means ± SEM for two to five technical replicates, normalized to the WT value at 5 h. (D) HAP1 clones were infected with EMCV or CV-B3, followed by crystal violet staining of surviving cells. The experiment was conducted twice with similar results.

Article Snippet: We performed such a screen with EMCV (strain Mengovirus, ATCC VR-1598) and identified, among others, genes involved in sialic acid biology ( SLC35A1 and CMAS ) and synthesis of sulfated glycosaminoglycans ( B3GAT3 and SLC35B2 ), two glycans that have previously been found to facilitate EMCV binding to cells ( ) ( ).

Techniques: Genome Wide, Infection, Control, Binding Assay, Staining, Clone Assay

Journal: mBio

Article Title: Identification of the Cell-Surface Protease ADAM9 as an Entry Factor for Encephalomyocarditis Virus

doi: 10.1128/mBio.01780-19

Figure Lengend Snippet: ADAM9 plays a role in an early step in EMCV infection. (A) HAP1 clones were infected with dNGluc-EMCV or Rluc-CV-B3. Luminescence was measured at 6 h postinfection. Dashed lines indicate the WT signal (top) and the signal in the presence of a replication inhibitor (bottom). Values are means plus SEM (error bars) from two independent experiments. Values were compared to the WT values, and statistical significance was calculated by an unpaired two-sided t test. Values that are significantly different from the WT value are indicated as follows: ** * , P < 0.001. Values that are not significantly different (ns) are indicated. (B) HAP1 clones were infected with dNGluc-EMCV in the presence of the replication inhibitor GPC-N114 (10 μM). Luminescence (in relative light units [RLU]) was measured at 6 h postinfection. Dashed lines indicate the WT signal (top) and the signal produced by uninfected cells (bottom). Values are means plus SEM (error bars) for three biological replicates. Statistical significance compared with the WT values were calculated by an unpaired two-sided t test of log-transformed data; ns, not significant; * , P < 0.05.

Article Snippet: We performed such a screen with EMCV (strain Mengovirus, ATCC VR-1598) and identified, among others, genes involved in sialic acid biology ( SLC35A1 and CMAS ) and synthesis of sulfated glycosaminoglycans ( B3GAT3 and SLC35B2 ), two glycans that have previously been found to facilitate EMCV binding to cells ( ) ( ).

Techniques: Infection, Clone Assay, Produced, Transformation Assay

Journal: mBio

Article Title: Identification of the Cell-Surface Protease ADAM9 as an Entry Factor for Encephalomyocarditis Virus

doi: 10.1128/mBio.01780-19

Figure Lengend Snippet: EMCV can use mouse ADAM9 to infect HAP1 cells, and this is independent of its metalloprotease activity. (A) HAP1 clones were pretreated for 30 min with batimastat and infected with Rluc-EMCV or Rluc-CV-B3 in the presence of batimastat. Luminescence was measured at 6 h postinfection. Dashed lines indicate the signal in the presence of the replication inhibitors dipyridamole (Dip) or guanidine hydrochloride (Gua). Values are means plus SEM (error bars) for four biological replicates. (B) HAP1 ADAM9 KO cells were transduced with murine leukemia virus particles harboring plasmid encoding catalytically inactive ADAM9 mutant E348A. Transduced cells were selected with hygromycin and subcloned. Cells were infected with EMCV, followed by staining of capsid proteins (green) and nuclei (blue). Representative confocal micrographs are shown. The values in the micrographs are mean ± SEM of the number of infected cells per field for two technical replicates.

Article Snippet: We performed such a screen with EMCV (strain Mengovirus, ATCC VR-1598) and identified, among others, genes involved in sialic acid biology ( SLC35A1 and CMAS ) and synthesis of sulfated glycosaminoglycans ( B3GAT3 and SLC35B2 ), two glycans that have previously been found to facilitate EMCV binding to cells ( ) ( ).

Techniques: Activity Assay, Clone Assay, Infection, Transduction, Virus, Plasmid Preparation, Mutagenesis, Staining

Journal: mBio

Article Title: Identification of the Cell-Surface Protease ADAM9 as an Entry Factor for Encephalomyocarditis Virus

doi: 10.1128/mBio.01780-19

Figure Lengend Snippet: ADAM9 is required for the entry phase of EMCV infection. (A) EMCV or CV-B3 were pretreated with the indicated concentrations of soluble ADAM9 protein or 100 μg/ml soluble FGFR1α/FGFR1β proteins for 1 h at 37°C and used to infect HAP1 cells. Capsid proteins (EMCV) or 3A protein (CV-B3) and nuclei (blue) were stained at 7 h postinfection. (B) HAP1 cells were pretreated with the indicated concentrations of antibody targeting ADAM9 and infected with virus for 7 h, followed by staining as described above for panel A. Representative confocal micrographs are shown in panels A, B, and C. Values (percentages) on the micrographs are mean ± SEM values for 3 or 4 (A) or 4 (B) technical replicates, normalized to the value for mock treatment. (C) WT, ADAM9 KO , or ADAM9 KO HAP1 cells overexpressing mouse ADAM9 mutant E348A (mADAM9) were incubated with EMCV or CV-B3 on ice, followed by qPCR analysis of bound virus. The values are means plus standard deviations (SD) (error bars) for three biological replicates. Experiments were conducted twice with similar results.

Article Snippet: We performed such a screen with EMCV (strain Mengovirus, ATCC VR-1598) and identified, among others, genes involved in sialic acid biology ( SLC35A1 and CMAS ) and synthesis of sulfated glycosaminoglycans ( B3GAT3 and SLC35B2 ), two glycans that have previously been found to facilitate EMCV binding to cells ( ) ( ).

Techniques: Infection, Staining, Virus, Mutagenesis, Incubation