Protein expression and histochemistry.

<p>(A) Amino acid homology of pigeon CoV S1 and partridge CoV S1 to chicken CoV S1 (strain M41-S1); (B) Purified recombinant proteins analyzed by SDS-PAGE and western blotting using an antibody against the <i>Strep</i>-tag; (C) Schematic presentation of protein/spike histochemistry using TMAs.</p

Development of avian tissue microarrays (TMA).

<p>(A) Tissue cores from similar organs from ten avian species were grouped in multispecies TMAs and tissues from one species were grouped in single-species TMA; (B) representative example of arrangement of tissues in multi-species TMA stained with H&E.</p

Host tissue specificity of S1 proteins of coronaviruses.

<p>Protein histochemistry was performed using chicken, pigeon and partridge CoV S1 on tissues from their respective hosts.</p

Host binding specificity of chicken CoV M41 S1.

<p>Protein histochemistry was performed by applying S1 proteins onto the trachea in the multispecies TMA. Binding affinity of chicken CoV S1 to different avian species is indicated as—(no signal), + (mild), ++ (moderate), +++ (strong). To elucidate the fine glycan specificity of S1 to sialylated glycans. S1 protein was premixed with either sialic acids (SA) type I or type II lactosamines before applying to tissues as described in materials and methods.</p

Host specificity of HA of influenza virus H5N1.

<p>HA proteins (1 μg/ml) were applied to multispecies TMA containing respiratory tissues. Binding of HA to trachea of avian species from order <i>Galliformes</i> (A), <i>Anseriformes</i> (B), and <i>Columbiformes</i> (C) is shown.</p

Tissue binding profiles of S1 of chicken, pigeon, and partridge CoV to the organ systems of the respective host.

<p>Attachment was graded as follows:—no signal, + mild, ++moderate, +++ strong</p><p>Tissue binding profiles of S1 of chicken, pigeon, and partridge CoV to the organ systems of the respective host.</p

Glycan binding specificities of pigeon, partridge and chicken CoV S1.

<p>Before applying S1, TMAs were either treated non treated (S1) or pretreated with neuraminidase (NA), or S1 proteins were mixed with sialic acids (SA) type I or type II lactosamines.</p

Binding profiles of chicken CoV S1 in respiratory tissues of various avian species.

<p>Attachment of S1 was graded as follows:—no signal, + mild, ++moderate, +++ strong</p><p>Binding profiles of chicken CoV S1 in respiratory tissues of various avian species.</p

Clathrin-mediated endocytosis and late endosome-to-lysosome trafficking is required for MHV fusion.

<p><b>A</b>) Fusion assay upon siRNA-mediated gene silencing. Three different siRNAs per gene were transfected individually into HeLa-mCC1a-ΔM15. 72 h post transfection, cells were pre-loaded with FDG by hypotonic shock. MHV-αN was allowed to bind to the cells on ice at MOI = 20 for 90 min. 100 min post warming to 37°C, cells were collected and analyzed by FACS. Fusion was determined relative to the number of FIC-positive cells observed upon mock treatment of infected cells (UNTR). Error bars represent SEM, n = 3. <b>B</b>) Fusion of MHV upon treatment of cells with different inhibitors was studied as in A. Cells were pretreated with ammonium chloride (NH4Cl), Bafilomycin A1 (BafA1), Chloroquine (Chloq), Chlorpromazine (Chlopro), Monensin (Mon), Dynasore, Dyngo-4A, EIPA, Latrunculin A, (LatA), Jasplakinolide (Jasp), Cytochalasin B (CytoB), Cytochalasin D (DytoD), Nocodazole (Noc), U18666A, MG132, Brefelding A (BrefA), as well as with the solvents dimethyl sulfoxide (DMSO) and methanol (MeOH), protein synthesis inhibitor cyclohexamide (CHX), and MHV fusion inhibitor HR2 peptide (HR2) for 30 min at 37°C. The inhibitors were kept present during binding of MHV-αN to cells and during warming to 37°C cells for 100 min. Fusion was determined relative to the number of FIC-positive cells after mock treatment (UNTR). Error bars represent SEM, n = 3.</p

RNAi-mediated downregulation of endocytosis-associated proteins affects MHV infection.

<p><b>A</b>) Confirmation of endocytosis-associated hits from druggable genome-wide siRNA screen. Gene silencing was performed using individual transfection of three different siRNAs per gene in HeLa-mCC1a cells. Cells were infected with MHV-EGFPM at MOI = 0.5 for 8 h and analyzed by FACS for cell viability and virus replication. The effect of downregulation of expression on MHV infection was studied for the actin cytoskeleton-associated proteins ACTR2 and ACTR3 (orange), late endosomal proteins RAB7A and RAB7B (turquoise), HOPS complex sububit VPS39 (light green), ER/Golgi secretion-associated protein SNX1, Integrin/Actin-associated protein VCL, and Serine/Threonine-protein kinase PAK1 (grey). Error bars represent SEM, n = 4. <b>B</b>) Confirmation of siRNA-mediated reduction in mRNA levels. mRNA levels at 72 h post transfection were measured by qRT-PCR in comparison to non-transfected cells. Error bars represent SEM, n = 3*3. <b>C</b>) The effect of the RNAi-mediated downregulation of an extended set of endocytosis-associated proteins on MHV infection. Infection of MHV-EGFPM was analyzed after downregulation of proteins associated with caveolae-mediated endocytosis (light blue), clathrin-mediated endocytosis (dark blue), early endosomes (cerulean), actin cytoskeleton (dark orange), microtubule cytoskeleton (orange), late endosomes (turquoise), and late endosome-to-lysosome trafficking (light green) as described above. Error bars represent SEM, n = 3. <b>A, C</b>) Dotted lines show the lower 95% confidence interval of the negative siRNA controls.</p