Journal: Nature Communications

Article Title: Human-type sialic acid receptors contribute to avian influenza A virus binding and entry by hetero-multivalent interactions

doi: 10.1038/s41467-022-31840-0

Figure Lengend Snippet: a Graphical summary of methods that were employed to establish density gradients on HEK ΔSia cells and LAMP I. The Sia repertoire exposed on HEK ΔSia cells was specifically tuned by transfection with different concentrations of ST6Gal1 and/or ST3Gal4. Co-transfected LAMP I will be secreted in a C-terminally biotinylated form that can be attached to streptavidin BLI sensors for comparing entry into HEK ΔSia cells with binding to LAMP I with a similar sialylation pattern in a BLI assay. b , c Comparison of virus binding rates to avian-type and human-type receptors. Absolute binding rates (nm/300 s), normalized to a particle number of 10 9 particles of each virus strain, were determined. b Binding to sensors fully loaded with LAMP I produced in HEK ΔSia cells co-transfected with 25 ng ST6Gal1 or 25 ng ST3Gal4 yielding high levels of LacNAc termini carrying 2-6Sia (magenta bars) or 2-3Sia (black bars). c Binding to sensors fully loaded with 2-6S(LN)2 (magenta bars) or 2-3S(LN)2 (black bars). Relative binding rates were plotted as a function of relative receptor density for human H3N2 strains BK79 ( d ) and WU95 ( e ), avian H5N1 strain HU02 ( f ) and human H1N1 strain PR8 ( g ). Black lines show binding to LAMP I proteins obtained by co-expression with a concentration range of sialyltransferases ST6Gal1 ( d , e ) or ST3Gal4 ( f , g ). Magenta lines show binding to LAMP I proteins obtained by co-expression of a concentration range of ST6Gal1 in combination with 25 ng ST3Gal4 ( d , e ) or a concentration range of ST3Gal4 in combination with 25 ng ST6Gal1 ( f , g ). 2-6Sia density or 2-3Sia density on LAMP I was quantified by SNA or MAL I binding respectively and plotted on the x-axis as relative SNA ( d , e ) or MAL I ( f , g ) signal reflecting the relative receptor density. Data from biological replicates were fitted into single curves of which R-sq values are indicated. ( b n = 7, 3, 8, 8, 3, 3, 4, 4, 3, 3, 9 and 4 for each bar from left to right; c n = 6, 6, 4, 8, 3, 3, 4, 7, 3, 3, 5 and 4 for each bar from left to right). Data are presented as mean values +/−S.D. Panel a was Created with BioRender.com. Source data are provided as a Source Data file.

Article Snippet: To specifically tune sialylation patterns on a secreted recombinant, C-terminally biotinylated, glycoprotein, specific sialyltransferases encoding expression vectors (pCDNA-ST3Gal4 and/or pCDNA-ST6Gal1) (Genscript, Piscataway, NJ, USA, NM_1254757.2; NM_173216.2) and an expression vector (pCD5) encoding a BirA biotin ligase were co-transfected with an expression vector coding for the extracellular domain of lysosomal membrane protein I (LAMP I) fused to a C-terminal biotinylation site.

Techniques: Transfection, Binding Assay, Comparison, Virus, Produced, Expressing, Concentration Assay

Journal: Nature Communications

Article Title: Human-type sialic acid receptors contribute to avian influenza A virus binding and entry by hetero-multivalent interactions

doi: 10.1038/s41467-022-31840-0

Figure Lengend Snippet: a – d Virus entry efficiency into HEK ΔSia cells transfected with a concentration range of sialyltransferases ST6Gal1 (magenta lines) or ST3Gal4 (black lines) was determined using a luciferase reporter and a single-round infection assay. Relative entry efficiency into cells (normalized to the highest value) was plotted as a function of transfected sialyltransferase quantities. Data are from four biological replicates and each point represents the mean of 3 measurements. Entry often reached a maximal efficiency followed by a decline upon further increase of transfected sialyltransferase concentration. Data points beyond maximal entry efficiency were not fitted to the curves. e Comparison of virus binding and entry kinetics. Relative entry efficiency (black line) and binding rate to LAMP I (solid magenta line) or 2-6 S(LN)2 (dotted magenta line) of human H3N2 strain BK79 was plotted as a function of receptor density. The lower x-axis shows the amount of ST6Gal1 that was used for transfection of HEK ΔSia cells into which entry was performed or in which LAMP I glycoprotein receptor was expressed. Top x -axis shows the density of 2-6S(LN)2 receptor as percentage of the maximal density of 0.6 pM/cm 2 (corresponding to ~0.0036 Sia moieties per nm 2 ). BLI binding data for BK79 come from experiments shown in Fig. (2-6S(LN)2) and Fig. (LAMP I). In addition relative SNA binding (performed to the same sensors as used for virus binding) was plotted in parallel (teal lines). Entry data for BK79 were derived from the experiment shown in Fig. 4a. Source data are provided as a Source Data file.

Article Snippet: To specifically tune sialylation patterns on a secreted recombinant, C-terminally biotinylated, glycoprotein, specific sialyltransferases encoding expression vectors (pCDNA-ST3Gal4 and/or pCDNA-ST6Gal1) (Genscript, Piscataway, NJ, USA, NM_1254757.2; NM_173216.2) and an expression vector (pCD5) encoding a BirA biotin ligase were co-transfected with an expression vector coding for the extracellular domain of lysosomal membrane protein I (LAMP I) fused to a C-terminal biotinylation site.

Techniques: Virus, Transfection, Concentration Assay, Luciferase, Infection, Comparison, Binding Assay, Derivative Assay