Differential cell tropism of EV71-VP1_97R167G and EV71-VP1_97L167E is linked to HS binding and correlates with its expression level at the cell surface.

<p><b>(A).</b> Binding of EV71-VP1_97R167G (left panel) and EV71-VP1_97L167E (right panel) in the presence or absence of HS. Binding <b>(B)</b> and replication <b>(C)</b> of a viral stock composed of equivalent amounts of EV71-VP1_97R167G and EV71-VP1_97L167E in different cell lines and under conditions preventing attachment to HS. Viral loads were measured by RT-qPCR (real-time quantitative reverse transcription PCR) in whole cell extracts for binding assay and both cell extracts and supernatants for replication assay, and expressed as the mean (±SEM, N≥2) relative to the EV71-VP1_97R167G load in non-treated RD cells (100%). <b>(D).</b> Quantification of HS-expressing (positive) cells was achieved through an analysis using MetaMorph software (left panel) based on the immunofluorescence labeling (right panel) of HS attachment receptors at the surface of Vero, RD, SH-SY5Y cells and Caco-2 cells. More than 500 cells were included in the analysis. Scale bar = 20 μm. <b>(E)</b> Binding of EV71-VP1_97R/L167G/E to heparin sepharose beads. Viral population present in the input, flow-through and elute population was characterized by sequencing. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. For B, C and E, the frequency plot [<a href="http://weblogo.berkeley.edu/" target="_blank">http://weblogo.berkeley.edu/</a>] of codons encoding VP1 aa 97, 98 and 167 are shown and are representative of biological replicates (N≥2). Arginine (R) at position 97 is encoded by CGC, Leucine (L) by CTC; Glutamate (E) at position 98 is encoded by GAA, Lysine (K) by AAA; Glycine (G) at position 167 is encoded by GGG, Glutamate (E) by CTC.