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1.
Figure 4

Figure 4. Alignment of EV71 VP1 sequences.. From: Enterovirus 71 Binding to PSGL-1 on Leukocytes: VP1-145 Acts as a Molecular Switch to Control Receptor Interaction.

The names of the EV71-PB and EV71-non-PB strains are in red and blue, respectively. 4AED (EV71-EQ) and 3VBS (EV71-KE) in black are presumably PB and non-PB viruses, respectively. The names are followed by their subgenogroups. The BC, DE, and HI loops (boxed) and the β barrels (shown with arrows) are defined as in reference 4. The amino acids where mutations were introduced are indicated by arrowheads. The conserved amino acids are shown with dots. The amino acid at VP1-145 (X) of EV71-KED005 could not be determined because there was a mixture of sequences at his position.

Yorihiro Nishimura, et al. PLoS Pathog. 2013 July;9(7):e1003511.
2.
Figure 1

Figure 1. Co-precipitation analysis of EV71 mutants with soluble PSGL-1-Fc.. From: Enterovirus 71 Binding to PSGL-1 on Leukocytes: VP1-145 Acts as a Molecular Switch to Control Receptor Interaction.

(A) Scheme of EV71 constructs with amino acid substitutions at VP1-98 and VP1-145. The original strains, which have the indicated amino acids at VP1-98 and VP1-145, are indicated in parentheses. (B, C) The co-precipitation of EV71 and PSGL-1-Fc was detected by western blotting using anti-VP1 mAb and an anti-Fc antibody. EV71-PB (C7-Osaka, 1095, 75-Yamagata) and EV71-non-PB (Nagoya and 02363) isolates (not derived from cDNA) were used as positive and negative controls for binding to PSGL-1-Fc, respectively. The data are representative of three independent experiments. (B) EV71 strains of genogroup B. (C) EV71 strains of genogroup C.

Yorihiro Nishimura, et al. PLoS Pathog. 2013 July;9(7):e1003511.
3.
Figure 6

Figure 6. The evolution of EV71-PB and EV71-non-PB. From: Enterovirus 71 Binding to PSGL-1 on Leukocytes: VP1-145 Acts as a Molecular Switch to Control Receptor Interaction.

. The amino acids at VP1-145 and their codons are shown. The numbers above or below the codons indicate their frequency in GenBank. EV71 with VP1-145G or VP1-145Q are the PSGL-1-binding (PB) phenotype and the minor populations in GenBank (red round rectangles). EV71 with VP1-145E is the PSGL-1-nonbinding (non-PB) phenotype and the major population in GenBank (blue round rectangles). The phenotypic change between PSGL-1-binding and PSGL-1-nonbinding can occur easily, because only one nucleotide substitution changes amino acid (white arrows). The amino acid change between G and Q would not occur easily, as their codons have two nucleotide differences. Neither GGC nor GGU was found to encode VP1-145G in GenBank.

Yorihiro Nishimura, et al. PLoS Pathog. 2013 July;9(7):e1003511.
4.
Figure 2

Figure 2. Replication of EV71 mutants in RD and Jurkat cells.. From: Enterovirus 71 Binding to PSGL-1 on Leukocytes: VP1-145 Acts as a Molecular Switch to Control Receptor Interaction.

(A) RD (left) or Jurkat (right) cells were infected with EV71-02363 mutants at 1 CCID50 per cell for 1 h, then washed, cultured, and harvested at the indicated days (d) post-infection. (B, C) EV71 replication in Jurkat cells incubated with anti-PSGL-1 mAb and isotype control. Cells were pretreated with antibodies for 1 h and then infected with 1 CCID50 per cell for 1 h, washed, cultured, and harvested at 3 days (d) post-infection. Viral titers are expressed as the mean, and error bars indicate s. d. for triplicate samples. (B) EV71-02363. (C) EV71-1095. Although arise in viral titer was observed for EV71-1095-EE at day 3 (asterisk), the recovered viruses were VP1-145E to G revertants.

Yorihiro Nishimura, et al. PLoS Pathog. 2013 July;9(7):e1003511.
5.
Figure 5

Figure 5. EV71–PSGL-1 binding assay using real-time RT-PCR.. From: Enterovirus 71 Binding to PSGL-1 on Leukocytes: VP1-145 Acts as a Molecular Switch to Control Receptor Interaction.

Viruses (5×107 viral genomes) were incubated with anti-VP1 mAb or PSGL-1-Fc and collected with protein G beads. Precipitated viruses were analyzed by real-time RT-PCR as described in Materials and Methods. Viral genome copies are expressed as the mean, and error bars indicate s. d. of three independent experiments. (A) Viruses were precipitated with anti-VP1 mAb to show the presence of virion in the sample. The amount of virus precipitated with nonspecific isotype control was considered as background binding (left). (B) Viruses were precipitated with PSGL-1-Fc. A control Fc chimeric protein (CTLA-4-Fc) was used as a negative control. Asterisks indicate a significant difference in specific binding to PSGL-1-Fc (P<0.01). Although there was a significant difference in EG-K244A, we consider it biologically meaningless.

Yorihiro Nishimura, et al. PLoS Pathog. 2013 July;9(7):e1003511.
6.
Figure 3

Figure 3. Comparison of EV71-PB and EV71-non-PB structures.. From: Enterovirus 71 Binding to PSGL-1 on Leukocytes: VP1-145 Acts as a Molecular Switch to Control Receptor Interaction.

Molecular surfaces of EV71-EQ (4AED; presumably PB, top of each figure section) and EV71-KE (3VBS; presumably non-PB, bottom of each figure section). (A) Electrostatic surface coloring of five icosahedral asymmetric units using UCSF Chimera software with ‘coulombic surface coloring’ function. Negatively charged surfaces are shown in red, whereas positively charged areas are shown in blue. Black-boxed areas are enlarged and shown in (B). (B) Molecular surfaces around the five-fold axis of symmetry. Dotted ovals indicate a set of VP1-145, VP1-242, and VP1-244. Left panels show electrostatic surface coloring. Arrowheads in EV71-KE show negatively charged patches around VP1-145E. Right panels show the positions of specific residues (dim gray, VP1-98; cyan, VP1-145Q; red, VP1-145E; cornflower blue, VP1-242K; blue, VP1-244K). (C) Oblique views of the five-fold axis. Dotted ovals indicate a set of VP1-145, VP1-242, and VP1-244. Specific residues are colored as in (B). (D) Loop structures of partial VP1-DE (yellow, VP1-142 to -145) and partial VP1-HI loops (cornflower blue, VP1-238 to -244) depicted in ribbon form. Side chains for the residues VP1-145, VP1-242, and VP1-244 are displayed as sticks (cyan, EV71-EQ; red, EV71-KE).

Yorihiro Nishimura, et al. PLoS Pathog. 2013 July;9(7):e1003511.

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