The evolution of EV71-PB and EV71-non-PB

<p>. 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.</p

Comparison of EV71-PB and EV71-non-PB structures.

<p>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).</p

Replication of EV71 mutants in RD and Jurkat cells.

<p>(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.</p

Enterovirus 71 Binding to PSGL-1 on Leukocytes: VP1-145 Acts as a Molecular Switch to Control Receptor Interaction

<div><p>Some strains of enterovirus 71 (EV71), but not others, infect leukocytes by binding to a specific receptor molecule: the P-selectin glycoprotein ligand-1 (PSGL-1). We find that a single amino acid residue within the capsid protein VP1 determines whether EV71 binds to PSGL-1. Examination of capsid sequences of representative EV71 strains revealed that the PSGL-1-binding viruses had either a G or a Q at residue 145 within the capsid protein VP1 (VP1-145G or Q), whereas PSGL-1-nonbinding viruses had VP1-145E. Using site-directed mutagenesis we found that PSGL-1-binding strains lost their capacity to bind when VP1-145G/Q was replaced by E; conversely, nonbinding strains gained the capacity to bind PSGL-1 when VP1-145E was replaced with either G or Q. Viruses with G/Q at VP1-145 productively infected a leukocyte cell line, Jurkat T-cells, whereas viruses with E at this position did not. We previously reported that EV71 binds to the N-terminal region of PSGL-1, and that binding depends on sulfated tyrosine residues within this region. We speculated that binding depends on interaction between negatively charged sulfate groups and positively charged basic residues in the virus capsid. VP1-145 on the virus surface is in close proximity to conserved lysine residues at VP1-242 and VP1-244. Comparison of recently published crystal structures of EV71 isolates with either Q or E at VP1-145 revealed that VP1-145 controls the orientation of the lysine side-chain of VP1-244: with VP1-145Q the lysine side chain faces outward, but with VP1-145E, the lysine side chain is turned toward the virus surface. Mutation of VP1-244 abolished virus binding to PSGL-1, and mutation of VP1-242 greatly reduced binding. We propose that conserved lysine residues on the virus surface are responsible for interaction with sulfated tyrosine residues at the PSGL-1 N-terminus, and that VP1-145 acts as a switch, controlling PSGL-1 binding by modulating the exposure of VP1-244K.</p></div

Structures of newly-synthesized NF449 analogues.

<p>Compounds NM2-16 are new. NM1 was resynthesized, but is identical to NF110.</p

NF449, NF110, and NM16 inhibit virus interactions with Jurkat cells.

<p>(A) Infection by EV-A71-1095, in the presence of inhibitors, measured by staining for VP1 expression. (B) Inhibition of virus attachment by NF449, NF110, and NM16. Results are indicated as the mean and S.D. for triplicate samples. Asterisks indicate <i>P</i> < 0.01 compared to the no inhibitor control.</p

Inhibition of EV-A71 attachment by newly-synthesized NF449 analogues.

<p>(A) Attachment of ³⁵S-labeled EV-A71-1095 to RD cells in the presence of compounds at 0.4 or 4 μM. Results from two experiments, one testing NM1-11 and one testing NM12-16, are combined, and are normalized to results for the no inhibitor control in each experiment. (B) Virus attachment to RD monolayers in the presence of NF449, NF110, and NM16. Results are indicated as the mean and S.D. for triplicate samples. Asterisks indicate <i>P</i> < 0.01 compared to the no inhibitor control.</p

NF449 and NF110 specifically prevent attachment of a monoclonal antibody to the viral 5-fold vertex.

<p>(A) An EV-A71 pentamer shown with the 5-fold vertex at the center. VP1-98 (red) was mutated in an NF449 escape mutant. VP1-242K (light blue) is involved in virus interaction with PSGL-1. VP1-244K (dark blue) is implicated both in virus interactions with both PSGL-1 and NF449. The MA28-7 footprint, as determined by cryo-electronmicroscopy, is outlined in black. The VP2 epitope recognized by mAb 10F0 is indicated in green. (B) NF449 inhibits immunoprecipitation of EV-A71-1095 by MA28-7 but not by 10F0. EV-A71-1095 concentrated from supernatants of infected cells was incubated with MA28-7 or 10F0 fixed to Protein G beads, in the presence of NF449 at the indicated concentrations, or 200 μM Pirodavir; beads were washed, and immunoprecipitated proteins were examined on Coomassie-stained gels. Molecular weight markers are shown at the left. Arrows at the right indicate capsid proteins VP0 and VP1, and brackets indicate antibody heavy (HC) and light chains (LC). (C) NF449 and NF110 inhibit attachment of MA28-7 to mature virions. Purified ³⁵S-labeled mature virions were incubated with MA28-7-coated beads, in the presence of inhibitors as indicated. (D) NF110 inhibits attachment of MA28-7, but not 10F0 or MA105, to procapsids. Purified ³⁵S-labeled procapsids were incubated with monoclonal antibodies fixed to Protein G beads, in the presence of 20 μM NF110. Results are indicated as the mean and S.D. for triplicate samples. Asterisks indicate <i>P</i> < 0.01 compared to the no inhibitor control.</p

NF449 inhibits EV-A71 interactions with RD cells.

<p>(A) RD cell monolayers were exposed to EGFP-expressing EV-A71-1095 in the presence of NF449 or the control compound fondaparinux at the indicated concentrations, then incubated at 37°C for 16 hr. Infected cells were identified by flow cytometry to detect EGFP expression. (B) ³⁵S-labeled EV-A71-1095 was exposed to NF449 in medium with or without 10% FBS, then attachment to RD monolayers was measured as described in Materials and Methods. (C) HeLa cell monolayers were exposed to ³⁵S-labeled CVB3-RD in the presence of NF449, the anti-DAF antibody IF7, or an isotype-matched control antibody, and attachment was measured. Results are indicated as the mean and S.D. for triplicate samples. Asterisks indicate <i>P</i> < 0.01 compared to results with the no inhibitor control.</p

Inhibition of EV-A71 attachment and infection by commercially-available NF449 analogues.

<p>(A) Structures of the tested compounds. (B) ³⁵S-labeled EV-A71-1095 was exposed to compounds at 0.4 or 4 μM, then virus attachment to RD monolayers was measured. (C) RD monolayers were exposed to EV-A71-1095 in the presence of inhibitors at the indicated concentrations. After 16 hrs at 37°C, infected cells were stained to detect intracellular VP1, and analyzed by flow cytometry. Results are indicated as the mean and S.D. of triplicate samples. Asterisks indicate <i>P</i> < 0.01 compared to the no inhibitor control.</p