Inhibition of enterovirus 71 entry by peptides targeting I β-sheet of VP1 protein

1. Inhibition of enterovirus 71 entry by peptides targeting I β-sheet of VP1 protein Ming-Liang He, Ph.D The Chinese University of Hong Kong

2. Enterovirus 71 (EV71) • A single stranded, non-enveloped RNA virus (~ 7.5 kb in size); • EV71 infection causes hand-foot-and-mouth disease (HFMD); • Children under 5 are highly susceptible to EV71 and easily develop central nervous system (CNS) symptoms; • As long as it causes CNS infection or symptoms, leading to neurologic sequelae, including neurological disorders, meningitis, and even death.

3. Hand, Foot & Mouth Disease an acute viral infection, commonly seen in infants and children

4. EV71 epidemiology in China The reported HFMD cases and related death in China from 2007 to 2012. No drugs, no vaccine available! Lu J et al., Crit. Rev. Microbiol. 2013

5. Principle of Antiviral • Block viral entry by peptides, small molecules or antibodies; • Block viral replication; • Block the assembly of virions; • Block the virion secretion; • Disturb the cellular signaling, and therefore block one or more processes in the life cycle of virus.

6. The 3D structure of VP1-VP4 complex of EV71 (PDB ID: 3VBS) virion. A C D VP1, VP2, VP3 and VP4 were represented in blue, red, green and yellow, respectively. I-β sheet is indicated by a big write arrow.

7. The VP1 structure canyon D-β a-helix, D-β and I-β sheet form the canyon, which binds EV71 receptors

8. The I β sheet is highly conserved among EV71 subtypes and suitable for drug design

9. Table 1 The physical and chemical parameters of the synthetic peptides

10. Peptides target I-β sheet inhibited cytopathic effects caused by EV71 infections

11. Peptides target I-β sheet protected the viability of cells infected by EV71 Cell viability (%)

12. Peptides target I-β sheet inhibited viral reproduction A Folds of inhibiting viral infection (RNA copies, control/peptide) B * * ** ** ** **

13. P1 P2 3CD? 2A, 3C? The structure of EV71 genome and its encoding proteins 1D 1B 1C 1A 2A 2B 2C 3A 3B 3C 3D 5’ IRES IRES guided translation VPg Polyprotein Structure Non-structure 2A 3CD? 3C? 3B VP1 VP0 VP3 3D 3C 3A 2A 2B 2C VPg ? P3 VP4 VP2 Helicase Polymerase Proteinase Cleavage

14. EV71 pseudovirus system

15. Peptides target I-β sheet inhibited viral infectivity A B Folds of inhibiting virus binding (Luc RNA copies of control/peptides) Folds of reducing Luciferase Activities (Control/peptides) Peptides target I-β sheet inhibited the binding of virions to host cells (binding at 4⁰C for 30 min; B. Peptides target I-β sheet inhibited viral entry as indicated By reduced translation expression of luciferase reporter. * * * * ** ** ** ** ** ** ** **

16. Table 2 The sequences and residue mutation rate of I-β in EV71 circulating worldwide The sequences of SP81-3 (same as the strain outbreaked in 2008 at Fuyang city, Anhui Province, China; GenBank: ACS12928.1). A total of 989 VP1 sequences was recorded in GeneBank and EMBL database by February 2013. The mutated residues are shown in red color.The approximately accumulated mutation rate: 247L to 247I/V, 0.3%; 248V to 248I, 0.7%; 37.4%; 251I to 251M/V/T, 0.6%.

17. Table 3 Identification of key residues for EV71 infectivity

18. The antiviral activity of I-β mutants Reduction folds of Luciferase Activities (Control/peptides)

19. Key residues canyon Arg250, Arg254, Met255 and Lys256are key residues which locate on the Surface and would involve interaction withEV71 receptors

20. Conclusion • The I β-sheet is an important structure to form the convoy of VP1, which involves receptor binding; • The residues in I β-sheet arehighly conserved among subtypes of EV71, therefore, it is an idea structure for drug target; • Peptide targeting I β-sheet potently inhibited EV71 infections; • Residues Arg250, Arg254, Met255 and Lys256 are critical for virion binding to host cells.

22. Thank you!