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

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.

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

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

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.

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

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

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

SequenceMWPIAntiviral SP40 (H)QMRRKVELFTYMRFD Strong SP45 (β)AEFTFVACTPTGEVV Weak SP81 (β)KSKYPLVVRIYMRMK Very strong SP81-3 KSKYPLVVRIYMRMKHVR AWI Very strong Table 1 The physical and chemical parameters of the synthetic peptides

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

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

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

3CD? 2A, 3C? Polyprotein Structure Non-structure IRES 5’5’ 1D1B1C1A2A2B2C3A3B3C3D P3 VPg IRES guided translation 2A3CD? VP0 VP3 VP1 VP4VP2 ? 3C? 2A2B2C 3A 3C 3D VPg 3B Proteinase Helicase Polymerase Cleavage P1 P2 The structure of EV71 genome and its encoding proteins

EV71 pseudovirus system

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 viral infectivity A.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.

SequenceNumberpercentage KSKYPLVVR I YMRMK % KSKYPLV I R I YMRMK % KSKYPLL I R I YMRMK 40.4% KSKYPLV I RMYMRMK 10.1% KSKYPL I VR I YMRMK 20.2% KSKYPL I I R I YMRMK 50.5% KSKYPI VVR I YMRMK 10.1% KSKYPVVVR IYMRMK 10.1% KSKYPLVVRVYMRMK 30.3% KSKYPLVVRTYMRMK 10.1% KSKYPVV I RIYMRMK 10.1% 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: ACS ). A total of 989 VP1 sequences was recorded in GeneBank and EMBL database by February The mutated residues are shown in red color.The approximately accumulated mutation rate: 247 L to 247 I/V, 0.3%; 248 V to 248 I, 0.7%; 37.4%; 251 I to 251 M/V/T, 0.6%.

SequenceAntiviral SP79RTVGSTKSKYPLVVR - SP80GSTKSKYPLVVRIYM+/- SP81 (β)KSKYPLVVRIYMRMK+++++ SP82YPLVVRIYMRMKHVR++++ SP83VVRIYMRMKHVRAWI++++ SP84VVRIYMRMKHVRAWIPRP- SPC1AAAVVRIYMRMK++ SPC2YPLAAAIYMRMK+ SPC3YPLVVRAAARMK++ SPC4YPLVVRIYMAAA- SPC5YPILIRMYMRMK++++ Table 3 Identification of key residues for EV71 infectivity

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

Key residues canyon Arg 250, Arg 254, Met 255 and Lys 256 are key residues which locate on the Surface and would involve interaction with EV71 receptors

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 are highly conserved among subtypes of EV71, therefore, it is an idea structure for drug target;  Peptide targeting I β-sheet potently inhibited EV71 infections;  Residues Arg 250, Arg 254, Met 255 and Lys 256 are critical for virion binding to host cells.

Thank you!