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

protein

Ming-Liang He, Ph.DThe 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 2012.

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

Sequence MW PI Antiviral

SP40 (H) QMRRKVELFTYMRFD 2020.4 9.98 Strong

SP45 (β) AEFTFVACTPTGEVV 1935.8 8.68 Weak

SP81 (β) KSKYPLVVRIYMRMK 1912.4 10.56 Very strong

SP81-3 KSKYPLVVRIYMRMKHVRAWI 2675.3 11.12 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

0

20

40

60

80

100

120

10 uM 50 uM 100 uM 200 uM

SPscr

SP40

SP81

SP81-3

Cel

l via

bil

ity

(%)

Peptides target I-β sheet protected the viability of cells infected by EV71

Fol

ds o

f in

hibi

ting

vir

al in

fect

ion

(R

NA

cop

ies,

con

trol

/pep

tide

)

A

B

10 uM 50 uM 100 uM 0

2

4

6

8

10

12

14

16

18

SPscr

SP40

SP81

SP81-3**

**

**

**

**

Peptides target I-β sheet inhibited viral reproduction

3CD? 2A, 3C?

Polyprotein

Structure Non-structure

IRES5’ 1D 1B 1C 1A 2A 2B 2C 3A 3B 3C 3D

P3

VPg

IRES guided translation

2A 3CD?

VP0 VP3 VP1

VP4 VP2

?

3C?

2A 2B 2C3A 3C 3D

VPg

3B

Proteinase Helicase PolymeraseCleavage

P1P2

The structure of EV71 genome and its encoding proteins

EV71 pseudovirus system

AB

10 uM 50 uM 100 uM0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

SPscr

SP40

SP81

SP81-3

Fo

lds

of

inh

ibit

ing

vir

us

bin

din

g

(Lu

c R

NA

co

pie

s o

f co

ntr

ol/p

epti

des

)

Fo

lds

of

red

uci

ng

Lu

cife

rase

A

ctiv

itie

s (C

on

tro

l/pep

tid

es)

****

****

*

****

****

*

*

*

10 uM 50 uM 100 uM0

5

10

15

20

25

30

35

40

SPscr

SP40

SP81

SP81-3

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 indicatedBy reduced translation expression of luciferase reporter.

Sequence Number percentage

KSKYPLVVR I YMRMK 611 61.8%

KSKYPLV I R I YMRMK 359 36.3%

KSKYPLL I R I YMRMK 4 0.4%

KSKYPLV I RMYMRMK 1 0.1%

KSKYPL I VR I YMRMK 2 0.2%

KSKYPL I I R I YMRMK 5 0.5%

KSKYPI VVR I YMRMK 1 0.1%

KSKYPVVVR IYMRMK 1 0.1%

KSKYPLVVRVYMRMK 3 0.3%

KSKYPLVVRTYMRMK 1 0.1%

KSKYPVV I RIYMRMK 1 0.1%

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: 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%.

Sequence Antiviral

SP79 RTVGSTKSKYPLVVR -

SP80 GSTKSKYPLVVRIYM +/-

SP81 (β) KSKYPLVVRIYMRMK +++++

SP82 YPLVVRIYMRMKHVR ++++

SP83 VVRIYMRMKHVRAWI ++++

SP84 VVRIYMRMKHVRAWIPRP -

SPC1 AAAVVRIYMRMK ++

SPC2 YPLAAAIYMRMK +

SPC3 YPLVVRAAARMK ++

SPC4 YPLVVRIYMAAA -

SPC5 YPILIRMYMRMK ++++

Table 3 Identification of key residues for EV71 infectivity

Red

uct

ion

fo

lds

of

Lu

cife

rase

A

ctiv

itie

s (

Co

ntr

ol/

pe

pti

des

)

SPscr SPC-1 SPC-2 SPC-3 SPC-4 SPC-5 SP810

3

6

9

12

15

18

21

The antiviral activity of I-β mutants

Key residues

canyon

Arg250, Arg254, Met255 and Lys256 are key residues which locate on theSurface 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 Arg250, Arg254, Met255 and Lys256 are critical for virion binding to host cells.

Thank you!