Transcript Inhibition of enterovirus 71 entry by peptides targeting I *

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 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.
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.
The VP1 structure
canyon
D-β
a-helix, D-β and I-β sheet form the canyon, which binds EV71 receptors
The I β sheet is highly conserved among EV71 subtypes and suitable for drug design
Table 1 The physical and chemical parameters of the synthetic peptides
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
KSKYPLVVRIYMRMKHVR
AWI
2675.3
11.12
Very strong
Peptides target I-β sheet inhibited cytopathic effects
caused by EV71 infections
Peptides target I-β sheet protected the viability of cells infected by EV71
120
100
SPscr
Cell viability (%)
80
SP40
SP81
60
SP81-3
40
20
0
10 uM
50 uM
100 uM
200 uM
Peptides target I-β sheet inhibited viral reproduction
A
**
**
Folds of inhibiting viral infection
(RNA copies, control/peptide)
18
16
14
SPscr
12
SP40
**
**
10
SP81
8
6
4
*
SP81-3
*
2
0
10 uM
B
50 uM
100 uM
The structure of EV71 genome and its
encoding proteins
5’
IRES
1D
1B
1C
Structure
VPg
1A
2A
2B
2C
3A
3B
3C
3D
Non-structure
IRES guided translation
Polyprotein
2A
3CD?
P1
P3
P2
3CD?
3C?
VP0
VP3
VP4
VP1
3C
3A
2A
?
2B
2C
VPg
VP2
Cleavage
Proteinase
2A, 3C?
3B
Helicase
Polymerase
3D
EV71 pseudovirus system
Peptides target I-β sheet inhibited viral infectivity
A
B
*
8.00
7.00
SPscr
6.00
SP40
5.00
4.00
3.00
**
**
**
**
SP81
SP81-3
2.00
1.00
0.00
**
**
40
*
Folds of reducing Luciferase
Activities (Control/peptides)
Folds of inhibiting virus binding
(Luc RNA copies of control/peptides)
9.00
35
30
**
**
25
SPscr
SP40
20
SP81
15
10
*
SP81-3
*
5
0
10 uM
50 uM
100 uM
10 uM
50 uM
100 uM
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.
Table 2 The sequences and residue mutation rate of I-β in EV71 circulating worldwide
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%
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%.
Table 3 Identification of key residues for EV71 infectivity
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
++++
The antiviral activity of I-β mutants
Reduction folds of Luciferase
Activities (Control/peptides)
21
18
15
12
9
6
3
0
SPscr
SPC-1
SPC-2
SPC-3
SPC-4
SPC-5
SP81
Key residues
canyon
Arg250, Arg254, Met255 and Lys256 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 Arg250, Arg254, Met255 and Lys256 are critical for virion binding to
host cells.
Thank you!