Dengue virus capsid protein functions and its
i hibiti by
inhibition
b pep14-23
14 23
Nuno C. Santos
Instituto de Medicina Molecular
Faculdade de Medicina da Universidade de Lisboa
Portugal
[email protected]
Dengue epidemics
Different clinical profiles
• No symptoms
• Mild symptoms
• Acute clinical cases
D
Dengue Hemorrhagic Fever
H
h i F
Dengue Shock Syndrome
Organs infected
• Liver
• Immune system
• Hearth, brain, muscle
Disease vectors
Aedes aegypti
gyp
Aedes albopictus
Worldwide yearly:
• 2.5 billion people at risk areas
• 390 millions infected
• 20 000 deaths
N
No specific treatment available
ifi t t
t
il bl
Senior (2010) Lancet Infect Dis 7:636
Dengue virus
Flaviviridae family
• Dengue virus (DENV)
Dengue virus (DENV)
• Zika virus • West Nile virus
West Nile virus
• Yellow fever virus
• Tick
Tick‐borne
borne encephalitis virus
encephalitis virus
• Hepatitis C virus
Yu et al. (2008) Science 319:1834
Polyprotein
Dengue is an enveloped virus with Dengue
is an enveloped virus with
a ss(+)RNA genome of ~11kb.
• 3 structural proteins
o Capsid (C)
o Membrane (M)
o Envelope (E)
Envelope (E)
• 7 non‐structural proteins
Dengue virus
Umareddy et al. (2007) Virol. J. 4:91
Polyprotein
Dengue is an enveloped virus with Dengue
is an enveloped virus with
a ss(+)RNA genome of 11kb.
• 3 structural proteins
o Capsid (C)
o Membrane (M)
o Envelope (E)
Envelope (E)
• 7 non‐structural proteins
Dengue virus
Mukhopadhyay et al. (2005)
Nat. Rev. Microbiol.3:13
Flaviviridae disrupt the host lipid homeostasis
Lipid droplets (LDs) size and number increased
Following DENV infection
Lipoproteins levels affected
Intracellular membrane network disorganization
DENV C protein in LDs surface
DENV C protein in LDs surface
LDs
C protein
merge
Samsa et al. (2009) PLoS Pathog 5:e1000632
Lipid droplets
Intracellular lipid storage and distribution
LDs structure
• Neutral lipids – internal core
• Polar lipids – surface monolayer
• Surface proteins
LDs proteins
•
•
•
•
Perilipin (Perilipin 1)
ADRP (Perilipin
(
l
2))
TIP47 (Perilipin 3)
…
Farese and Walther (2009) Cell 139:855
Krahmer et al. (2009) Cell 139:1024
Force spectroscopy
DENV C protein ‐ AFM tip functionalization
Vacuum chamber APTES deposition – 1h
Glutaraldehyde 2.5 % (v/v) – 20 min
DENV C protein C 167 M – 30 min
https:/.../news_releases/2005/NR‐05‐10‐07p.html
DENV capsid
protein
Guedes et al. (2016) Nature Nanotechnol. 11: 687
Carvalho et al. (2010) ACS Nano 4: 4609
Barattin et al (2008) Chem. Commun. 13: 1513
Force spectroscopy
approach
retract
Carvalho & Santos (2012) IUBMB Life 64: 465
DENV C interaction with LDs requires K+
Force rupture Force
rupture
histograms
Force mapping images
Force mapping images height map adhesion map
TEE buffer with KCl 10 mM
TEE buffer with KCl
100 mM
TEE buffer with KCl 400 mM
Carvalho et al.
(2012) J. Virol. 86:
2096
DENV C interaction with LDs requires K+
Buffer conditions
C1/2 (nM)
∆ζmax (mV)
TEE + 10 mM
M KCl
5 9  2.2
5.9
22
27 9  0.4
27.9
04
TEE + 100 mM KCl
85.7  17.6
34.4  1.3
TEE + 400 mM KCl
188 4  68.8
188.4
68 8
27 6  2.3
27.6
23
Carvalho et al. (2012) J. Virol. 86: 2096
DENV C interaction with LDs requires K+
Lipid droplets
Lipid droplets
DENV C
DENV C
Effects on different steps of the DENV replication cycle of Na+/K+‐ATPase partial inhibition (decreased [K+]int)
For DENV‐infected HepG2 cells For
DENV infected HepG2 cells
treated with ouabain, a redistribution of DENV C protein to a diffuse pattern in the cytosol
y
was observed (D‐F).
( )
Treatment with ouabain
resulted in an up to 50‐fold
inhibition of infectious virus production (G), followed by a recovery from the loss of viability caused by infection (H).
No significant difference was No
significant difference was
found in the DENV RNA content at (I), indicating that the alterations in the [ +]] inhibit viral intracellular [K
assembly without interfering with RNA replication.
Control experiments ensured that these ouabain
concentrations do not affect the viability of noninfected cells.
DENV C interaction with LDs requires LDs protein(s)
F
Force‐rupture
t
hi t
histograms
Trypsin 0 µM
0 µM
Trypsin 1 µM
Trypsin 1 µM
Trypsin 5 µM
Trypsin 10 µM
Carvalho et al. (2012) J. Virol. 86: 2096
DENV C interaction with LDs requires LDs protein(s)
Carvalho et al. (2012) J. Virol. 86: 2096
DENV C protein regions involved in the interaction with LDs
Nuclear magnetic resonance (NMR)
Nuclear magnetic resonance (NMR)
Martins et al. (2012) Biochem. J., 444: 405
Sequence conservation of the Flavivirus C proteins
Computational predictions
Computational predictions
N‐terminus L1‐2 α2‐α2’
Conserved regions on the Flavivirus
C proteins sequences
C proteins sequences
Martins et al. (2012) Biochem. J., 444: 405
Inhibition of the interaction between DENV C and LDs
NMR
pep5‐26
(RKKTGRPSFNMLKRARNRVSTV)
pep14‐23
(NMLKRARNRV)
Zeta potential
p
DENV C protein
pep14‐23
pep14‐23 + trypsin
pep5‐26
Martins et al. (2012) Biochem. J., 444: 405
Faustino et al. (2015) ACS Chem. Biol., 10: 517
Inhibition of the interaction between DENV C and LDs
Martins et al. (2012) Biochem. J., 444: 405
Inhibition of the interaction between DENV C and LDs
Lipoproteins
Distribution and recovery of lipids in the bloodstream
Distribution and recovery of lipids in the bloodstream
Lipoproteins structure
• Neutral lipids – internal core
• Polar lipids – surface monolayer
• Surface proteins
Farese and Walther (2009) Cell 139:855
Cushley and Okon (2002) Annu Rev BBS 31:177
Lipoproteins in Flaviviridae infections
Lipoproteins in Flaviviridae
• Altered bloodstream levels
• LDL receptor involved in viral entry
LDL receptor involved in viral entry
• HCV forms lipo‐viro‐particles
DENV C interaction with lipoproteins
Force spectroscopy
Force spectroscopy
VLDL with KCl
VLDL with NaCl
LDL with KCl
VLDL in KCl
VLDL
in KCl
without DENV C
DENV C‐
lipoproteins interaction is
interaction is dependent on K+ and on and on
VLDL intrinsic components
Catching a Catching
a
ride?
Faustino et al. (2014) Nanomedicine, 10: 247
DENV C interaction with lipoproteins
Dynamic light scattering 45
*
Lipop
protein diame
eter (nm)
*
40
*
*
VLDL
6 nm
* p < 0.001
* p
< 0 001
35
Mann–Whitney U test
30
VLDL
LDL
25
20
0,0
0,5
1,0
1,5
2,0
2,5
3,0
[DENV C] (µM)
Surface interaction between DENV C and VLDL
NV C d VL L
Faustino et al. (2014) Nanomedicine, 10: 247
DENV C interaction with lipoproteins
DENV LVP
Apolipoprotein E
DENV C
DENV C target on VLDL
VLDL
Hickenbottom et al. (2004)
Structure 12:1199
DENV C ApoE
ApoE N-terminus
1
Per3 C-terminus
215
ApoE-Per3 consensus
KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVT
ATSLDGFDVASVQQQRQEQSYFVRLGSLSERLRQHAYEHSLGKLRATKQRAQEAL
hXQQXEXQS
L
TL
EXX
57
269
ApoE N-terminus
58
Per3 C-terminus
270
ApoE-Per3 consensus
QELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQ
LQLSQVLSLMETVKQGVDQKLVEGQEKLHQMWLSWNQKQLQGPEKEPPKPEQVESRALTM
XELXXhh
LXE
hXE
KELQXXE
E
LXX
117
329
ApoE N-terminus
118
Per3 C-terminus
330
ApoE-Per3 consensus
YRGEVQAMLGQSTEELR VRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLS
FRDIAQQLQATCTSLGSSIQGLPTNVKDQVQQARRQVEDLQATFSSIHS FQDLSSSILA
XR
QXh
T
LXTXh+
RXX-DLQXXh
LX
175
388
ApoE N-terminus
176
Per3 C-terminus
389
ApoE-Per3 consensus
AIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEE
QSRERVASAREALDHMVEYVAQNTPVTWLVGPFAPGITE KAPEEKK
XXRERh
E
V
AXG
+XXE
220
434
Faustino et al. (2014) Nanomedicine, 10: 247
Faustino et al. (2015) Sci. Rep., 5: 10592
DENV C interaction with lipoproteins
NMR
pep14‐23 inhibits
DENV C‐VLDL binding
DLS
AFM
Martins et al. (2012) Biochem. J., 444: 405
Faustino et al. (2015) ACS Chem. Biol., 10: 517
Faustino et al. (2015) Sci. Rep., 5: 10592
Understanding the inhibition by pep14-23
NMR with DPC micelles
Tensiometry with
POPG monolayers
Zeta potential
with POPG LUVs
CD with POPG LUVs
POPC (zwitterionic)
POPC
(zwitterionic)
POPG:POPC 1:1
POPG (anionic)
Faustino et al. (2015) ACS Chem. Biol., 10: 517
Understanding the inhibition by pep14-23
DENV C protein N‐terminal conserved region……….14NMLKRERNRVS24
Importin α N‐terminal autoinhibition region………..46QMLKR-RN-VS54
• Anionic phospholipids (POPG) trigger the α‐helical conformation conversion of pep14‐23
• The interaction and inhibition mechanisms may involve phospholipids from LDs and VLDL
• DENV C may have an auto‐inhibition mechanism similar to importin
DENV C may have an auto‐inhibition mechanism similar to importin α
Faustino et al. (2015) ACS Chem. Biol., 10: 517
Nuno C. Santos
Sónia Gonçalves
Filomena Carvalho
Ivo Martins
Axel Hollmann
Marco Domingues
André Faustino
Ana Filipa Guedes
Marcelo Augusto
Bárbara Gomes
á
Mário Felício
Ana Martins
Patrícia Carvalho
Patrícia Silva
Marcin Makowski
Catarina Lopes
André Nascimento
Teresa Freitas
Acknowledgments
Funding
Federal University of Rio de J
Janeiro, Brazil
i B il
• Andrea Da Poian
• Fábio Almeida
• Ronaldo
R
ld M
Mohana‐Borges
h
B
Other Groups from IMM
• Miguell Castanho
h
• Francisco Enguita
A*STAR Si
A*STAR, Singapore
• Peter Bond
• Roland Huber