Localisation and Replication of Venezuelan Equine Encephalitis Virus and Related New World Alphaviruses Kylie M Wagstaff Lindsay Lundberg Chelsea Pinkham Ashwini Benedict ID: 754690
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Slide1
Nuclear Transport Inhibitors Alter Capsid Subcellular Localisation and Replication of Venezuelan Equine Encephalitis Virus and Related New World Alphaviruses
Kylie M.
Wagstaff
,
Lindsay
Lundberg,
Chelsea
Pinkham,
Ashwini
Benedict,
Nazly
Shafagati
,
Moushimi
Amaya,
Aarthi
Narayanan,
David
Thomas,
Aaron
DeBono
,
Jonathan
Baell
,
David A.
Jans
,
Sharon
Tamir
,
Kylene
Kehn-
HallSlide2
Zacks
, M.A.,
Paessler
, S., Encephalitic
alphaviruses. Vet. Microbiol. (2009)
Enveloped,
Icosahedral symmetry Positive ssRNA genomeCytoplasmic replicating virus
Family: Togaviridae; Genus: AlphavirusCategory B select agentOverlap priority pathogen for CDC and USDA
Venezuelan Equine
Encephalitis
VirusSlide3
VEEV Capsid is a major pathogenicity factor
Capsid protein has multiple functions
Packaging of the viral genome
Protease activity for processing the structural proteins
Host cell transcriptional
inhibitor
Causes extensive CPECellular transcriptional inhibition is: Independent of its protease activity and RNA binding domainDependent on a short amino-terminal peptidePresent at the nuclear envelope embedded nuclear pore complex (NPC) and is a potent inhibitor of
nucleo-cytoplasmic traffickingSlide4
Nuclear transport
NLS
, nuclear
localisation
signal
(Lys-
Arg rich)NES, nuclear export signal(generally hydrophobic)
Wagstaff and Jans (2009) European Journal of Pharmacology, invited reviewSlide5
Inhibition of nuclear transport by VEEV capsid protein
CRM-1
NES
RANGTP
b
a
NLS
CRM-1
b
a
Capsid
NPC
Blocks Nuclear Import
Blocks Nuclear Export
Target for inhibitorsSlide6
Capsid binds to both Impa and Impb1 directly
SV40 T-
ag
CapsidSlide7
In Vitro Nuclear Transport Assay
Fluorescent IN
+/- DNA etc.
+ ATP/GTP
+/- Cytosol
+/- antibodies inhibitors
Analysis of Nuclear import by CLSM
Mechanically perforated
HTC cells
IN
Time (mins)
0
5
10
15
20
Fn/c
0
2
4
6
8Slide8
Nuclear Import of Capsid is dependent on both Impa and Impb1
Capsid alone
+ CHAPS
Capsid
+ CHAPS
Capsid alone
+ BSA
+ anti-Impb2
Capsid
+ BSA
+ anti-Imp
b
2Slide9
Nuclear Import of Capsid is dependent on both Impa and Impb1
Capsid alone
+ anti-
Imp
a
+ anti-Imp
b1
Capsid+ anti-Impb1+ anti-ImpaT-ag
+ anti-Imp
b
1
T-
ag
alone
+ anti-
Imp
a
+ anti-Imp
b
1
+ anti-
Imp
a
Capsid nuclear transport appears to be mediated by multiple pathways.Slide10
Depletion of Imps inhibits Capsid nuclear accumulation in infected cellssiNeg
Capsid
Importin
α
Merge
siImpα/β1
Lundberg et al.,
Antiviral Research 100 (2013) 662–672
****
****
****Slide11
Depletion of CRM-1 enhances Capsid nuclear accumulation in infected cells
Lundberg et al.,
Antiviral Research 100 (2013) 662–672
siNeg
Capsid
Importin
α
Merge
siImp
α
/
β
1
****Slide12
Nuclear import and export inhibitors alter Capsid nuclear accumulationSlide13
Nuclear import and export inhibitors alter Capsid nuclear accumulationSlide14
Nuclear transport inhibitors increase cell survival following VEEV infectionSlide15
Nuclear import and export inhibitors inhibit VEEV replication
**
**
**
Lundberg et al.,
Antiviral Research 100 (2013) 662–672
TC-83
**TC-83********TrDSlide16
Nuclear transport inhibitors also inhibit other important new world alphaviruses
**
**
**
**
**
**
****EEEVWEEVSlide17
ConclusionsVEEV capsid undergoes active nuclear transport via multiple importin-mediated pathwaysInhibitors of Nuclear import and export efficiently alter Capsid nuclear accumulation in transfected and infected cells
Nuclear transport inhibitors reduce VEEV infection, increase cell survival following infection and reduce viral
titre
Similar mechanisms are observed in the related WEEV and EEEV.Slide18
AcknowledgementsMonash UniversityDavid Jans
(Dept.
B
iochemistry)David ThomasJonathan Baell (MIPS)Aaron DeBono
George Mason UniversityKylene
Kehn-HallLindsay LundbergChelsea PinkhamAshwini BenedictNazly Shafagati,Moushimi AmayaAarthi NarayananKaryopharm TherapeuticsSharon
TamirSchecter Computational SolutionsSharon SchecterDefense Threat Reduction Agency