rotavirus strategies to take over the host cell response susana... · 2014-09-17 · -rbps like:...
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Rotavirus strategies to take over the host cell response
Instituto de BiotecnologíaUniversidad Nacional Autónoma de México
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Virus Host
Host cell defenses
Viral counter-measures
Battle between viruses and host cells
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During rotavirus infection the cellularprotein synthesis is shutoff
+Virus-
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PABPAAAA
Cap
mRNA
eIF4F
eIF4G
4A
4E
NSP3
During infection NSP3 displaces PABP fromeIF4G and prevents cellular protein synthesis
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a-PABP a-eIF4GI merge
control
During RV infection, PABP relocalizes tothe nucleus, and this depends on NSP3
a-NSP2
siNSP3
siIrr
+ virus
- virus
Montero H et al, 2006, J Virol. 80:9031-9038
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No probe probe :Oligo dT
No VirussiIrr
aNSP3 Merge
+ VirussiIrr
+ VirussiNSP3
polyA+ mRNAs are also relocalized in the nucleus of the cell during infection
Rubio RM et al, 2013. J Virol.
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GRP78
Mock 6h 12h0
1
2
3
4
5Cytop
Nuc
Hours postinfection
Rela
tive a
mo
un
t o
f
RN
A (
arb
itra
ry u
nit
s)
GRP94
s/v 6h 12h0.0
0.5
1.0
Hours postinfection
Rela
tive a
mo
un
t o
f
RN
A (
arb
itra
ry u
nit
s)
During RV infection cellular mRNAs accumulate in the nucleus
Rotavirus prevents host translation by blocking the nucleo-cytoplasmic transport of poly(A)-containing mRNAs
Rubio RM et al, 2013. J Virol
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eIF2a is phosphorylated during rotavirus infection
s/v 1 2 3 4 5 6 7 8 10 12hr post-infection:
anti-eIF2ap
Rojas M et al, 2010. J Virol.
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GDP
Translation factor eIF2
a
GTP
a
Met
a
GTP
Translation initiation
GTPeIF2B
GDP
GDP
a
GDPeIF2B
Global translationinhibition
Selective synthesis
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α-eIF2α-P
PERK-/-
- Tg Ar
eIF2a-P
eIF2a
MEFs:
Rotavirus infection induces phosphorylation of
eIF2a in a PKR-dependent manner
-Tg Tg -
WT PKR-/-
Irr siPKR
MA104
PKRRojas M et al, 2010. J Virol.
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Rotavirus strategies to control cell-protein synthesis:
1.- NSP3 interacts with eIF4G and prevents thebinding of PABP-bound mRNAs
2.- Rotavirus prevents host translation by blocking the nucleo-cytoplasmic transport of poly(A)-containing mRNAs
3.- eIF2a is phosphorylated by PKR in rotavirus-infected cells
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Stress granulesare formed when eIF2a is phosphorylated
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Composition of Stress Granules
-mRNAs
-Translation factorseIF3eIF4GeIF4E (partially)PABP
-Small ribosomal subunit
-RBPs like: TIA, TIAR, HuR, TTP, G3BP, FragileX, SMN, etc.
Ars
Ctrol
PABP eIF4G merge
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virus
aTIA aNSP2 merge
control
arsenite
virus & arsenite
Rotavirus infection prevents the formation of stress granules
Mon
tero
H e
t al
, 2
00
8. J
Vir
ol.
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SGs and P-bodies are sites of RNA triage.
Li Y R et al. J Cell Biol 2013;201:361-372
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aeIF4E a-NSP2 merge
+virus
-virus
P-Bodies are reduced in infected cells
Oceguera A. et al, unpublished
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Activation of 2´5´OAS
dsRNAThe OAS/RNAse L
complex
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1 10
RRVM
28 S
18 S
During RV infection there is no degradationof rRNAs
Sanchez et al, unpublished
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Summary
-Rotavirus infection causes a severe inhibition of cellular protein synthesis
-The OAS-RNAse L pathway is blocked during RV-infection, VP3 participates in this control
- The number of P-bodies is reduced
-The formation of stress granules is prevented.
-NSP3 prevents translation of polyA mRNAs, -The nucleo-cytoplasmic transport of polyA mRNAs is blocked, -eIF2a is phosphorylated by PKR in RV-infected cells
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Grupo Arias/López
CONACyT, and DGAPA-UNAM
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Gracias!!
PKR and dsRNAMargarito Rojas
Stress GranulesHilda Montero
P-BodiesAlfonso Oceguera
OAS/RNAse LLiliana Sánchez
Nucleo-Cytoplasmic transportof mRNAsRosa Ma Rubio
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a-dsRNA a-NSP5 Merge
Control
What is the nature of the dsRNA that activates PKR in infected cells?
RNAse III
RNAse A
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a-vimentin
a-NSP3
Ctrol NSP3
VP1VP2VP4
VP6VP7
NSP3
NSP4
siRNA
M
Silencing NSP3 protein does not affect the synthesis of the remaining viral proteins
5 10 15 20 250
1
2
3
4
5
6siRNANSP3
siRNACtrol
time post-infection (hr)