Rotavirus strategies to take over the host cell response

Instituto de BiotecnologíaUniversidad Nacional Autónoma de México

Virus Host

Host cell defenses

Viral counter-measures

Battle between viruses and host cells

During rotavirus infection the cellularprotein synthesis is shutoff

+Virus-

PABPAAAA

Cap

mRNA

eIF4F

eIF4G

4A

4E

NSP3

During infection NSP3 displaces PABP fromeIF4G and prevents cellular protein synthesis

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

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.

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

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.

GDP

Translation factor eIF2

a

GTP

a

Met

a

GTP

Translation initiation

GTPeIF2B

GDP

GDP

a

GDPeIF2B

Global translationinhibition

Selective synthesis

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.

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

Stress granulesare formed when eIF2a is phosphorylated

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

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.

SGs and P-bodies are sites of RNA triage.

Li Y R et al. J Cell Biol 2013;201:361-372

aeIF4E a-NSP2 merge

+virus

-virus

P-Bodies are reduced in infected cells

Oceguera A. et al, unpublished

Activation of 2´5´OAS

dsRNAThe OAS/RNAse L

complex

1 10

RRVM

28 S

18 S

During RV infection there is no degradationof rRNAs

Sanchez et al, unpublished

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

Grupo Arias/López

CONACyT, and DGAPA-UNAM

Gracias!!

PKR and dsRNAMargarito Rojas

Stress GranulesHilda Montero

P-BodiesAlfonso Oceguera

OAS/RNAse LLiliana Sánchez

Nucleo-Cytoplasmic transportof mRNAsRosa Ma Rubio

a-dsRNA a-NSP5 Merge

Control

What is the nature of the dsRNA that activates PKR in infected cells?

RNAse III

RNAse A

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)