Rotavirus strategies to take over the host cell response Instituto de Biotecnología Universidad Nacional Autónoma de México Battle between viruses and host cells Viral counter-measures Virus Host Host cell defenses During rotavirus infection the cellular protein synthesis is shutoff Virus - + During infection NSP3 displaces PABP from eIF4G and prevents cellular protein synthesis mRNA AAAA NSP3PABP 4A eIF4G Cap 4E eIF4F During RV infection, PABP relocalizes to the nucleus, and this depends on NSP3 a-PABP a-eIF4GI merge - virus control a-NSP2 + virus siIrr siNSP3 Montero H et al, 2006, J Virol. 80:9031-9038 polyA+ mRNAs are also relocalized in the nucleus of the cell during infection probe :Oligo dT No probe No Virus siIrr aNSP3 Merge + Virus siIrr + Virus siNSP3 Rubio RM et al, 2013. J Virol. During RV infection cellular mRNAs accumulate in the nucleus GRP78 GRP94 Cytop 5 Nuc 4 1.0 3 2 0.5 1 Relative amount Relative of amount Relative amount Relative of amount RNA units)(arbitrary RNA RNA units)(arbitrary RNA 0 0.0 Mock 6h 12h s/v 6h 12h Hours postinfection Hours postinfection 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 hr post-infection: s/v 1 2 3 4 5 6 7 8 10 12 anti-eIF2ap Rojas M et al, 2010. J Virol. Translation factor eIF2 a GTP eIF2B GTP GDP a GDP GDP Met a a eIF2B GTP GDP Global translation inhibition Selective synthesis Translation initiation Rotavirus infection induces phosphorylation of eIF2a in a PKR-dependent manner MEFs: PERK-/- WT PKR-/- MA104 - Tg Ar Tg - Tg - Irr siPKR α-eIF2α-P eIF2a-P eIF2a PKR Rojas M et al, 2010. J Virol. Rotavirus strategies to control cell- protein synthesis: 1.- NSP3 interacts with eIF4G and prevents the binding 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 granules are formed when eIF2a is phosphorylated Composition of Stress Granules -mRNAs Ctrol -Translation factors PABP eIF4G merge eIF3 Ars eIF4G eIF4E (partially) PABP -Small ribosomal subunit -RBPs like: TIA, TIAR, HuR, TTP, G3BP, FragileX, SMN, etc. Rotavirus infection prevents the formation of stress granules aTIA aNSP2 merge control arsenite . Virol virus virus & arsenite Montero H et al, al, etMontero2008. J H SGs and P-bodies are sites of RNA triage. Li Y R et al. J Cell Biol 2013;201:361-372 P-Bodies are reduced in infected cells aeIF4E a-NSP2 merge -virus +virus Oceguera A. et al, unpublished The OAS/RNAse L dsRNA complex Activation of 2´5´OAS During RV infection there is no degradation of rRNAs RRV M 1 10 28 S 18 S Sanchez et al, unpublished Summary -Rotavirus infection causes a severe inhibition of cellular protein synthesis -NSP3 prevents translation of polyA mRNAs, -The nucleo-cytoplasmic transport of polyA mRNAs is blocked, -eIF2a is phosphorylated by PKR in RV-infected cells -The formation of stress granules is prevented . - The number of P-bodies is reduced -The OAS-RNAse L pathway is blocked during RV-infection, VP3 participates in this control Grupo Arias/López CONACyT, and DGAPA-UNAM Nucleo-Cytoplasmic transport of mRNAs Rosa Ma Rubio PKR and dsRNA Margarito Rojas Stress Granules Hilda Montero P-Bodies Alfonso Oceguera Gracias!! OAS/RNAse L Liliana Sánchez What is the nature of the dsRNA that activates PKR in infected cells? a-dsRNA a-NSP5 Merge Control RNAse A RNAse III Silencing NSP3 protein does not affect the synthesis of the remaining viral proteins siRNA Ctrol NSP3 a-vimentin M a-NSP3 VP1 VP2 VP4 6 siRNANSP3 5 4 VP6 3 siRNACtrol VP7 2 NSP3 1 0 NSP4 5 10 15 20 25 time post-infection (hr).