Viral strategies to control cellular translation Rodney Colina Munioz

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1 Viral strategies to control cellular translation Rodney Colina Munioz Department of Biochemistry, McGill University, Montreal, Canada

2 Initiation of eukaryotic translation GMPPNP Cycloheximide

3 La?

4 Internal Ribosome Entry Site Picornavirus genomes fail to meet the inclusion criteria for 40S ribosomal subunit scanning from the 5' end of the mrna on four counts: 1) they are uncapped mrnas, 2) contains highly structured 5'UTRs 3) The 5 UTR are approximately 750 nt. long, 4) which harbour about 10 cryptic AUG codons

5 Viral and cellular IRES

6 Gold standard measure of IRES activity Intercistronic region Assay System Cistron 1 expression Cistron 2 expression 5 Cistron 1 Cistron 2 RRL 5 Cistron 1 Cistron 2 RRL Cistron 1 Cistron 2 Cistron 1 Cistron 2 Cistron 1 Cistron 2 PV-infected HeLa cells RRL RRL Adapted from Hellen and Pestova, 1999.

7 Picornaviruses Type I Enteroviruses/ Rhinoviruses Type II Cardiovirus/ Aphtoviruses Type II Hepatitis A virus

8 Cricket Paralysis Virus RNA Naturally Occurring Dicistronic Cricket Paralysis Virus RNA Is Regulated by Two Internal Ribosome Entry Sites. Special structural characteristics of cricket paralysis-like virus RNAs. (a) In contrast to picornaviruses, the parts of the RNA coding for non-structural and structural proteins are separated into separate open reading frames, ORF1 and ORF2, respectively. (b) It has been proposed that the intergenic internal ribosome entry site (IRES) region interacts with the 40S ribosomal subunit in such a way as to mimic an occupied P-site. The 3' stem-loop structure is thought to form a pseudoknot, together with the overlined nucleotides just upstream of the CAA codon used to initiate translation of the Plautia stali intestine virus capsid protein.

9 A cell cycle-dependent internal ribosome entry site Pyronnet et al., 2000, Mol Cell

10 List of IRES-trans-acting-factors (ITAFs) Functional data *PCBP1 and PCBP2 Poliovirus 5 UTR *PTB binds to EMCV, FMDV, hrhinovirus, HAV, HCV IRES -Apaf-1 and *unr binds to human rhinovirus IRES *La poliovirus, HCV and coxsackievirus B3, HIV-1 leader, Human T-lymphotropic virus type I - XIAP and BIP and IRESs. *ITAF 45 also known as murine proliferation-associated protein (Mpp1) binds to IRES FMDV Without functional data *hnrnpl binds to HCV IRES *GADPH HAV IRES *Nucleolin Poliovirus IRES

11 Control of translation in HeLa cells infected with Poliovirus

12 Host Cell Shutoff 1. trna Cleavage (T-even phages) 2. eif4f Complex Formation a) eif4g Cleavage (Poliovirus, FMDV). b) eif4e Dephosphorylation (Influenza virus, VSV). c) 4E-BPs Dephosphorylation (VSV, EMCV). 3. PABP inactivation (Poliovirus, Reovirus) 4. eif2 Phosphorylation (VSV, SV, EMCV) 5. Interferon system a) Activation of PKR and Rnase L

13 eif4f Cap-Binding Protein Complex: A Regulator of Virus Infection Efficiency?

14 eif4f cap-binding protein complex eif4g eif4e eif4a

15 Initiation of eukaryotic translation

16 Translation Initiation 60S 40S 4E eif3 eif4g 4A m 7 Gppp 2A GTP AUG PABP PABP AAAAAAAAAAAAAAAAAAAAAAAA 43S 80S

17 eif4g inactivation

18 Cleavage of eif4g by Picornaviruses

19 Inhibition of Host Cell Protein Synthesis after Poliovirus Infection: Correlation with the Cleavage of eif4gi and eif4gii Gradi, Alessandra et al. (1998) Proc. Natl. Acad. Sci. USA 95,

20 4A3 is required for NMD but not for bulk translation

21 eif4gi/ii are required for poliovirus-ires-dependent translation Costa-Mattioli and Sonenberg, Unpublished data

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23 Costa-Mattioli and Sonenberg, Unpublished data

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25 eif4e phosphorylation

26 eif4e is phosphorylated on Ser209 by Mnk1/2 stress growth factors hormones mitogens cytokines P38 MAPK RAS RAF MEK ERK P eif4e Mnk1/2 eif4g

27 Adenovirus reduces eif4e phosphorylation Cuesta et al., 2000 Feigenblum and Schneider, 1993; Zhan et al., 1994

28 VSV reduces eif4e but not MNK1 phosphorylation Connor and Lyles; 2002

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31 4E-BPs phosphorylation

32 The 4E-BPs regulate eif4f formation eif4f complex eif4e 4E-BP eif4e eif4e eif4g eif4a hormones growth factors mitogens cytokines GPCR agonists adenovirus P P P P 4E-BP serum starvation amino acid deprivation EMCV poliovirus environmental stress

33 Adenovirus inactivates the translation inhibitors 4E-BP1 and 4E-BP2 Feigenblum and Schneider, 1996

34 Adenovirus inactivates the translation inhibitors 4E-BP1 and 4E-BP2 Gingras et al., 1997

35 Phosphorylation of 4E-BP1 decreases in parallel with the inhibition of host synthesis in EMCV-infected cells Gingras et al., 1996

36 Signaling pathway to 4E-BPs phosphorylation growth factors wortmannin PI3K Akt/PKB rapamycin FRAP/mTOR 4E-BP1

37 Rapamycin enhances EMCV translation Beretta et al., 1996

38 Rapamycin and Wortmannin rescue an EMCV defective mutant Svitkin et al., 1998

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40 4E-BP1 and eif4e Oppositely Regulate EMCV RNA Translation and Replication in Untreated Extract

41 4E-BP1 and eif4e oppositely regulate EMCV synthesis in an untreated Krebs-2 cell extract PFU/ml X control GST GST-4E-BP1 GST-4E-BP1-4EBS GST-4E-BP2 GST-eIF4E GST-eIF4EW73A eif4e GDP m 7 GDP eif4a 2A 2A+GSP-4E-BP1 2A+eIF4E eif4g-ct

42 4E-BP1 and eif4e Regulate EMCV RNA Translation in A Nuclease-Treated Extract Only in the Presence of Competing Cellular mrnas

43 eif4e Knockdown Stimulates EMCV Infection

44 Characterization of eif4e-knockdown Cells

45 eif4e Knockdown Stimulates PV Infection

46 eif4e Knock Down Stimulates EMCV Infection sirna not infected EMCV (h p.i.) kd S-Met WB: β actin WB: eif4e

47 eif4e Knock Down Enhances EMCV Yields (Plaque Assay) sirna eif4e control Mock 6 hours 4 hours post infection 6 hours post infection

48 sirna not infected Poliovirus (h p.i.) kd eif4e Knock Down Stimulates Poliovirus Infection S-Met WB: eif4e WB: β actin

49 A model for regulation of EMCV replication by 4E-BPs

50 Picornaviruses IRES type I and II require all the canonical initiation factors but not eif4e. What about IRES type III?

51 VSV reduces 4E-BP1 phosphorylation Connor and Lyles, 2002

52 PABP

53 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA PABP PABP PABP cap Closed loop model of translation initiation complex 40s ribosome eif4g eif3 eif4e eif4a Cap

54 Closed Loop Model 4E 4E 4G 4G CAP CAP 4A4A PABP

55 eif2alpha kinases

56

57 eif2α KINASES FAMILY GCN K618 m1 m2 m N-terminal uinasa parcial Dominio eif2α quinasa K199 HRM1 HRM2 HisRS-like nión a ribosoma HRI Dominio eif2α quinasa PKR M1 M2 K nó ominio eif2α quinasa PERK K618 SP TM Dominio lumenal Dominio eif2α quinasa

58 PKR

59 Dey et al., 2005

60 Overexpression of PKR render cells resistant to EMCV infection (Meurs et al., 1992). Reduction of PKR convert EMCV infection into a permanent infection (Yeung et al., 1999).

61 PKR / MEFs are more permissive for VSV infection than are control MEFs. Stojdl et al., 2000

62 EMCV titers in brain and serum of treated and untreated mice determined 72 h after infection. Yang et al., 1995

63 Stojdl et al., 2000

64 PERK

65 Baltzis et al., 2004

66 Baltzis et al., 2004

67 Activation of PKR 1) induction of interferon response through the IRF-1, NF-KB, 2) Inhibition of protein synthesis, 3) induction of apoptosis.

68 GCN2

69 A NEW ROLE OF THE eif2a KINASE, GCN2, IN THE CELLULAR RESPONSE TO VIRAL INFECTION. Juan José Berlanga, Iván Ventoso, Luis Carrasco, César de Haro Centro de Biología Molecular Severo Ochoa (CSIC-UAM)

70 TRANSLATIONAL CONTRO BY THE eif2alpha KINASES

71 CONTROL TRADUCCIONAL DE LA EXPRESIÓN DE GCN4 EN S. cerevisiae Y ATF4 EN MAMÍFEROS Respuesta a estrés en mamíferos Respuesta a estrés en levaduras Señales de estrés Escasez de nutrientes eif2α (fosforilación) eif2α (fosforilación) ATF4 (traducción) GCN4 (traducción) Genes de respuesta a estrés (transcripción) Enzimas biosintéticas (transcripción)

72 The genomic RNA of Sindbis virus stimulates phosphorylation of GCN2 eif2a through

73 LA ACTIVACIÓN DE GCN2 POR EL RNA DEL VIRUS SINDBIS IMPLICA DOS REGIONES NO ADYACENTES DEL EXTREMO 5 DEL RNA VIRAL Y EL DOMINIO HIS-RS DE GCN

74 CINÉTICA DE SÍNTESIS DE PROTEÍNAS VIRALES EN FIBROBLASTOS EMBRIONARIOS OBTENIDOS DE RATONES CONTROL Y GCN2 -/-

75 SÍNTESIS DE PROTEÍNAS VIRALES Y EFECTO CITOPÁTICO EN -/- -/- FIBROBLASTOS EMBRIONARIOS OBTENIDOS DE RATONES CONTROL, GCN2 Y PKR

76 EFECTO DE LA SOBREEXPRESIÓN DE GCN2 EN LA REPLICACIÓN Y EN LA SÍNTESIS DE PROTEÍNAS VIRALES Producción viral en un ciclo de infección Clon 3T3 Título viral (log pfu/ml) EMCV SFV SV VSV CONTROL 7.16 ± ± ± ± 0.12 GCN2-K618R 7.36 ± ± ± ± 0.02 GCN2-WT 7.10 ± ± ± ± 0.01