Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP)

Svitkin, Yuri V.; Costa-Mattioli, Mauro; Herdy, Barbara; Perreault, Sandra; Sonenberg, Nahum

doi:10.1261/rna.606407

Cited by 22 publications

(30 citation statements)

References 58 publications

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“…3B, treatment of the translation reaction mixtures with glutathione S-transferase (GST)-Paip2 but not GST alone inhibited translation of both capped, polyadenylated RNA and PV5Ј-FLuc-3Ј-A 71 but not PV5Ј-FLuc-3Ј-A 0 , which has no poly(A) tail. The degree of inhibition approached 40%, similar to previously reported effects of GST-Paip2 on IRES translation in vitro (32,56). To test the importance of PABP to translation of these reporters, recombinant PABP was added to supplement endogenous PABP in these reactions.…”

Section: C Pro Inhibits Ires-mediated Translation Of Poly(a) ؉ and Psupporting

confidence: 79%

“…The previously described shutoff mechanisms of cleavage of PTB, PCBP2, binding of PCBP2 to the cloverleaf, and now PABP cleavage are not mutually exclusive and are likely complementary. More recently, PABP was reported to not be required for viral RNA replication in PABP depletion experiments, though depletion did decrease viral IRES translation (56). In other work, we have shown that PABP inhibits 3D pol activity on viral RNA poly(A) templates, which complements these results.…”

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confidence: 86%

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Cleavage of Poly(A)-Binding Protein by Poliovirus 3C Proteinase Inhibits Viral Internal Ribosome Entry Site-Mediated Translation

Bonderoff

LaRey

Lloyd³

2008

J Virol

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Poliovirus (PV) is a medically important member of the picornavirus genus Enterovirus, which is collectively responsible for millions of human infections per year worldwide. Enteroviruses contain a single-stranded RNA genome that is a direct template for translation by the host cell's translational machinery, and this translation step is required for host morbidity and progeny virus production. The ϳ7.5-kb PV genome is comprised of a single open reading frame encoding a 247-kDa polyprotein which is processed to 11 final products, a short 3Ј untranslated region (UTR) with defined RNA structure, a poly(A) tail of strain-specific length that is approximately 80 to 120 nucleotides long, and a ϳ700-nucleotide 5Ј UTR containing an internal ribosome entry site (IRES) for facilitation of viral translation. The extreme 5Ј end of the PV genome, known as the cloverleaf or oriL, has roles in nucleic acid stability (5), translation (54), and viral genomic RNA replication (43,59). Notably, the viral genome has no 7-methylguanosine cap.The viral IRES is responsible for driving translation, and the 7-methylguanosine cap-binding protein, eukaryotic initiation factor 4E (eIF4E), is not required for IRES-dependent translation. The large scaffolding protein eIF4G is cleaved early in PV infection, and the fragments produced cannot support capdependent translation (40); however, cleaved eIF4G stimulates IRES-driven translation more than intact eIF4G (12,25,41). The PV IRES is thought to bind eIF4G directly, as it does to related viral IRESs such as that of encephalomyocarditis virus (34). The C-terminal fragment of eIF4G that functions in IRES-dependent translation (p100) is able to recruit eIF3 and the 40S ribosomal subunit to the viral RNA. Other canonical translation factors involved in PV IRES-mediated translation include the DEAD box helicase eIF4A and its activator eIF4B, which also directly binds the IRES (47). The poly(A) tail/ poly(A)-binding protein (PABP) interaction with eIF4G is also proposed to stimulate PV IRES translation by mediating a closed-loop structure and enhanced interactions between PABP and eIF4GI (7,13,45,57).In addition to these canonical translation factors, other cellular proteins, IRES trans-activating factors (ITAFs), stimulate IRES translation. These include polypyrimidine tract-binding protein (PTB) (26, 50), poly r(C)-binding protein 2 (PCBP2) (9, 10), lupus autoantigen (La) (44), and upstream of N-ras (Unr) (16). The ITAFs map to multiple binding sites in enteroviral IRES structures (9,18,23) and are proposed to have roles as chaperones to hold the IRES in a translation-competent conformation and possibly to also act as de facto signals for the switch from translation to replication. Additionally, ITAFs may recruit effector proteins to the IRES, as seen in the recent discovery that SRp20 binds PCBP2 and is required for IRES-mediated translation (6).The two viral proteinases, 2A proteinase (2A pro ) and 3C proteinase (3C pro ), cleave several cellular proteins during infection in addition to viral p...

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Section: C Pro Inhibits Ires-mediated Translation Of Poly(a) ؉ and Psupporting

confidence: 79%

supporting

confidence: 86%

Cleavage of Poly(A)-Binding Protein by Poliovirus 3C Proteinase Inhibits Viral Internal Ribosome Entry Site-Mediated Translation

Bonderoff

LaRey

Lloyd³

2008

J Virol

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“…The potential contribution of PABP bound to poly(A) sequences during the initiation of negative-strand RNA synthesis has been considered previously (21,35). Nevertheless, recent evidence suggests that picornavirus RNA replication does not require PABP (43). For this reason and for simplicity, we chose to exclude PABP from the diagrams in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…7B, note 3Ј poly(A) tail lengths of ϳ28 to 43 bases]. The PV A (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) RNA templates were transcribed into 5Ј VPglinked polyU RNA products (Fig. 7C).…”

Section: Vpg-linked Poly(u) From Pv a (84) A (51)mentioning

confidence: 99%

Poly(A) at the 3′ End of Positive-Strand RNA and VPg-Linked Poly(U) at the 5′ End of Negative-Strand RNA Are Reciprocal Templates during Replication of Poliovirus RNA

Steil¹,

Kempf²,

Barton

2010

J Virol

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A 3 poly(A) tail is a common feature of picornavirus RNA genomes and the RNA genomes of many other positive-strand RNA viruses. We examined the manner in which the homopolymeric poly(A) and poly(U) portions of poliovirus (PV) positive-and negative-strand RNAs were used as reciprocal templates during RNA replication. Poly(A) sequences at the 3 end of viral positive-strand RNA were transcribed into VPg-linked poly(U) products at the 5 end of negative-strand RNA during PV RNA replication. Subsequently, VPg-linked poly(U) sequences at the 5 ends of negative-strand RNA templates were transcribed into poly(A) sequences at the 3 ends of positive-strand RNAs. The homopolymeric poly(A) and poly(U) portions of PV RNA products of replication were heterogeneous in length and frequently longer than the corresponding homopolymeric sequences of the respective viral RNA templates. The data support a model of PV RNA replication wherein reiterative transcription of homopolymeric templates ensures the synthesis of long 3 poly(A) tails on progeny RNA genomes.Many positive-strand RNA viruses (e.g., members of the Picornavirales, Nidovirales, Togaviridae, Caliciviridae, and Astroviridae) have long poly(A) sequences at the 3Ј termini of their RNA genomes (16,25), yet the mechanisms by which 3Ј poly(A) sequences are derived during viral replication are unclear. Picornavirus RNA genomes, including that of poliovirus (PV), have a covalently linked 5Ј-terminal protein called VPg (viral protein, genome linked), a 5Ј untranslated region (UTR), a single large open reading frame, a 3Ј UTR, and a poly(A) tail of variable length (ϳ20 to 150 adenosine residues) (1). RNA polymerases encoded by viruses in the order Picornavirales utilize viral proteins (and their nucleotidylylated intermediates) to prime the initiation of RNA replication at the 3Ј termini of viral RNA templates (28,29,31,34). In this mechanism, the viral protein VPg becomes covalently linked to the 5Ј ends of both positive-and negative-strand RNAs during viral RNA replication (30, 32). PV, which is commonly studied to elucidate mechanisms of picornavirus replication, is viable when the 3Ј UTR of the genome is deleted (12, 44); however, the 3Ј poly(A) tail is essential for RNA replication (33, 39). The length of the 3Ј poly(A) tail required for virus viability and for efficient negative-strand RNA synthesis has been examined in some detail (35,45). PV RNAs with 3Ј poly(A) tails less than 9 bases long support less than 1% of wild-type negative-strand RNA synthesis, whereas poly(A) tails Ն20 bases long support wild-type levels of negative-strand RNA synthesis (35).In this investigation, we programmed PV RNAs with defined 3Ј 84-, 51-, and 32-base poly(A) sequences [designated poly(A) (84) , poly(A) (51) , and poly(A) (32) , respectively] into cell-free reactions that faithfully reconstitute all of the metabolic steps of viral mRNA translation (11,22,23) and viral RNA replication (5, 7, 27). A significant advantage of this experimental system is the ability to study one cycle of seq...

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“…3A). First, the IRES element in EMCV recruits the translation initiation complex directly, through interactions with eIF4G, and that process can be stimulated by the poly(A) tail (Humphreys et al 2005) and PABP, the poly(A) binding protein (Bergamini et al 2000;Svitkin et al 2007). Second, in contrast to capped mRNAs, the AUG is positioned at the site of ribosome recruitment, so there is no scanning and AUG recognition occurs in a spatially restricted manner (Kaminski et al 1994).…”

Section: Implications For Mirna-mediated Repression Of Ires-driven Trmentioning

confidence: 99%

Computational analysis of miRNA-mediated repression of translation: Implications for models of translation initiation inhibition

Nissan¹,

Parker²

2008

RNA

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The mechanism by which miRNAs inhibit translation has been under scrutiny both in vivo and in vitro. Divergent results have led to the suggestion that miRNAs repress translation by a variety of mechanisms including blocking the function of the cap in stimulating translation. However, these analyses largely only examine the final output of the multistep process of translation. This raises the possibility that when different steps in translation are rate limiting, miRNAs might show different effects on protein production. To examine this possibility, we modeled the process of translation initiation and examined how the effects of miRNAs under different conditions might be explained. Our results suggest that different effects of miRNAs on protein production in separate experiments could be due to differences in rate-limiting steps. This analysis does not rule out that miRNAs directly repress the function of the cap structure, but it demonstrates that the observations used to argue for this effect are open to alternative interpretations. Taking all the data together, our analysis is consistent with the model that miRNAs may primarily repress translation initiation at a late step.

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Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP)

Cited by 22 publications

References 58 publications

Cleavage of Poly(A)-Binding Protein by Poliovirus 3C Proteinase Inhibits Viral Internal Ribosome Entry Site-Mediated Translation

Cleavage of Poly(A)-Binding Protein by Poliovirus 3C Proteinase Inhibits Viral Internal Ribosome Entry Site-Mediated Translation

Poly(A) at the 3′ End of Positive-Strand RNA and VPg-Linked Poly(U) at the 5′ End of Negative-Strand RNA Are Reciprocal Templates during Replication of Poliovirus RNA

Computational analysis of miRNA-mediated repression of translation: Implications for models of translation initiation inhibition

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