scholarly journals Detailed Analysis of the Requirements of Hepatitis A Virus Internal Ribosome Entry Segment for the Eukaryotic Initiation Factor Complex eIF4F

2001 ◽  
Vol 75 (17) ◽  
pp. 7864-7871 ◽  
Author(s):  
Andrew M. Borman ◽  
Yanne M. Michel ◽  
Katherine M. Kean
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Nonstructural Protein ◽  
Binding Protein ◽  
Encephalomyocarditis Virus ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Internal Ribosome Entry ◽  
Eif4f Complex ◽  
Internal Ribosome Entry Segment

ABSTRACT The hepatitis A virus (HAV) internal ribosome entry segment (IRES) is unique among the picornavirus IRESs in that it is inactive in the presence of either the entero- and rhinovirus 2A or aphthovirus Lb proteinases. Since these proteinases both cleave eukaryotic initiation factor 4G (eIF4G) and HAV IRES activity could be rescued in vitro by addition of eIF4F to proteinase-treated extracts, it was concluded that the HAV IRES requires eIF4F containing intact eIF4G. Here, we show that the inability of the HAV IRES to function with cleaved eIF4G cannot be attributed to inefficient binding of the cleaved form of eIF4G by the HAV IRES. Indeed, the binding of both intact eIF4F and the C-terminal cleavage product of eIF4G to the HAV IRES was virtually indistinguishable from their binding to the encephalomyocarditis virus IRES, as assessed by UV cross-linking and filter retention assays. Rather, we show that HAV IRES activity requires, either directly or indirectly, components of the eIF4F complex which interact with the N-terminal fragment of eIF4G. Effectively, HAV IRES activity, but not that of the human rhinovirus IRES, was sensitive to the rotavirus nonstructural protein NSP3 [which displaces poly(A)-binding protein from the eIF4F complex], to recombinant eIF4E-binding protein (which prevents the association of the cap binding protein eIF4E with eIF4G), and to cap analogue.

scholarly journals Cellular cap-binding protein, eIF4E, promotes picornavirus genome restructuring and translation

2017 ◽  
Vol 114 (36) ◽  
pp. 9611-9616 ◽  
Author(s):  
Brian C. Avanzino ◽  
Gabriele Fuchs ◽  
Christopher S. Fraser
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Protein A ◽  
Typical Feature ◽  
Initiation Factor ◽  
Internal Ribosome Entry ◽  
High Affinity ◽  
Eif4f Complex ◽  
Cap Binding Complex ◽  
2A Protease

Picornaviruses use internal ribosome entry sites (IRESs) to translate their genomes into protein. A typical feature of these IRESs is their ability to bind directly to the eukaryotic initiation factor (eIF) 4G component of the eIF4F cap-binding complex. Remarkably, the hepatitis A virus (HAV) IRES requires eIF4E for its translation, but no mechanism has been proposed to explain this. Here we demonstrate that eIF4E regulates HAV IRES-mediated translation by two distinct mechanisms. First, eIF4E binding to eIF4G generates a high-affinity binding conformation of the eIF4F complex for the IRES. Second, eIF4E binding to eIF4G strongly stimulates the rate of duplex unwinding by eIF4A on the IRES. Our data also reveal that eIF4E promotes eIF4F binding and increases the rate of restructuring of the poliovirus (PV) IRES. This provides a mechanism to explain why PV IRES-mediated translation is stimulated by eIF4E availability in nuclease-treated cell-free extracts. Using a PV replicon and purified virion RNA, we also show that eIF4E promotes the rate of eIF4G cleavage by the 2A protease. Finally, we show that cleavage of eIF4G by the poliovirus 2A protease generates a high-affinity IRES binding truncation of eIF4G that stimulates eIF4A duplex unwinding independently of eIF4E. Therefore, our data reveal how picornavirus IRESs use eIF4E-dependent and -independent mechanisms to promote their translation.

scholarly journals Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes.

1996 ◽  
Vol 16 (12) ◽  
pp. 6870-6878 ◽  
Author(s):  
T V Pestova ◽  
I N Shatsky ◽  
C U Hellen
Keyword(s):  
Binding Protein ◽  
Encephalomyocarditis Virus ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Structural Element ◽  
Entry Site ◽  
Independent Manner ◽  
Central Domain ◽  
Nontranslated Region ◽  
Carboxy Terminal

Eukaryotic translation is initiated following binding of ribosomes either to the capped 5' end of an mRNA or to an internal ribosomal entry site (IRES) within its 5' nontranslated region. These processes are both mediated by eukaryotic initiation factor 4F (eIF4F), which consists of eIF4A (helicase), eIF4E (cap-binding protein), and eIF4G subunits. Here we present a functional analysis of eIF4F which defines the subunits and subunit domains necessary for its function in initiation mediated by the prototypical IRES element of encephalomyocarditis virus. In an initiation reaction reconstituted in vitro from purified translation components and lacking eIF4A and -4F, IRES-mediated initiation did not require the cap-binding protein eIF4E but was absolutely dependent on eIF4A and the central third of eIF4G. This central domain of eIF4G bound strongly and specifically to a structural element within the encephalomyocarditis virus IRES upstream of the initiation codon in an ATP-independent manner and with the same specificity as eIF4F. The carboxy-terminal third of eIF4G did not bind to the IRES. The central domain of eIF4G was itself UV cross-linked to the IRES and strongly stimulated UV cross-linking of eIF4A to the IRES in conjunction with either eIF4B or with the carboxy-terminal third of eIF4G.

scholarly journals Insulin-stimulated kinase from rat fat cells that phosphorylates initiation factor 4E-binding protein 1 on the rapamycin-insensitive site (serine-111)

1998 ◽  
Vol 336 (1) ◽  
pp. 39-48 ◽  
Author(s):  
Kate J. HEESOM ◽  
Matthew B. AVISON ◽  
Tricia A. DIGGLE ◽  
Richard M. DENTON
Keyword(s):  
Binding Protein ◽  
Initiation Factor ◽  
Fat Cells ◽  
Two Dimensional ◽  
Eukaryotic Initiation Factor ◽  
Peptide Substrates ◽  
Eukaryotic Initiation Factor 4E ◽  
Eif4f Complex ◽  
Layer Analysis ◽  
Rat Fat Cells

The effects of insulin and rapamycin on the phosphorylation of the translation regulator, initiation factor 4E-binding protein 1 (4E-BP1) have been studied in rat fat cells by following changes in the incorporation of 32P from [32P]Pi under steady-state conditions. Both unbound 4E-BP1 and 4E-BP1 bound to eukaryotic initiation factor 4E (eIF4E) were isolated from the cells and then digested with trypsin and other proteases; the radiolabelled phosphopeptides were then separated by two-dimensional thin- layer analysis and HPLC. The results provide confirmation of the conclusion of Fadden, Haystead and Lawrence [J. Biol. Chem. (1997) 272, 10240–10247] that insulin increases the phosphorylation of four sites that fit a Ser/Thr-Pro motif (Thr-36, Thr-45, Ser-64 and Thr-69) and that taken together these phosphorylations result in the dissociation of 4E-BP1 from eIF4E. The effects of insulin on the phosphorylation of these sites, and hence dissociation from eIF4E, are blocked by rapamycin. However, the present study also provides evidence that insulin increases the phosphorylation of 4E-BP1 bound to eIF4E on a further site (Ser-111) and that this is by a rapamycin-insensitive mechanism. Extraction of rat epididymal fat cells followed by chromatography on Mono-S and Superose 12 columns resulted in the separation of both an insulin-stimulated eIF4E kinase and an apparently novel kinase that is highly specific for Ser-111 of 4E-BP1. The 4E-BP1 kinase was activated more than 10-fold by incubation of the cells with insulin and was markedly more active towards 4E-BP1 bound to eIF4E than towards unbound 4E-BP1. The effects of insulin were blocked by wortmannin, but not by rapamycin. A 14-mer peptide based on the sequence surrounding Ser-111 of 4E-BP1 was also a substrate for the kinase, but peptide substrates for other known protein kinases were not. The kinase is quite distinct from casein kinase 2, which also phosphorylates Ser-111 of 4E-BP1. The possible importance of these kinases in the phosphorylation of 4E-BP1 in fat cells is discussed. It is suggested that the phosphorylation of Ser-111 might be a priming event that facilitates the subsequent phosphorylation of Thr-36, Thr-45, Ser-64 and Thr69 by a rapamycin-sensitive process that initiates the dissociation of 4E-BP1 from eIF4E and hence the formation of the eIF4F complex.

scholarly journals Canonical Initiation Factor Requirements of the Myc Family of Internal Ribosome Entry Segments

2009 ◽  
Vol 29 (6) ◽  
pp. 1565-1574 ◽  
Author(s):  
Keith A. Spriggs ◽  
Laura C. Cobbold ◽  
Catherine L. Jopling ◽  
Rebecca E. Cooper ◽  
Lindsay A. Wilson ◽  
...  
Keyword(s):  
Untranslated Region ◽  
Ternary Complex ◽  
Initiation Factor ◽  
Start Codon ◽  
Structural Element ◽  
Internal Ribosome Entry ◽  
Ribosomal Subunits ◽  
Eif4f Complex ◽  
Internal Ribosome Entry Segment ◽  
Minimum Requirements

ABSTRACT Initiation of protein synthesis in eukaryotes requires recruitment of the ribosome to the mRNA and its translocation to the start codon. There are at least two distinct mechanisms by which this process can be achieved; the ribosome can be recruited either to the cap structure at the 5′ end of the message or to an internal ribosome entry segment (IRES), a complex RNA structural element located in the 5′ untranslated region (5′-UTR) of the mRNA. However, it is not well understood how cellular IRESs function to recruit the ribosome or how the 40S ribosomal subunits translocate from the initial recruitment site on the mRNA to the AUG initiation codon. We have investigated the canonical factors that are required by the IRESs found in the 5′-UTRs of c-, L-, and N-myc, using specific inhibitors and a tissue culture-based assay system, and have shown that they differ considerably in their requirements. The L-myc IRES requires the eIF4F complex and the association of PABP and eIF3 with eIF4G for activity. The minimum requirements of the N- and c-myc IRESs are the C-terminal domain of eIF4G to which eIF4A is bound and eIF3, although interestingly this protein does not appear to be recruited to the IRES RNA via eIF4G. Finally, our data show that all three IRESs require a ternary complex, although in contrast to c- and L-myc IRESs, the N-myc IRES has a lesser requirement for a ternary complex.

scholarly journals Eukaryotic Initiation Factor 4G-Poly(A) Binding Protein Interaction Is Required for Poly(A) Tail-Mediated Stimulation of Picornavirus Internal Ribosome Entry Segment-Driven Translation but Not for X-Mediated Stimulation of Hepatitis C Virus Translation

2001 ◽  
Vol 21 (13) ◽  
pp. 4097-4109 ◽  
Author(s):  
Yanne M. Michel ◽  
Andrew M. Borman ◽  
Sylvie Paulous ◽  
Katherine M. Kean
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Initiation Factor ◽  
Translation System ◽  
Eukaryotic Initiation Factor ◽  
Internal Ribosome Entry ◽  
Internal Ribosome Entry Segment ◽  
Eukaryotic Initiation Factor 4G ◽  
Hcv Ires ◽  
Stimulation Of

ABSTRACT Efficient translation of most eukaryotic mRNAs results from synergistic cooperation between the 5′ m7GpppN cap and the 3′ poly(A) tail. In contrast to such mRNAs, the polyadenylated genomic RNAs of picornaviruses are not capped, and translation is initiated internally, driven by an extensive sequence termed IRES (for internal ribosome entry segment). Here we have used our recently described poly(A)-dependent rabbit reticulocyte lysate cell-free translation system to study the role of mRNA polyadenylation in IRES-driven translation. Polyadenylation significantly stimulated translation driven by representatives of each of the three types of picornaviral IRES (poliovirus, encephalomyocarditis virus, and hepatitis A virus, respectively). This did not result from a poly(A)-dependent alteration of mRNA stability in our in vitro translation system but was very sensitive to salt concentration. Disruption of the eukaryotic initiation factor 4G-poly(A) binding protein (eIF4G-PABP) interaction or cleavage of eIF4G abolished or severely reduced poly(A) tail-mediated stimulation of picornavirus IRES-driven translation. In contrast, translation driven by the flaviviral hepatitis C virus (HCV) IRES was not stimulated by polyadenylation but rather by the authentic viral RNA 3′ end: the highly structured X region. X region-mediated stimulation of HCV IRES activity was not affected by disruption of the eIF4G-PABP interaction. These data demonstrate that the protein-protein interactions required for synergistic cooperativity on capped and polyadenylated cellular mRNAs mediate 3′-end stimulation of picornaviral IRES activity but not HCV IRES activity. Their implications for the picornavirus infectious cycle and for the increasing number of identified cellular IRES-carrying mRNAs are discussed.

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