scholarly journals Hepatitis C Virus Internal Ribosome Entry Site-Dependent Translation in Saccharomyces cerevisiae Is Independent of Polypyrimidine Tract-Binding Protein, Poly(rC)-Binding Protein 2, and La Protein

2005 ◽  
Vol 79 (16) ◽  
pp. 10126-10137 ◽  
Author(s):  
Amy B. Rosenfeld ◽  
Vincent R. Racaniello
Keyword(s):  
Mammalian Cells ◽  
Binding Protein ◽  
Polypyrimidine Tract ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Reading Frame ◽  
Polypyrimidine Tract Binding Protein ◽  
La Protein ◽  
Internal Initiation ◽  
Hcv Ires

ABSTRACT Translation initiation of some viral and cellular mRNAs occurs by ribosome binding to an internal ribosome entry site (IRES). Internal initiation mediated by the hepatitis C virus (HCV) IRES in Saccharomyces cerevisiae was shown by translation of the second open reading frame in a bicistronic mRNA. Introduction of a single base change in the HCV IRES, known to abrogate internal initiation in mammalian cells, abolished translation of the second open reading frame. Internal initiation mediated by the HCV IRES was independent of the nonsense-mediated decay pathway and the cap binding protein eIF4E, indicating that translation is not a result of mRNA degradation or 5′-end-dependent initiation. Human La protein binds the HCV IRES and is required for efficient internal initiation. Disruption of the S. cerevisiae genes that encode La protein orthologs and synthesis of wild-type human La protein in yeast had no effect on HCV IRES-dependent translation. Polypyrimidine tract-binding protein (Ptb) and poly-(rC)-binding protein 2 (Pcbp2), which may be required for HCV IRES-dependent initiation in mammalian cells, are not encoded within the S. cerevisiae genome. HCV IRES-dependent translation in S. cerevisiae was independent of human Pcbp2 protein and stimulated by the presence of human Ptb protein. These findings demonstrate that the genome of S. cerevisiae encodes all proteins necessary for internal initiation of translation mediated by the HCV IRES.

scholarly journals A Peptide Derived from RNA Recognition Motif 2 of Human La Protein Binds to Hepatitis C Virus Internal Ribosome Entry Site, Prevents Ribosomal Assembly, and Inhibits Internal Initiation of Translation

2005 ◽  
Vol 79 (15) ◽  
pp. 9842-9853 ◽  
Author(s):  
Renuka Pudi ◽  
Sudhamani S. Ramamurthy ◽  
Saumitra Das
Keyword(s):  
Internal Ribosome Entry Site ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Recognition Motif ◽  
La Protein ◽  
Initiation Of Translation ◽  
Internal Initiation ◽  
Hcv Ires

ABSTRACT Human La protein is known to interact with hepatitis C virus (HCV) internal ribosome entry site (IRES) and stimulate translation. Previously, we demonstrated that mutations within HCV SL IV lead to reduced binding to La-RNA recognition motif 2 (RRM2) and drastically affect HCV IRES-mediated translation. Also, the binding of La protein to SL IV of HCV IRES was shown to impart conformational alterations within the RNA so as to facilitate the formation of functional initiation complex. Here, we report that a synthetic peptide, LaR2C, derived from the C terminus of La-RRM2 competes with the binding of cellular La protein to the HCV IRES and acts as a dominant negative inhibitor of internal initiation of translation of HCV RNA. The peptide binds to the HCV IRES and inhibits the functional initiation complex formation. An Huh7 cell line constitutively expressing a bicistronic RNA in which both cap-dependent and HCV IRES-mediated translation can be easily assayed has been developed. The addition of purified TAT-LaR2C recombinant polypeptide that allows direct delivery of the peptide into the cells showed reduced expression of HCV IRES activity in this cell line. The study reveals valuable insights into the role of La protein in ribosome assembly at the HCV IRES and also provides the basis for targeting ribosome-HCV IRES interaction to design potent antiviral therapy.

scholarly journals Internal initiation of translation from the human rhinovirus-2 internal ribosome entry site requires the binding of Unr to two distinct sites on the 5′ untranslated region

2007 ◽  
Vol 88 (11) ◽  
pp. 3043-3052 ◽  
Author(s):  
Emma C. Anderson ◽  
Sarah L. Hunt ◽  
Richard J. Jackson
Keyword(s):  
Internal Ribosome Entry Site ◽  
Binding Sites ◽  
Cold Shock ◽  
Human Rhinovirus ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding Protein ◽  
Initiation Of Translation ◽  
Cold Shock Domain ◽  
Internal Initiation

Internal initiation of translation from the human rhinovirus-2 (HRV-2) internal ribosome entry site (IRES) is dependent upon host cell trans-acting factors. The multiple cold shock domain protein Unr and the polypyrimidine tract-binding protein have been identified as synergistic activators of HRV-2 IRES-driven translation. In order to investigate the mechanism by which Unr acts in this process, we have mapped the binding sites of Unr to two distinct secondary structure domains of the HRV-2 IRES, and have identified specific nucleotides that are involved in the binding of Unr to the IRES. The data suggest that Unr acts as an RNA chaperone to maintain a complex tertiary IRES structure required for translational competency.

scholarly journals The hnRNA-Binding Proteins hnRNP L and PTB Are Required for Efficient Translation of the Cat-1 Arginine/Lysine Transporter mRNA during Amino Acid Starvation

2009 ◽  
Vol 29 (10) ◽  
pp. 2899-2912 ◽  
Author(s):  
Mithu Majumder ◽  
Ibrahim Yaman ◽  
Francesca Gaccioli ◽  
Vladimir V. Zeenko ◽  
Chuanping Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Binding Protein ◽  
Mrna Translation ◽  
Amino Acid Starvation ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding Protein ◽  
Global Protein Synthesis

ABSTRACT The response to amino acid starvation involves the global decrease of protein synthesis and an increase in the translation of some mRNAs that contain an internal ribosome entry site (IRES). It was previously shown that translation of the mRNA for the arginine/lysine amino acid transporter Cat-1 increases during amino acid starvation via a mechanism that utilizes an IRES in the 5′ untranslated region of the Cat-1 mRNA. It is shown here that polypyrimidine tract binding protein (PTB) and an hnRNA binding protein, heterogeneous nuclear ribonucleoprotein L (hnRNP L), promote the efficient translation of Cat-1 mRNA during amino acid starvation. Association of both proteins with Cat-1 mRNA increased during starvation with kinetics that paralleled that of IRES activation, although the levels and subcellular distribution of the proteins were unchanged. The sequence CUUUCU within the Cat-1 IRES was important for PTB binding and for the induction of translation during amino acid starvation. Binding of hnRNP L to the IRES or the Cat-1 mRNA in vivo was independent of PTB binding but was not sufficient to increase IRES activity or Cat-1 mRNA translation during amino acid starvation. In contrast, binding of PTB to the Cat-1 mRNA in vivo required hnRNP L. A wider role of hnRNP L in mRNA translation was suggested by the decrease of global protein synthesis in cells with reduced hnRNP L levels. It is proposed that PTB and hnRNP L are positive regulators of Cat-1 mRNA translation via the IRES under stress conditions that cause a global decrease of protein synthesis.

scholarly journals Transient Expression of Cellular Polypyrimidine-Tract Binding Protein Stimulates Cap-Independent Translation Directed by Both Picornaviral and Flaviviral Internal Ribosome Entry Sites In Vivo

2000 ◽  
Vol 20 (5) ◽  
pp. 1583-1595 ◽  
Author(s):  
Rainer Gosert ◽  
Ki Ha Chang ◽  
Rene Rijnbrand ◽  
MinKyung Yi ◽  
David V. Sangar ◽  
...  
Keyword(s):  
Binding Protein ◽  
Fold Increase ◽  
Null Mutant ◽  
Polypyrimidine Tract ◽  
Reporter Protein ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Independent Translation

ABSTRACT The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37- to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule.

scholarly journals Interaction of Eukaryotic Initiation Factor eIF4B with the Internal Ribosome Entry Site of Foot-and-Mouth Disease Virus Is Independent of the Polypyrimidine Tract-Binding Protein

1999 ◽  
Vol 73 (7) ◽  
pp. 6111-6113 ◽  
Author(s):  
René C. Rust ◽  
Kerstin Ochs ◽  
Karsten Meyer ◽  
Ewald Beck ◽  
Michael Niepmann
Keyword(s):  
Disease Virus ◽  
Translation Initiation ◽  
Internal Ribosome Entry Site ◽  
Foot And Mouth Disease ◽  
Initiation Factor ◽  
Polypyrimidine Tract ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding Protein ◽  
Mouth Disease Virus

ABSTRACT Eukaryotic translation initiation factor 4B (eIF4B) binds directly to the internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV). Mutations in all three subdomains of the IRES stem-loop 4 reduce binding of eIF4B and translation efficiency in parallel, indicating that eIF4B is functionally involved in FMDV translation initiation. In reticulocyte lysate devoid of polypyrimidine tract-binding protein (PTB), eIF4B still bound well to the wild-type IRES, even after removal of the major PTB-binding site. In conclusion, the interaction of eIF4B with the FMDV IRES is essential for IRES function but independent of PTB.

scholarly journals Protein Factor Requirements of the Apaf-1 Internal Ribosome Entry Segment: Roles of Polypyrimidine Tract Binding Protein and upstream of N-ras

2001 ◽  
Vol 21 (10) ◽  
pp. 3364-3374 ◽  
Author(s):  
Sally A. Mitchell ◽  
Emma C. Brown ◽  
Mark J. Coldwell ◽  
Richard J. Jackson ◽  
Anne E. Willis
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Cell Types ◽  
Polypyrimidine Tract ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Different Cell Types

ABSTRACT It has been reported previously that the 5′ untranslated region of the mRNA encoding Apaf-1 (apoptotic protease-activating factor 1) has an internal ribosome entry site (IRES), whose activity varies widely among different cell types. Here it is shown that the Apaf-1 IRES is active in rabbit reticulocyte lysates, provided that the system is supplemented with polypyrimidine tract binding protein (PTB) and upstream of N-ras (unr), two cellular RNA binding proteins previously identified to be required for rhinovirus IRES activity. In UV cross-linking assays and electrophoretic mobility shift assays with individual recombinant proteins, the Apaf-1 IRES binds unr but not PTB; however, PTB binding occurs if unr is present. Over a range of different cell types there is a broad correlation between the activity of the Apaf-1 IRES and their content of PTB and unr. In cell lines deficient in these proteins, overexpression of PTB and unr stimulated Apaf-1 IRES function. This is the first example where an IRES in a cellular mRNA has been shown to be functionally dependent, both in vitro and in vivo, on specific cellular RNA binding proteins. Given the critical role of Apaf-1 in apoptosis, these results have important implications for the control of the apoptotic cascade.

scholarly journals Tissue-Specific Replicating Capacity of a Chimeric Poliovirus That Carries the Internal Ribosome Entry Site of Hepatitis C Virus in a New Mouse Model Transgenic for the Human Poliovirus Receptor

2003 ◽  
Vol 77 (19) ◽  
pp. 10479-10487 ◽  
Author(s):  
Akiko Yanagiya ◽  
Seii Ohka ◽  
Noriyasu Hashida ◽  
Masahito Okamura ◽  
Choji Taya ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mouse Model ◽  
Internal Ribosome Entry Site ◽  
Mammalian Cells ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Tissue Specific ◽  
Hcv Ires ◽  
The Brain

ABSTRACT Nucleotides (nt) 108 to 742 of an infectious cDNA clone of poliovirus (PV) Mahoney strain, including the corresponding region of the internal ribosome entry site (IRES), was replaced by nt 28 to 710 of hepatitis C virus (HCV) cDNA corresponding to the whole HCV IRES. A chimeric PV (2A-369) was generated by transfecting mammalian cells with an RNA transcribed in vitro from the cDNA. To examine replicating capacity of virus 2A-369 in the brain and liver of a mouse model for poliomyelitis, a new mouse model (MPVRTg25-61) that is transgenic for human PV receptor (hPVR; CD155) was generated in order to obtain a higher expression level of hPVR in the liver than those of hPVRTg mouse lines generated by us so far. The transgene used was constructed by combining a putative regulatory region of the mouse PVR homolog and the whole structural region of the hPVR gene. Virus 2A-369 replicated well in the liver of MPVRTg25-61 but not in the brain, whereas control Mahoney virus replicated well both in the liver and in the brain. The data suggest that the HCV IRES works more efficiently in the liver than in the brain and that PV IRES works well both in the liver and in the brain. The results support the notion that tissue-specific activity of IRES may be reflected in tissue tropism of a virus whose specific translation initiation is driven by IRES, that is, an IRES-dependent virus tropism.

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