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Demonstration of functional requirement of polypyrimidine tract-binding protein by SELEX RNA during hepatitis C virus internal ribosome entry site-mediated translation initiation.
J Biol Chem. 2000 Nov 03; 275(44):34231-5.JB

Abstract

Polypyrimidine tract-binding protein (PTB) has been previously shown to physically interact with the hepatitis C virus (HCV) RNA genome at its 5'- and 3'-noncoding regions. Using high affinity SELEX RNA molecules, we present evidence for the functional requirement of PTB during HCV internal ribosome entry site (IRES)-controlled translation initiation. This study was carried out in rabbit reticulocyte translation lysates in which the HCV IRES-driven reporter RNA was introduced along with the PTB-specific SELEX RNA molecules. The SELEX RNAs specifically inhibited the HCV IRES function in the context of mono- and dicistronic mRNAs. The cap-dependent translation of a reporter (chloramphenicol acetyltransferase) RNA or naturally capped brome mosaic virus RNA, however, was not affected by the presence of SELEX during in vitro translation assays. The SELEX-mediated inhibition of the HCV IRES is shown to be relieved by the addition of recombinant human PTB in an add-back experiment. The in vivo requirement of PTB was further confirmed by cotransfection of Huh7 cells with reporter RNA and PTB-specific SELEX RNA. The HCV IRES activity was inhibited by the SELEX RNA in these cells, but not by an unrelated control RNA. Together, these results demonstrate the functional requirement of cellular PTB in HCV translation and further support the feasible use of SELEX RNA strategy in demonstrating the functional relevance of cellular protein(s) in complex biological processes.

Authors+Show Affiliations

Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10938288

Citation

Anwar, A, et al. "Demonstration of Functional Requirement of Polypyrimidine Tract-binding Protein By SELEX RNA During Hepatitis C Virus Internal Ribosome Entry Site-mediated Translation Initiation." The Journal of Biological Chemistry, vol. 275, no. 44, 2000, pp. 34231-5.
Anwar A, Ali N, Tanveer R, et al. Demonstration of functional requirement of polypyrimidine tract-binding protein by SELEX RNA during hepatitis C virus internal ribosome entry site-mediated translation initiation. J Biol Chem. 2000;275(44):34231-5.
Anwar, A., Ali, N., Tanveer, R., & Siddiqui, A. (2000). Demonstration of functional requirement of polypyrimidine tract-binding protein by SELEX RNA during hepatitis C virus internal ribosome entry site-mediated translation initiation. The Journal of Biological Chemistry, 275(44), 34231-5.
Anwar A, et al. Demonstration of Functional Requirement of Polypyrimidine Tract-binding Protein By SELEX RNA During Hepatitis C Virus Internal Ribosome Entry Site-mediated Translation Initiation. J Biol Chem. 2000 Nov 3;275(44):34231-5. PubMed PMID: 10938288.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Demonstration of functional requirement of polypyrimidine tract-binding protein by SELEX RNA during hepatitis C virus internal ribosome entry site-mediated translation initiation. AU - Anwar,A, AU - Ali,N, AU - Tanveer,R, AU - Siddiqui,A, PY - 2000/8/12/pubmed PY - 2001/2/28/medline PY - 2000/8/12/entrez SP - 34231 EP - 5 JF - The Journal of biological chemistry JO - J Biol Chem VL - 275 IS - 44 N2 - Polypyrimidine tract-binding protein (PTB) has been previously shown to physically interact with the hepatitis C virus (HCV) RNA genome at its 5'- and 3'-noncoding regions. Using high affinity SELEX RNA molecules, we present evidence for the functional requirement of PTB during HCV internal ribosome entry site (IRES)-controlled translation initiation. This study was carried out in rabbit reticulocyte translation lysates in which the HCV IRES-driven reporter RNA was introduced along with the PTB-specific SELEX RNA molecules. The SELEX RNAs specifically inhibited the HCV IRES function in the context of mono- and dicistronic mRNAs. The cap-dependent translation of a reporter (chloramphenicol acetyltransferase) RNA or naturally capped brome mosaic virus RNA, however, was not affected by the presence of SELEX during in vitro translation assays. The SELEX-mediated inhibition of the HCV IRES is shown to be relieved by the addition of recombinant human PTB in an add-back experiment. The in vivo requirement of PTB was further confirmed by cotransfection of Huh7 cells with reporter RNA and PTB-specific SELEX RNA. The HCV IRES activity was inhibited by the SELEX RNA in these cells, but not by an unrelated control RNA. Together, these results demonstrate the functional requirement of cellular PTB in HCV translation and further support the feasible use of SELEX RNA strategy in demonstrating the functional relevance of cellular protein(s) in complex biological processes. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/10938288/Demonstration_of_functional_requirement_of_polypyrimidine_tract_binding_protein_by_SELEX_RNA_during_hepatitis_C_virus_internal_ribosome_entry_site_mediated_translation_initiation_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=10938288 DB - PRIME DP - Unbound Medicine ER -