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.
Highlights
§ American Liver Foundation Scholar; supported by the Cancer League of Colorado and the Milheim Foundation for Cancer Research
It has been suggested that hepatitis C virus (HCV) RNA translation is regulated by at least three distinct elements: 1) the global structure of the viral internal ribosome entry site (IRES), which includes almost the entire sequence within the 5Ј-noncoding region (5Ј-NCR) and a few nucleotides downstream of the AUG initiator codon [5,6,7,8,9,10,11,12,13]; 2) the 3Ј-terminal region, which has been shown to enhance IRES activity [14]; and 3) trans-acting cellular factors that interact with the viral IRES element and assist in internal initiation of translation [15,16,17,18]
In an attempt to understand the biological significance of such unusual binding, we demonstrated dramatic reduction in the HCV IRES activity in HeLa and rabbit reticulocyte translation lysates that were immunodepleted of PTB [15]
Summary
§ American Liver Foundation Scholar; supported by the Cancer League of Colorado and the Milheim Foundation for Cancer Research. Analysis of the conserved and divergent regions of the HCV genomes far cloned from various geographical locations indicates that the 5Ј-noncoding region (5Ј-NCR) that precedes the open reading frame and the 3Ј terminus of the genome are highly conserved among all subtypes [1, 4]. These elements function as promoters of HCV gene expression. Notable among the non-canonical translation initiation factors that regulate the viral IRES elements are the polypyrimidine tract-binding protein (PTB) and La antigen (for review, see Ref. 19). The PTB requirement for HCV IRES function was established by schemes of RNA cotransfections and add-back experiments
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
