Regulation of Hepatitis C Virus translation by the viral internal ribosome entry site and the 3´-untranslated region
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In this study, the regulation of translation of Hepatitis C Virus (HCV) by the internal ribosome entry site (IRES) and the 3´-untranslated region (3´-UTR) was investigated. The 3´-UTR stimulates HCV IRES-directed translation, and some known cellular RNA-binding proteins as well as a newly discovered 210 kDa protein specifically binding to the 3´-UTR may be involved in HCV translation regulation. HCV, the main causative agent of non-A, non-B hepatitis (NANBH), belongs to the unique genus Hepacivirus in the family Flaviviridae. HCV has infected more than 170 million people worldwide, about 80 % of whom are unable to eliminate the virus, and those are at high risk to develop chronic liver diseases including cirrhosis and hepatocellular carcinoma. The recent development of replicon systems has largely accelerated HCV research, but there is still no tissue culture system supporting a complete replication cycle of HCV, a circumstance that has slowed down studies on the basic understanding of the viral life cycle as well as drug and vaccine development. The interactions of some known cellular RNA-binding proteins, including polypyrimidine tract-binding protein (PTB), heterogeneous nuclear ribonucleoprotein L (hnRNP L) and the protein encoded upstream of N-ras (Unr), with the HCV IRES and the 3´-UTR were examined. There is no direct interaction of PTB with the HCV IRES, but PTB binds specifically to the 3´-UTR. In contrast, hnRNP L binds to the IRES only. In addition, the binding of PTB to the 3´-UTR can be strengthened by hnRNP L, indicating that there is a synergistic interaction between PTB and hnRNP L. However, only a very slight positive effect of hnRNP L on HCV translation was observed in vitro. The recombinant Unr protein used in this work binds to the HCV 3´-UTR, but no significant effect of Unr on HCV translation could be observed in vitro. Considering previous conflicting reports on a possible function of the HCV 3´-UTR in translation stimulation, it was found that reporter construct design is an important parameter in experiments testing 3´-UTR function. A translation enhancer function of the HCV 3´-UTR was detected only after transfection of monocistronic reporter RNAs and depends on a precise 3´-terminus of the HCV 3´-UTR. The 3´-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in non-liver cell lines. Within the 3´-UTR the variable region, the poly(U/C)-tract and the most 3´-terminal stem-loop 1 of the highly conserved 3´-X region contribute significantly to translation enhancement, whereas the stem-loops 2 and 3 of the 3´-X region are involved only to minor extents. Thus, the signals for translation enhancement and the initiation of RNA minus-strand synthesis in the HCV 3´-UTR partially overlap, supporting the idea that these sequences along with viral and possibly also cellular factors may be involved in an RNA 3´-5´-end interaction and in a switch between translation and RNA replication. In an initial attempt to search for trans-acting factors possibly involved in the translation initiation of HCV, no new protein was detected to bind to the HCV IRES. From the finding that the 3´-UTR stimulates translation, it was assumed that the translation initiation could be positively regulated by proteins binding to the 3´-UTR. This gave rise to a reconsideration of the experimental design of the HCV RNA constructs used for the search for new proteins, finally resulting in the discovery of a novel, yet unknown protein that binds to the HCV RNA. This unknown 210 kDa protein binds to the variable region of the HCV 3´-UTR only when a reporter RNA with exact 3´-terminus of 3´-UTR was used. This suggests that the protein may be involved in the regulation of translation stimulation by interacting, perhaps together with other yet undiscovered proteins, with the 3´-end of the HCV 3´-UTR, and the protein(s) may be involved in a switch from translation to negative-strand RNA synthesis in the life cycle of HCV.Verknüpfung zu Publikationen oder weiteren Datensätzen
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RNA, 11 (2005), S. 1809-1824