Involvement of the 5′ Proximal Coding Sequences of Hepatitis C Virus with Internal Initiation of Viral Translation (original) (raw)
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Unique features of internal initiation of hepatitis C virus RNA translation
The EMBO journal, 1995
The question of whether hepatitis C virus (HCV) RNA is translated by a mechanism of internal ribosome entry has been examined by testing whether insertion of HCV sequences between the two cistrons of a dicistronic mRNA promotes translation of the downstream cistron in rabbit reticulocyte lysates. Deletion analysis showed that efficient internal initiation required a segment of the HCV genome extending from about nucleotides 40-370 and that deletions from the 3'-end of this element were highly deleterious. As the authentic initiation codon for HCV polyprotein synthesis is at nucleotide 342, this demonstrates that, besides 5'-UTR sequences, a short length of HCV coding sequences is required for internal initiation. This finding was confirmed in transfection assays of BT7-H cells and was shown to be independent of the nature of the downstream reporter cistron. The strong requirement for coding sequences is in sharp contrast to internal initiation of picornavirus RNA translation...
Nucleic Acids Research, 1997
We recently compared the efficiency of six picornaviral internal ribosome entry segments (IRESes) and the hepatitis C virus (HCV) IRES for their ability to drive internal initiation of translation in vitro. Here we present the results of a similar comparison performed in six different cultured cell lines infected with a recombinant vaccinia virus expressing the T7 polymerase and transfected with dicistronic plasmids. The IRESes could be divided into three groups: (i) the cardiovirus and aphthovirus IRESes (and the HCV element) direct internal initiation efficiently in all cell lines tested; (ii) the enterovirus and rhinovirus IRESes are at least equally efficient in several cell lines, but are extremely inefficient in certain cell types; and (iii) the hepatitis A virus IRES is incapable of directing efficient internal initiation in any of the cell lines used (including human hepatocytes). These are the same three groups found when IRESes were classified according to their activities in vitro, or according to sequence homologies. In a mouse neuronal cell line, the poliovirus and other type I IRESes were not functional in an artificial bicistronic context. However, infectious poliovirions were produced efficiently after transfection of these cells with a genomic length RNA. Furthermore, activity of the type I IRESes was dramatically increased upon co-expression of the poliovirus 2A proteinase, demonstrating that while IRES efficiency may vary considerably from one cell type to another, at least in some cases viral proteins are capable of overcoming cellspecific translational defects.
Translational Enhancement by the 3'-untranslated Region of Hepatitis C Virus RNA
The Showa University Journal of Medical Sciences, 2003
Hepatitis C virus (HCV) RNA has an internal ribosome entry site (IRES) in the 5'-untranslated region (UTR) and an X region in the 3'-UTR. The IRES and the X-region regulate HCV translation. HCV translation is enhanced by the X region through interaction with the polypyrimidine-tractbinding protein (PTB). Recently, several cellular factors that bind both ends of HCV RNA were identified. However, the function of these binding factors remains unclear. HCV RNA also contains a long poly (u/c) region just upstream of the X region. The function of this poly (U/C) region on HCV translation is also uncertain. In this study, we analyzed the role of the entire 3'-UTR on HCV translation using various translation systems. We constructed T7-based plasmids containing the 5'-UTR, the luciferase (Luc) gene as a reporter, and the entire 3'-UTR containing the variable region, poly (U/ C) stretch, and the X region (HCV 5'L3'). We also prepared plasmids in which the core (HCV 5'CL3') or the core-to-E2 (HCV 5'CE 1 E2L3') region was inserted preceding the LUC gene. In vitro translation of HCV 5'CE1E2L3' using rabbit reticulocyte lysates (RRL) showed a 25-fold enhancement by the entire 3'-UTR. We also demonstrated this translational enhancement by the 3'-UTR in animal cell culture systems using Huh7, HeLa, and HepG2 cells. In conclusion, the entire 3'-UTR can enhance translation of HCV RNA in both rabbit reticulocyte lysates and in animal cell culture systems.
RNA, 2008
The initiation of protein synthesis on mRNAs within eukaryotic cells is achieved either by a 59 cap-dependent mechanism or through internal initiation directed by an internal ribosome entry site (IRES). Picornavirus IRES elements, located in the 59 untranslated region (59UTR), contain extensive secondary structure and multiple upstream AUG codons. These features can be expected to inhibit cap-dependent initiation of translation. However, we have now shown that certain mutant hepatitis C virus-like picornavirus IRES elements (from porcine teschovirus-1 and avian encephalomyelitis virus), which are unable to direct internal initiation, are not significant barriers to efficient translation of capped monocistronic mRNAs that contain these defective elements within their 59UTRs. Moreover, the translation of these mRNAs is highly sensitive to the expression of an enterovirus 2A protease (which induces cleavage of eIF4G) and is also inhibited by hippuristanol, a specific inhibitor of eIF4A function, in contrast to their parental wild-type IRES elements. These results provide a possible basis for the evolution of viral IRES elements within the context of functional mRNAs that are translated by a cap-dependent mechanism.
Keywords: Hepatitis C virus Porcine teschovirus 3 0-untranslated region Poly(A) Picornavirus Internal ribosome entry site a b s t r a c t Translation of hepatitis C virus (HCV) genomic RNA is directed by an internal ribosome entry site (IRES) in the 5 0-untranslated region (5 0-UTR), and the HCV 3 0-UTR enhances IRES activity. Since the HCV 3 0-UTR has a unique structure among 3 0-UTRs, we checked possible communication between the 5 0-and the 3 0-UTR of HCV during translation using chimeric reporter RNAs. We show that translation directed by the HCV IRES and by the HCV-like IRES of porcine teschovirus (PTV) which belongs to a quite distinct family of viruses (picornaviruses) or by the EMCV IRES is also enhanced by the HCV 3 0-UTR or by a poly(A)-tail in different cell types.
FEBS Letters, 2010
Keywords: Hepatitis C virus Porcine teschovirus 3 0 -untranslated region Poly(A) Picornavirus Internal ribosome entry site a b s t r a c t Translation of hepatitis C virus (HCV) genomic RNA is directed by an internal ribosome entry site (IRES) in the 5 0 -untranslated region (5 0 -UTR), and the HCV 3 0 -UTR enhances IRES activity. Since the HCV 3 0 -UTR has a unique structure among 3 0 -UTRs, we checked possible communication between the 5 0 -and the 3 0 -UTR of HCV during translation using chimeric reporter RNAs. We show that translation directed by the HCV IRES and by the HCV-like IRES of porcine teschovirus (PTV) which belongs to a quite distinct family of viruses (picornaviruses) or by the EMCV IRES is also enhanced by the HCV 3 0 -UTR or by a poly(A)-tail in different cell types.
Determinants of Hepatitis C Translational Initiation in Vitro, in Cultured Cells and Mice
Molecular Therapy, 2002
Hepatitis C virus (HCV) is an RNA virus infecting 1 in every 40 people worldwide. Development of new therapeutics for treating HCV has been hampered by the lack of small-animal models. We have adapted existing hydrodynamic transfection methods to optimize the delivery of RNAs to the cytoplasm of mouse liver cells in vivo. Transfected HCV genomic RNA failed to replicate in mouse liver, suggesting a post-entry block to viral replication. Real-time imaging of HCV internal ribosome entry site (IRES) firefly luciferase reporter mRNA translation in living mice demonstrated that the HCV IRES was functional in mouse liver. We then used this system as a model for studying HCV RNA translation in mice.
Journal of virology, 1999
The 5' noncoding regions of the genomes of picornaviruses form a complex structure that directs cap-independent initiation of translation. This structure has been termed the internal ribosome entry site (IRES). The efficiency of translation initiation was shown, in vitro, to be influenced by the binding of cellular factors to the IRES. Hence, we hypothesized that the IRES might control picornavirus tropism. In order to test this possibility, we made a bicistronic construct in which translation of the luciferase gene is controlled by the IRES of Theiler's murine encephalomyelitis virus. In vitro, we observed that the IRES functions in various cell types and in macrophages, irrespective of their activation state. In vivo, we observed that the IRES is functional in different tissues of transgenic mice. Thus, it seems that the IRES is not an essential determinant of Theiler's virus tropism. On the other hand, the age of the mouse could be critical for IRES function. Indeed, ...