Involvement of the 5'Proximal Coding Sequences of Hepatitis C Virus with Internal Initiation of Viral Translation* 1 (original) (raw)
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Biochemical and Biophysical Research Communications, 1998
The 5 nontranslated region (NTR) of hepatitis C virus (HCV) consists of 341 nucleotides (nt). This region comprises the majority of the internal ribosome entry site (IRES) which controls the efficiency of viral translation. Previous studies of the 3 boundary of the HCV IRES yielded conflicting data regarding the involvement of viral coding sequences in IRES activity. We therefore studied the functional significance of the 5 proximal coding sequences of the HCV core gene on IRES activity. We constructed monocistronic and bicistronic DNAs that contained either a chloramphenicol acetyl transferase (CAT) gene or a luciferase (Luc) gene as the reporter. Results from both in vitro and in vivo experiments indicated that the optimal IRES ranged within nt 1-371. Further mutational analyses of sequences surrounding the initiation codon revealed that primary sequences downstream of the AUG initiator rather than the secondary structure are important in regulating optimal IRES function. We are also able to demonstrate that a non-AUG codon could be used to initiate the synthesis of a reporter protein, albeit with lower efficiency. These findings bear important implications for the HCV IRES secondary structures.
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...
Journal of Virology, 2006
The positive-strand RNA genome of the hepatitis C virus (HCV) is flanked by 5-and 3-untranslated regions (UTRs). Translation of the viral RNA is directed by the internal ribosome entry site (IRES) in the 5-UTR, and subsequent viral RNA replication requires sequences in the 3-UTR and in the 5-UTR. Addressing previous conflicting reports on a possible function of the 3-UTR for RNA translation in this study, we 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 or complete RNA genomes having a 3-UTR with a precise 3 terminus. The 3-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in nonliver cell lines. 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 stem-loops 2 and 3 of the 3 X region are involved only to a minor extent. Thus, the signals for translation enhancement and for the initiation of RNA minusstrand 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 a switch between translation and RNA replication.
Nucleic acids research, 2009
The HCV internal ribosome entry site (IRES) spans a region of~340 nt that encompasses most of the 5' untranslated region (5'UTR) of the viral mRNA and the first 24-40 nt of the core-coding region. To investigate the implication of altering the primary sequence of the 5'UTR on IRES activity, naturally occurring variants of the 5'UTR were isolated from clinical samples and analyzed. The impact of the identified mutations on translation was evaluated in the context of RLuc/FLuc bicistronic RNAs. Results show that depending on their location within the RNA structure, these naturally occurring mutations cause a range of effects on IRES activity. However, mutations within subdomain IIId hinder HCV IRES-mediated translation. In an attempt to explain these data, the dynamic behavior of the subdomain IIId was analyzed by means of molecular dynamics (MD) simulations. Despite the loss of function, MD simulations predicted that mutant G266A/G268U possesses a structure similar to the wt-RNA. This prediction was validated by analyzing the secondary structure of the isolated IIId RNAs by circular dichroism spectroscopy in the presence or absence of Mg 2+ ions. These data strongly suggest that the primary sequence of subdomain IIId plays a key role in HCV IRES-mediated translation.
Journal of Biological Chemistry, 2001
The hepatitis C virus (HCV) 5-untranslated region and, in particular, domains II to IV are involved in the internal ribosome entry site (IRES) structure. Recent structural evidence has shown that the function of domain II may be to hold the coding RNA in position until the translational machinery is correctly assembled on the decoding site. However, a comprehensive mutational and functional study concerning the importance of the different RNA regions that compose domain II is not yet available. Therefore, we have taken advantage of the recently proposed secondary structure of domain II to design a series of specific mutants. The bulge regions present in the latest secondary structure prediction of domain II were selectively deleted, and the effects of these mutations on IRES translation efficiency were analyzed. Our results show that the introduction of these mutations can variably affect the degree of HCV translation, causing a moderate to total loss of translation ability that correlates with the severity of changes induced in the RNA secondary structure and degree of p25 ribosomal protein UV cross-linking, but not with the ability of the 40S ribosomal subunit to bind the IRES. These findings support the proposed structural role of domain II in HCV translation.
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.
2002
, but the precise role of these sequences is still unknown. In this study, the formation of RNA-RNA complexes in the HCV IRES was evaluated. Using transcripts that contain the sequences of the structural HCV IRES domains II, IIIabcd, IIIabc, IV and IIIef-IV, specific long-range interactions between domains II and IV, as well as domains II and IIIabcd, have been found. These interactions were readily detected in a gel mobility-shift assay and required the presence of magnesium ions. A high concentration of nonspecific competitors, an 80 nt fragment of 18S rRNA or poly(I : C), did not interfere with the formation of RNA complexes.
Journal of virology, 1998
Translation initiation of hepatitis C virus (HCV) RNA occurs by internal entry of a ribosome into the 5' nontranslated region in a cap-independent manner. The HCV RNA sequence from about nucleotide 40 up to the N terminus of the coding sequence of the core protein is required for efficient internal initiation of translation, though the precise border of the HCV internal ribosomal entry site (IRES) has yet to be determined. Several cellular proteins have been proposed to direct HCV IRES-dependent translation by binding to the HCV IRES. Here we report on a novel cellular protein that specifically interacts with the 3' border of the HCV IRES in the core-coding sequence. This protein with an apparent molecular mass of 68 kDa turned out to be heterogeneous nuclear ribonucleoprotein L (hnRNP L). The binding of hnRNP L to the HCV IRES correlates with the translational efficiencies of corresponding mRNAs. This finding suggests that hnRNP L may play an important role in the translati...