Domains I and II in the 5′ Nontranslated Region of the HCV Genome Are Required for RNA Replication

Kim, Yoon Ki; Kim, Chon Saeng; Lee, Song Hee; Jang, Sung Key

doi:10.1006/bbrc.2001.6167

Cited by 59 publications

(49 citation statements)

References 36 publications

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“…While such interactions could be important for viral packaging and assembly, the presence of RNA replication signals within domain III would be consistent with observations made in cell culture systems using subgenomic HCV RNAs. 24,35,36 Our observations thus significantly extend these previous studies of 5ЈNTR RNA replication signals using HCV replicons, 24,35,36 as they reflect requirements for specific 5ЈNTR sequences for completion of the entire viral life cycle in vivo, within the livers of small primates. Altogether, they provide strong support for the presence of virus-specific replication signals within domains I-II of the 5ЈNTR.…”

Section: Discussionsupporting

confidence: 77%

A chimeric GB virus B with 5′ nontranslated RNA sequence from hepatitis C virus causes hepatitis in tamarins

et al. 2005

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H epatitis C virus (HCV) infection contributes significantly to human morbidity and mortality worldwide, and new and better therapeutics are urgently needed. 1 However, the evaluation of candidate antiviral compounds is hindered by the absence of readily available animal models. Chimpanzees (Pan troglodytes) are susceptible to HCV infection, but are endangered as a species and increasingly difficult to access for experimental studies. More recently developed murine models with chimeric human livers are technically challenging and, moreover, do not recapitulate pathogenesis because of underlying immunodeficiency. 2,3 GB virus B (GBV-B), a hepatotropic member of the Flaviviridae family with close phylogenetic relatedness to HCV (genus Hepacivirus), 4,5 thus provides a potentially useful surrogate for animal studies of hepatitis C. It is capable of infecting and causing hepatitis in several different nonendangered species of New World primates 6-9 and, like HCV, can establish a persistent infection associated with chronic liver disease. 10 The GBV-B and HCV genomes share many similarities and

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Section: Discussionsupporting

confidence: 77%

A chimeric GB virus B with 5′ nontranslated RNA sequence from hepatitis C virus causes hepatitis in tamarins

et al. 2005

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“…10). These results suggest an interesting symmetry in the 5Ј and 3Ј terminal RNA replication signals, as studies by others indicate that the 5Ј-most domains I and II of the 5ЈNTR are essential for replication, while sequences lying further downstream within the 5ЈNTR and comprising domain III help to facilitate replication but are not absolutely required (12,23) (Fig. 10).…”

Section: Vol 77 2003 3јntr Signals For Hepatitis C Virus Rna Replicmentioning

confidence: 71%

“…This conservation is likely due to the need to maintain RNA structures that are required either for the initiation of translation of the viral polyprotein or for recognition of the positive-and negative-strand forms of the viral RNA by the replicase during the process of RNA replication. In support of the latter possibility, several recent studies suggest that both the 3Ј-and 5Ј-terminal RNA segments contain specific RNA sequences that are required for viral RNA replication, either in chimpanzees inoculated with synthetic genome-length RNA (25,48) or in cell cultures supporting the replication of subgenomic viral RNAs (11,12,23).…”

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confidence: 98%

“…Domains II to IV comprise an internal ribosome entry site (IRES) that is responsible for cap-independent initiation of the translation of the viral polyprotein (36,47), while domains I, II, and III contain overlapping replication signals that are required for optimal replication of the viral RNA in cultured cells (12,23). The essential 5Ј RNA replication signals in HCV thus appear to extend over a considerably longer segment of the 5Ј-terminal sequences than has been shown to be the case in pestiviruses and flaviviruses, other members of the family Flaviviridae (13,21,52).…”

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confidence: 99%

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3′ Nontranslated RNA Signals Required for Replication of Hepatitis C Virus RNA

Lemon

2003

J Virol

186

182

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We describe a mutational analysis of the 3 nontranslated RNA (3NTR) signals required for replication of subgenomic hepatitis C virus (HCV) RNAs. A series of deletion mutants was constructed within the background of an HCV-N replicon that induces the expression of secreted alkaline phosphatase in order to examine the requirements for each of the three domains comprising the 3NTR, namely, the highly conserved 3 terminal 98-nucleotide (nt) segment (3X), an upstream poly(U)-poly(UC) [poly(U/UC)] tract, and the variable region (VR) located at the 5 end of the 3NTR. Each of these domains was found to contribute to efficient replication of the viral RNA in transiently transfected hepatoma cells. Replication was not detected when any of the three putative stem-loop structures within the 3X region were deleted. Similarly, complete deletion of the poly(U/UC) tract abolished replication. Replacement of a minimum of 50 to 62 nt of poly(U/UC) sequence was required for detectable RNA replication when the native sequence was restored in a stepwise fashion from its 3 end. Lengthier poly(U/UC) sequences, and possibly pure homopolymeric poly(U) tracts, were associated with more efficient RNA amplification. Finally, while multiple deletion mutations were tolerated within VR, each led to a partial loss of replication capacity. The impaired replication capacity of the deletion mutants could not be explained by reduced translational activity or by decreased stability of the RNA, suggesting that each of these mutations may impair recognition of the RNA by the viral replicase during an early step in negative-strand RNA synthesis. The results indicate that the 3-most 150 nt of the HCV-N genome [the 3X region and the 3 52 nt of the poly(U/UC) tract] contain RNA signals that are essential for replication, while the remainder of the 3NTR plays a facilitating role in replication but is not absolutely required.Hepatitis C virus (HCV) is prevalent throughout the world. Most acute infections with this hepatotropic human virus lead to persistent infection and various forms of associated liver injury, including chronic hepatitis, cirrhosis, and even hepatocellular carcinoma (1, 38). Although recombinant alpha interferon, either alone or in combination with ribavirin, is widely used to treat HCV infection, the efficacy of these drugs in eliminating the infection is less than 50% overall (27, 31). The development of more effective drugs for the treatment of chronic hepatitis C is urgently awaited but has been hampered by the lack of cell culture systems that are fully permissive for viral replication and by the absence of small-animal models of the disease. Recently, however, the development of subgenomic HCV replicons that are capable of replication in human hepatoma (Huh7) cells has facilitated drug discovery efforts while providing new approaches to characterizing the molecular processes involved in replication of the viral RNA (28).HCV is the sole member of the Hepacivirus genus within the family Flaviviridae. The genome of this virus is a sin...

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“…In the case of HCV, the minimal signals that are essential for RNA replication have been mapped within the NTRs. The 5Ј NTR is composed of four distinct domains, with domains 1 and 2 being sufficient for RNA replication (10,19). The 3Ј NTR has a tripartite structure and is composed of a highly variable region immediately downstream of the stop codon of the polyprotein, a polypyrimidine [poly(U/UC)] tract of variable length, and a highly conserved 98-nt-long RNA element designated the X-tail (22,41,42,52).…”

mentioning

confidence: 99%

Kissing-Loop Interaction in the 3′ End of the Hepatitis C Virus Genome Essential for RNA Replication

Friebe

Boudet

Simorre

et al. 2005

J Virol

328

435

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The hepatitis C virus (HCV) is a positive-strand RNA virus belonging to the Flaviviridae. Its genome carries at either end highly conserved nontranslated regions (NTRs) containing cis-acting RNA elements that are crucial for replication. In this study, we identified a novel RNA element within the NS5B coding sequence that is indispensable for replication. By using secondary structure prediction and nuclear magnetic resonance spectroscopy, we found that this RNA element, designated 5BSL3.2 by analogy to a recent report (S. You, D. D. Stump, A. D. Branch, and C. M. Rice, J. Virol. 78:1352-1366, 2004), consists of an 8-bp lower and a 6-bp upper stem, an 8-nucleotide-long bulge, and a 12-nucleotide-long upper loop. Mutational disruption of 5BSL3.2 structure blocked RNA replication, which could be restored when an intact copy of this RNA element was inserted into the 3 NTR. By using this replicon design, we mapped the elements in 5BSL3.2 that are critical for RNA replication. Most importantly, we discovered a nucleotide sequence complementarity between the upper loop of this RNA element and the loop region of stem-loop 2 in the 3 NTR. Mismatches introduced into the loops inhibited RNA replication, which could be rescued when complementarity was restored. These data provide strong evidence for a pseudoknot structure at the 3 end of the HCV genome that is essential for replication. The hepatitis C virus (HCV) is the only member of theHepacivirinae within the Flaviviridae family (46). These viruses possess an RNA genome of positive polarity that in the case of HCV has a length of about 9,600 nucleotides (nt). Its genome carries a single long open reading frame (ORF) that is flanked at either end by highly conserved nontranslated regions (NTRs) (2, 37). The 5Ј NTR harbors an internal ribosome entry site (IRES) directing the synthesis of the viral polyprotein which is cleaved into 10 different products. RNA replication takes place in the cytoplasm in a distinct compartment (12) and requires viral proteins NS3 to NS5B as well as host cell factors (28,43). In addition, cisacting replication elements (CREs) are important for the synthesis of RNA progeny. Such CREs may reside at various positions in an RNA genome but are most often found in the NTRs. In the case of HCV, the minimal signals that are essential for RNA replication have been mapped within the NTRs. The 5Ј NTR is composed of four distinct domains, with domains 1 and 2 being sufficient for RNA replication (10, 19). The 3Ј NTR has a tripartite structure and is composed of a highly variable region immediately downstream of the stop codon of the polyprotein, a polypyrimidine [poly(U/UC)] tract of variable length, and a highly conserved 98-nt-long RNA element designated the X-tail (22,41,42,52). The latter has the potential to form three stemloop (SL) structures, of which the most 3Ј terminal, designated SL1, has been confirmed experimentally (5,15). In contrast, the structures of the two other SLs that are located upstream are much less clear (5,15,56). Both the X-tai...

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Domains I and II in the 5′ Nontranslated Region of the HCV Genome Are Required for RNA Replication

Cited by 59 publications

References 36 publications

A chimeric GB virus B with 5′ nontranslated RNA sequence from hepatitis C virus causes hepatitis in tamarins

A chimeric GB virus B with 5′ nontranslated RNA sequence from hepatitis C virus causes hepatitis in tamarins

3′ Nontranslated RNA Signals Required for Replication of Hepatitis C Virus RNA

Kissing-Loop Interaction in the 3′ End of the Hepatitis C Virus Genome Essential for RNA Replication

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