Structure–function analysis of the 3′ stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication

Yi, Min Kyung; Lemon, Stanley M.

doi:10.1261/rna.2144203

Cited by 90 publications

(68 citation statements)

References 38 publications

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“…We examined whether mutations in the NTPibinding site affected specific recognition of the T1 by comparing the amount of product from LE19 to that of a version of LE19 in which the 3Ј-most C was changed to a U ( Table 1). The HCV RdRp has been reported to initiate RNA synthesis (23), and the HCV-N replicon may even switch to using an initiation uridylate preferentially (27). In the absence of Mn 2ϩ , the only mutant that retained RNA synthesis was S367A.…”

Section: Resultsmentioning

confidence: 99%

De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

Ranjith-Kumar

Sarisky

Gutshall

et al. 2004

J Virol

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The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has several distinct biochemical activities, including initiation of RNA synthesis by a de novo mechanism, extension from a primed template, nontemplated nucleotide addition, and synthesis of a recombinant RNA product from two or more noncovalently linked templates (template switch). All of these activities require specific interaction with nucleoside triphosphates (NTPs). Based on the structure of the HCV RdRp bound to NTP (S. Bressanelli, L. Tomei, F. A. Rey, and R. DeFrancesco, J. Virol. 76:3482-3492, 2002), we mutated the amino acid residues that contact the putative initiation GTP and examined the effects on the various activities. Although all mutations retained the ability for primer extension, alanine substitution at R48, R158, R386, R394, or D225 decreased de novo initiation, and two or more mutations abolished de novo initiation. While the prototype enzyme had a K m for GTP of 3.5 M, all of the mutations except one had K m s that were three-to sevenfold higher. These results demonstrate that the affected residues are functionally required to interact with the initiation nucleotide. Unexpectedly, many of the mutations also affected the addition of nontemplated nucleotide, indicating that residues in the initiating NTP (NTPi)-binding pocket are required for nontemplated nucleotide additions. Interestingly, mutations in D225 are dramatically affected in template switch, indicating that this residue of the NTPi pocket also interacts with components in the elongation complex. We also examined the interaction of ribavirin triphosphate with the NTPi-binding site.Hepatitis C Virus (HCV), a member of the flaviviridae family of positive-strand RNA viruses, infects up to 3% of the world's population (1). A prime target for antiviral treatment in HCV is the NS5B protein, the RNA-dependent RNA polymerase (RdRp) responsible for viral RNA synthesis. The RdRp is one of the subunits expected to participate in HCV replication and is responsible for the initiation and elongation of viral RNA synthesis. Initiation of RNA synthesis in infected cells likely starts de novo; that is, by use of a 1-nucleotide (nt) primer (3,14). This mode of RNA synthesis requires a specialized site in the RdRp, called the I site, that specifically recognizes the initiating nucleoside triphosphate (designated NTPi, with GTP being preferred by the recombinant HCV RdRp) (14,23,25). A second NTP-binding pocket, the I ϩ 1 site, binds a nucleotide for phosphoryl transfer as dictated by the template base (14). Cocrystal structures of the HCV RdRp and nucleotides have been reported previously (3, 21), identifying a number of residues at D225, R48, R158, R386, R394, and S367 that interact with specific moieties of the initiation GTP (Fig. 1A). In this structure, it is not known whether the GTP binds to the I site or the I ϩ 1 site. NTPi binding to the I site is base specific, while binding of the second NTP to the I ϩ 1 site should be directed by the template (25). Because the RdRp-GTP st...

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Section: Resultsmentioning

confidence: 99%

De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

Ranjith-Kumar

Sarisky

Gutshall

et al. 2004

J Virol

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“…To overcome this limitation, we attempted to rescue replication of an RNA with scrambled 5BSLs by insertion of an intact copy of these elements or only 5BSL3.2 at different positions of the RNA. For this purpose, we chose three different positions: the region upstream of the EMCV IRES, a region in the coding sequence on NS5A that was found to be dispensable for RNA replication (4), and the variable region in the 3Ј NTR that is nonessential for RNA replication (9,10,(54)(55)(56). As shown in Fig.…”

Section: Functional Rescue Of Hcv Replicons With Disturbed Sls In Ns5bmentioning

confidence: 99%

“…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-tail and a minimal poly(U/UC) tract of about 40 nt are essential for RNA replication in cell culture and in vivo (9,23,(54)(55)(56).…”

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confidence: 99%

“…Both the X-tail and a minimal poly(U/UC) tract of about 40 nt are essential for RNA replication in cell culture and in vivo (9,23,(54)(55)(56). Removal of part of or the complete variable region from HCV genomes or replicons is tolerated but significantly reduces RNA replication and in vivo infectivity (9,23,(54)(55)(56).For several positive-strand RNA viruses, in addition to RNA signals located in the NTRs, CREs residing within the coding sequence have been described. One of the best-studied examples with respect to RNA replication is the CRE of picornaviruses, including the human rhinovirus-14 (HRV-14), the poliovirus (PV), the Mengo virus, Theiler's virus, and the foot-and-mouth disease virus (FMDV) (11,25,30,31,34).…”

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confidence: 99%

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confidence: 99%

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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

329

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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In VitroReplication Models

Bartenschlager¹,

Sparacio²

2005

Viral Hepatitis

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Structure–function analysis of the 3′ stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication

Cited by 90 publications

References 38 publications

De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

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

In VitroReplication Models

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