Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus

Ago, Hideo; Adachi, Tsutomu; Yoshida, Akinori; Yamamoto, Masaki; Habuka, Noriyuki; Yatsunami, Kimio; Miyano, Masashi

doi:10.1016/s0969-2126(00)80031-3

Cited by 377 publications

(321 citation statements)

References 42 publications

Supporting

Mentioning

313

Contrasting

Order By: Relevance

“…The dependence of polymerase activity upon divalent metal ions has been demonstrated for other viral RNA polymerases (29)(30)(31). Interestingly, Mg 2ϩ , the most common divalent metal ion used by many RNA polymerases, cannot support ZIKV NS5 primer extension activity.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

Bluemling

Collop

et al. 2017

Antimicrob Agents Chemother

View full text Add to dashboard Cite

Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primertemplate pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn 2ϩ is required for enzymatic activity, while Mg 2ϩ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5=-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2=-C-methyl-and 2=-C-ethynyl-substituted analog 5=-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

show abstract

Section: Discussionmentioning

confidence: 99%

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

Bluemling

Collop

et al. 2017

Antimicrob Agents Chemother

View full text Add to dashboard Cite

show abstract

“…Substrates (0.5 nM) were composed of a 5Ј-fluorescein-labeled 15-mer DNA of the sequence (dT) 15 . Binding buffer consisted of 50 mM MOPS-K ϩ , pH 7.0, and 50 M EDTA.…”

Section: Methodsmentioning

confidence: 99%

“…The focal point of such investigations has been NS5B, which possesses an RNA-dependent RNA polymerase (RdRp) activity and is believed to be the key enzyme catalyzing HCV RNA synthesis (7)(8)(9)(10)(11)(12)(13)(14). Its crystal structure reveals that it contains the classical finger, palm, and thumb subdomains of the polymerases with the unique feature of a more fully enclosed active site tunnel (15)(16)(17), and a recent report by Bressanelli and colleagues (18) has provided additional information about the complex of NS5B with ribonucleotides. Recombinant NS5B protein from different sources has been shown to replicate a range of natural and synthetic RNA templates, both in a primer-dependent and primer-independent fashion (19 -25).…”

mentioning

confidence: 99%

Modulation of the Hepatitis C Virus RNA-dependent RNA Polymerase Activity by the Non-Structural (NS) 3 Helicase and the NS4B Membrane Protein

Piccininni¹,

Varaklioti²,

Nardelli³

et al. 2002

Journal of Biological Chemistry

135

124

View full text Add to dashboard Cite

The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is believed to be the central catalytic enzyme responsible for HCV replication but there are many unanswered questions about how its activity is controlled. In this study we reveal that two other HCV proteins, NS3 (a protease/helicase) and NS4B (a hydrophobic protein of unknown function), physically and functionally interact with the NS5B polymerase. We describe a new procedure for generating highly pure NS4B, and use this protein in biochemical studies together with NS5B and NS3. To study the functional effects of the protein-protein interactions, we have developed an in vitro replication assay using the natural noncoding 3 regions of the respective positive ((؉)-3 -untranslated region) and negative ((؊)-3 -terminal region) RNA strands of the HCV genome. Our studies show that NS3 dramatically modulates template recognition by NS5B and changes the synthetic products generated by this enzyme. The use of an NTPase-deficient mutant form of NS3 demonstrates that the NTPase activity (and thus helicase activity) of this protein is specifically required for these effects. Moreover, NS4B is found to be a negative regulator of the NS3-NS5B replication complex. Overall, these results reveal that NS3, NS4B, and NS5B can interact to form a regulatory complex that could feature in the process of HCV replication.Hepatitis C virus (HCV) 1 is a major pathogen of parenterally transmitted non-A, non-B hepatitis (1) and often causes the development of malignant chronic disease, including liver cirrhosis and hepatocellular carcinoma (2). With nearly 3% of the population of the world infected with HCV and no protective vaccine available at present, this disease has emerged as a serious global health problem since the virus was first identified (3, 4). HCV is a positive-stranded RNA virus with a genome of ϳ9400 bp. This genomic RNA initially directs the synthesis of at least 10 structural and nonstructural viral proteins (5). Following that, it is utilized by the viral RNA-dependent RNA polymerase (the nonstructural protein 5B; NS5B) as template to generate a complementary negative-stranded RNA. Once synthesized, the negative strands are transcribed into new molecules of positive-stranded genomic RNA, which in turn provide additional templates for viral protein synthesis as well as genomic RNA for the production of progeny virus (5, 6). However, the molecular events that mediate this process remain largely unclear.Several attempts to dissect the mechanistic details of the viral replication cycle have been reported to date. The focal point of such investigations has been NS5B, which possesses an RNA-dependent RNA polymerase (RdRp) activity and is believed to be the key enzyme catalyzing HCV RNA synthesis (7-14). Its crystal structure reveals that it contains the classical finger, palm, and thumb subdomains of the polymerases with the unique feature of a more fully enclosed active site tunnel (15)(16)(17), and a recent report by Bressanelli and colleagues (18) has provided add...

show abstract

“…Recombinant forms of NS5B have been expressed and purified from bacterial and insect cells (9 -16). It belongs to a large family of nucleic acid-dependent nucleic acid polymerases (NdNp) sharing finger and thumb subdomain structures with conserved motifs (17)(18)(19). NS5B also has several unique properties, including two loops connecting fingers and a thick thumb that may be the major elements responsible for the closed conformation of HCV NS5B, and may also play a role in the "clamping" motion of this enzyme (42).…”

mentioning

confidence: 99%

Mutational Analysis of Hepatitis C Virus NS5B in the Subgenomic Replicon Cell Culture

Ma¹,

Shimakami

Luo³

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

The hepatitis C virus (HCV) NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme of HCV RNA replication. We previously identified five novel residues of NS5B in a JK-1 isolate indispensable for RdRP activity in vitro (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001) Hepatology 33, 728 -737). We addressed the role of these residues in HCV RNA replication using a HCV replicon system derived from an M1LE isolate (Kishine, H., Sugiyama, K., Hijikata, M., Kato, N., Takahashi, H., Noshi, T., Nio, Y., Hosaka, M., Miyanari, Y., and Shimotohno, K. (2002) Biochem. Biophys. Res. Commun. 293, 993-999). The five residues of NS5B in M1LE were found to be critical for HCV replication in vivo and also indispensable for RdRP activity in vitro along with purified bacterial recombinant proteins. We also found a chimeric replicon of JK-1 and M1LE in which only the NS5B sequence derived from JK-1 could not replicate in Huh-7 cells. The residues responsible for the phenomenon were mapped by several chimeric and substituted forms of NS5B M1LE and/or JK-1 isolates in the HCV RNA replicon. Two residues, amino acids 220 and 288, were critical, and two residues, amino acids 213 and 231, were important for efficient HCV replication. Mutant JK-1 NS5B harboring all four residues of M1LE was replication-competent in the chimeric replicon and was as efficient as the original M1LE replicon. By comparing the replication competence in vivo and RdRP activity in vitro with various chimeric and mutated versions of NS5B, the HCV replication ability was found to correlate well with the RdRP activity. However, heat-and dilution-sensitive NS5Bs exhibiting weaker RdRP activity in vitro were found to be replication-incompetent, suggesting that HCV replication requires RdRP activity higher than a certain critical threshold.Hepatitis C virus (HCV), 1 a member of the Flaviviridae family, is the major causative agent of non-A and non-B hepatitis (1). Persistent infection with HCV is an important cause of chronic liver disease around the world. Infections with this hepatotropic flavivirus are associated with inflammatory liver injury, progressive fibrosis, and, in the most severely affected patients, cirrhosis and hepatocellular carcinoma (2, 3). The HCV genome is a single-stranded RNA molecule 9.6 kb in length with positive-sense polarity (1, 5). The genome consists of a 5Ј-untranslated region, an open reading frame, and a 3Ј-untranslated region (1, 4, 5). The order of the gene products of the single open reading frame is NH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. This polyprotein precursor can be processed by the host and virally encoded proteases to generate mature structural and nonstructural proteins required for virus replication and assembly (6 -8).Studies on HCV replication focusing on the inhibition of HCV replication may have therapeutic significance for chronic hepatitis and also could reduce the incidence of or even prevent hepatocellular carcinoma. NS5B is an RNA-dependent RNA polymerase ...

show abstract

Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus

Cited by 377 publications

References 42 publications

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

Modulation of the Hepatitis C Virus RNA-dependent RNA Polymerase Activity by the Non-Structural (NS) 3 Helicase and the NS4B Membrane Protein

Mutational Analysis of Hepatitis C Virus NS5B in the Subgenomic Replicon Cell Culture

Contact Info

Product

Resources

About