A Novel Mechanism to Ensure Terminal Initiation by Hepatitis C Virus NS5B Polymerase

Hong, Zhi; Cameron, Craig Ε.; Walker, Michelle; Castro, Christian; Yao, Nanhua; Lau, Johnson Y.N.; Zhong, Weidong

doi:10.1006/viro.2001.0948

Cited by 175 publications

(171 citation statements)

References 21 publications

Supporting

Mentioning

164

Contrasting

Order By: Relevance

“…Similar steric mechanisms to ensure specificity for the ends of a genome have been employed by RNA viruses such as hepatitis C (Lesburg et al, 1999;Hong et al, 2001) and f6 (Butcher et al, 2001).…”

Section: Specificity For 3 0 Template Endsmentioning

confidence: 90%

The φ29 DNA polymerase:protein-primer structure suggests a model for the initiation to elongation transition

Kamtekar

Berman

Wang

et al. 2006

EMBO J

160

View full text Add to dashboard Cite

The absolute requirement for primers in the initiation of DNA synthesis poses a problem for replicating the ends of linear chromosomes. The DNA polymerase of bacteriophage /29 solves this problem by using a serine hydroxyl of terminal protein to prime replication. The 3.0 Å resolution structure shows one domain of terminal protein making no interactions, a second binding the polymerase and a third domain containing the priming serine occupying the same binding cleft in the polymerase as duplex DNA does during elongation. Thus, the progressively elongating DNA duplex product must displace this priming domain. Further, this heterodimer of polymerase and terminal protein cannot accommodate upstream template DNA, thereby explaining its specificity for initiating DNA synthesis only at the ends of the bacteriophage genome. We propose a model for the transition from the initiation to the elongation phases in which the priming domain of terminal protein moves out of the active site as polymerase elongates the primer strand. The model indicates that terminal protein should dissociate from polymerase after the incorporation of approximately six nucleotides.

show abstract

Section: Specificity For 3 0 Template Endsmentioning

confidence: 90%

The φ29 DNA polymerase:protein-primer structure suggests a model for the initiation to elongation transition

Kamtekar

Berman

Wang

et al. 2006

EMBO J

160

View full text Add to dashboard Cite

show abstract

“…3c). In addition, a ȋ-hairpin structure protrudes from the thumb toward the active site and is likely to be involved in correct positioning of the template 44 . The overall structure of NS5B is remarkably similar to the RdRP of bacteriophage Ƞ6 (ref.…”

Section: Dissecting the Structure And Function Of Hcv Ns Proteinsmentioning

confidence: 99%

Unravelling hepatitis C virus replication from genome to function

Lindenbach

Rice

2005

Nature

743

561

View full text Add to dashboard Cite

Since the discovery of the hepatitis C virus over 15 years ago, scientists have raced to develop diagnostics, study the virus and find new therapies. Yet virtually every attempt to dissect this pathogen has met with roadblocks that impeded progress. Its replication was restricted to humans or experimentally infected chimpanzees, and efficient growth of the virus in cell culture failed until very recently. Nevertheless hard-fought progress has been made and the first wave of antiviral drugs is entering clinical trials.virus life cycle: first, as a messenger RNA (mRNA) for translation of the viral proteins; second as a template for RNA replication; and third, as a nascent genome packaged within new virus particles. Virions presumably form by budding into the endoplasmic reticulum (ER) and leave the cell through the secretory pathway.Researchers have followed each aspect of the virus life cycle in turn. Just as infection starts from an HCV genome entering the cytoplasm and progressing through translation, replication and particle production, our understanding has progressed from having a genome sequence to understanding translation and the viral gene products, characterizing RNA replication, and establishing systems to characterize virus particles and infectivity. In this review, we summarize the current understanding of HCV replication with special emphasis on recent developments. As space is limited, we cannot be comprehensive; readers may wish to consult other reviews for detail and breadth 5,13,14. Initial studies: HCV translation and polyprotein processingThe identification of HCV yielded a partial viral genome sequence 5 . Research in the early 1990s focused on dissecting HCV gene expression and characterizing the gene products. Much has been learned about the biochemistry of three key enzymes, and structural information is now available at the atomic level for roughly half of the proteincoding region.Translation of the HCV genome, which lacks a 5፱ cap, depends on an internal ribosome entry site (IRES) within the 5፱-noncoding region (NCR). The HCV IRES binds 40S ribosomal subunits directly and avidly, bypassing the need for pre-initiation factors, and inducing an mRNA-bound conformation in the 40S subunit 15 . The IRES-40S complex then recruits eukaryotic initiation factor (eIF) 3 and the ternary complex of Met-tRNA-eIF2-GTP to form a non-canonical 48S intermediate, before a kinetically slow transition to the translationally active 80S complex 16,17 .Once initiated, translation of the HCV genome produces a large polyprotein that is proteolytically cleaved to produce 10 viral proteins (Fig. 2a). The amino-terminal one-third of the polyprotein encodes the virion structural proteins: the highly basic core (C) protein, and glycoproteins E1 and E2. After the structural region comes a small integral membrane protein, p7, which seems to function as an ion channel 18,19 . The remainder of the genome encodes the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B, which coordinate the intracellular process...

show abstract

“…1), Glu-18 is located in the middle of a unique A1-loop that provides many contact points during the proposed "closed" conformation of the enzyme (42). His-502 is in helix T, the part of the thumb most distal to the catalytic center, and close to the second GTPbinding pocket, which may be critical for a conformation change in NS5B (37).…”

Section: Figmentioning

confidence: 99%

“…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). Similar to other viral RdRPs, purified HCV NS5B is able to synthesize RNA using various RNAs as templates in vitro (9 -16), and two RNA synthesis reaction modes have been described: RNA elongation using a pre-annealed primer, and RNA initiation through a de novo mechanism (14, 15, 20 -22).…”

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

A Novel Mechanism to Ensure Terminal Initiation by Hepatitis C Virus NS5B Polymerase

Cited by 175 publications

References 21 publications

The φ29 DNA polymerase:protein-primer structure suggests a model for the initiation to elongation transition

The φ29 DNA polymerase:protein-primer structure suggests a model for the initiation to elongation transition

Unravelling hepatitis C virus replication from genome to function

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

Contact Info

Product

Resources

About