Construction, expression, and characterization of a novel fully activated recombinant single‐chain hepatitis C virus protease

Taremi, S. Shane; Beyer, Brian M.; Maher, Maureen; Yao, Nanhua; Prosise, W.W.; Weber, Patricia C; Malcolm, Bruce A.

doi:10.1002/pro.5560071011

Cited by 113 publications

(133 citation statements)

References 29 publications

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“…All structural studies were carried out with the highly soluble, single-chain construct of the NS3/4A protease domain described previously (33), which contains a fragment of the essential cofactor NS4A covalently linked at the N terminus by a flexible linker. A similar protease construct was shown to retain comparable catalytic activity to the authentic protein complex (34). Crystallization trials were initially carried out using the inactive (S139A) protease variant in complex with substrate peptides spanning P7-P5′.…”

Section: Structure Determination Of Inhibitor and Substrate Complexesmentioning

confidence: 99%

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Romano

Ali

Royer

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

182

245

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Hepatitis C virus infects an estimated 180 million people worldwide, prompting enormous efforts to develop inhibitors targeting the essential NS3/4A protease. Resistance against the most promising protease inhibitors, telaprevir, boceprevir, and ITMN-191, has emerged in clinical trials. In this study, crystal structures of the NS3/4A protease domain reveal that viral substrates bind to the protease active site in a conserved manner defining a consensus volume, or substrate envelope. Mutations that confer the most severe resistance in the clinic occur where the inhibitors protrude from the substrate envelope, as these changes selectively weaken inhibitor binding without compromising the binding of substrates. These findings suggest a general model for predicting the susceptibility of protease inhibitors to resistance: drugs designed to fit within the substrate envelope will be less susceptible to resistance, as mutations affecting inhibitor binding would simultaneously interfere with the recognition of viral substrates.drug design | hepatitis C | substrate envelope D rug resistance is a major obstacle in the treatment of quickly evolving diseases. Hepatitis C virus (HCV) is a genetically diverse Hepacivirus of the Flaviviridae family infecting an estimated 180 million people worldwide (1). The viral RNA genome is translated as a single polyprotein and subsequently processed by host-cell and viral proteases into structural (C, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (2). The viral RNA-dependent RNA polymerase, NS5B, is inherently inaccurate and misincorporation of bases accounts for a very high mutation rate (3). While some mutations are neutral, others will alter the viability of the virus and propagate with varying efficiencies in each patient. Thus HCV infected individuals will develop a heterogeneous population of virus variants known as quasispecies (4). As patients begin treatment, the selective pressures of antiviral drugs will favor drug resistant variants (5). Therefore, an inhibitor must not only recognize one protein variant, but an ensemble of related enzymes. A detailed understanding of the atomic mechanisms of resistance is essential to effectively combat drug resistance against HCV antivirals.The essential HCV NS3/4A protease is an attractive therapeutic target responsible for cleaving at least four sites along the viral polyprotein. These sites share little sequence homology except for an acid at position P6, Cys or Thr at P1, and Ser or Ala at P1′ (Table S1). The first cleavage event at the 3-4A junction occurs in cis as a unimolecular process, while the remaining substrates are processed bimolecularly in trans. The NS3/4A protease also cleaves the human cellular targets TRIF and MAVS, which confounds the innate immune response to viral infection (6-8).Early drug design efforts were hampered by the relatively shallow, featureless architecture of the protease active site. The eventual observation of N-terminal product inhibition served as a stepping stone f...

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Section: Structure Determination Of Inhibitor and Substrate Complexesmentioning

confidence: 99%

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Romano

Ali

Royer

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

182

245

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“…The viral NS3 protease in complex with the cofactor NS4A cleaves the polyprotein at four junctions releasing the NS proteins 4A, 4B, 5A, and 5B, and therefore is essential for viral replication (3,4). A central, hydrophobic 14-mer peptide of the 54-residue NS4A, comprising residues 21-32, is necessary and sufficient for maximal activation in vitro (5). NS3/4A has also been shown to cleave cellular proteins leading to inhibition of interferon production, thereby impairing the innate immune response against viral infections (6).…”

mentioning

confidence: 99%

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

Schiering

D’Arcy

Villard

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Hepatitis C virus (HCV) infection is a global health burden with over 170 million people infected worldwide. In a significant portion of patients chronic hepatitis C infection leads to serious liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV NS3 protein is essential for viral polyprotein processing and RNA replication and hence viral replication. It is composed of an N-terminal serine protease domain and a C-terminal helicase/NTPase domain. For full activity, the protease requires the NS4A protein as a cofactor. HCV NS3/4A protease is a prime target for developing direct-acting antiviral agents. First-generation NS3/4A protease inhibitors have recently been introduced into clinical practice, markedly changing HCV treatment options. To date, crystal structures of HCV NS3/4A protease inhibitors have only been reported in complex with the protease domain alone. Here, we present a unique structure of an inhibitor bound to the full-length, bifunctional protease-helicase NS3/4A and show that parts of the P4 capping and P2 moieties of the inhibitor interact with both protease and helicase residues. The structure sheds light on inhibitor binding to the more physiologically relevant form of the enzyme and supports exploring inhibitor-helicase interactions in the design of the next generation of HCV NS3/4A protease inhibitors. In addition, small angle X-ray scattering confirmed the observed proteasehelicase domain assembly in solution.structure-based drug design | X-ray structure | solution scattering | medicinal chemistry C hronic hepatitis C virus (HCV) infection affects more than 3% of the world's population and is a leading cause of chronic liver diseases (1). Therapeutic options have been suboptimal, especially for HCV genotype 1, the most prevalent genotype in developed countries. Recently, the addition of direct-acting antiviral agents (DAAs) to the previous standard of care (combination therapy with pegylated interferon and ribavirin) have demonstrated considerable improvement in sustained virological response rates in patients infected with HCV genotype 1 (2). With the first DAAs now having been introduced into clinical practice, it is to be expected that in the near future standard therapy will change to a triple therapy including an HCV NS3/4A protease inhibitor in combination with pegylated interferon and ribavirin.The positive-strand RNA genome of HCV encodes a polyprotein precursor, which is proteolytically processed by host and viral proteases into 10 individual structural and nonstructural (NS) proteins. The viral NS3 protease in complex with the cofactor NS4A cleaves the polyprotein at four junctions releasing the NS proteins 4A, 4B, 5A, and 5B, and therefore is essential for viral replication (3, 4). A central, hydrophobic 14-mer peptide of the 54-residue NS4A, comprising residues 21-32, is necessary and sufficient for maximal activation in vitro (5). NS3/4A has also been shown to cleave cellular proteins leading to inhibition of interferon production, thereby impairin...

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“…Attempts to express NS3 and NS4A as separated proteins for reconstitution of NS3-NS4A complex did not lead to efficient protease activity and complementation of the protease domain of NS3, with the central region of the NS4A protein showing low protease activity Shimizu et al 1996;Morgenstern et al 1997;Beran and Pyle 2008). Expression of the NS3 (1-631) -NS4A complex in eukaryotic systems yields small amounts of protein that are insufficient for extensive in vitro analysis (Sali et al 1998;Taremi et al 1998). Taremi et al (1998) developed a single-chain form of the NS3 protease fused to residues 21-GSVVIVGRIILS-32 of NS4A, through a linker (GSGS).…”

mentioning

confidence: 99%

“…Expression of the NS3 (1-631) -NS4A complex in eukaryotic systems yields small amounts of protein that are insufficient for extensive in vitro analysis (Sali et al 1998;Taremi et al 1998). Taremi et al (1998) developed a single-chain form of the NS3 protease fused to residues 21-GSVVIVGRIILS-32 of NS4A, through a linker (GSGS). This linker makes NS4A able to interact with NS3 through a beta-turn as in the natural full-length NS3 (1-631) -NS4A complex.…”

mentioning

confidence: 99%

“…This linker makes NS4A able to interact with NS3 through a beta-turn as in the natural full-length NS3 (1-631) -NS4A complex. The single-chain protein (NS4A (21-32) -GSGS-NS3 (3-181) ) expressed in Escherichia coli shows enzymatic parameters equivalent to those of the wild type NS3 (1-631) -NS4A protein generated in eukaryotic cells and can be over expressed to high levels as a soluble protein (Dimasi et al 1998;Taremi et al 1998;Howe et al 1999;Leyssen et al 2000;Wang et al 2004;Kou et al 2007). In this study we identified, by DNA sequencing of the 4870-5902 nucleotide region, NS4A amino acid variants from serum samples of HCV infected patients; moreover, we performed amino acid modifications on the 21-32 NS4A sequence in order to understand better the role of NS4 as regulator in HCV replication.…”

mentioning

confidence: 99%

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Identification of Amino Acid Variants in the Hepatitis C Virus Non-Structural Protein 4A

Bermúdez-Aguirre

Padilla-Noriega

Zenteno

et al. 2009

Tohoku J. Exp. Med.

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Construction, expression, and characterization of a novel fully activated recombinant single‐chain hepatitis C virus protease

Cited by 113 publications

References 29 publications

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

Identification of Amino Acid Variants in the Hepatitis C Virus Non-Structural Protein 4A

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