Crystal Structure of the Hepatitis C Virus NS3 Protease Domain Complexed with a Synthetic NS4A Cofactor Peptide

Kim, J.L; Morgenstern, Kurt; Lin, Chao; Fox, Ted; Dwyer, M.D.; Landro, James A.; Chambers, Stephen P.; Markland, William; Lepre, Christopher A.; O'Malley, Ethan T.; Harbeson, Scott L.; Rice, Charles M.; Murcko, Mark A.; Caron, Paul L.; Thomson, John A.

doi:10.1016/s0092-8674(00)81351-3

Cited by 699 publications

(625 citation statements)

References 74 publications

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“…Furthermore, the pocket was predicted to be very hydrophobic and closed by the aromatic ring of the phenylalanine. While this article was in preparation the crystal structure of NS3 protease has been solved independently by two groups (20,21). In the published structures the side chain of Phe 213 is indeed pointing inside the S1 pocket, thereby confirming the model.…”

Section: Discussionsupporting

confidence: 53%

“…The homology model has been used to successfully redesign the enzyme's specificity, thereby increasing its validity. Very recentyl the three-dimensional structure of the protease has been solved by two different groups (20,21), confirming the presence of a phenylalanine ring pointing into the pocket. The sequences of the four NS3 cleavage sites are listed in Table I, indicating that the intramolecular cleavage site between NS3 and NS4A differs from the consensus having a Thr in the P1 position.…”

mentioning

confidence: 94%

“…Availability of quantitative data using peptidic substrates has been so far hampered by difficulties in expressing and purifying sufficient amounts of enzymatically active recombinant NS3 protease. We (17,26) and others (15,20,21,(27)(28)(29)(30)(31)(32)(33) have recently described efficient heterologous expression and purification of the enzyme and were able to define optimized conditions for the determination of protease activity (26).…”

mentioning

confidence: 99%

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Substrate Specificity of the Hepatitis C Virus Serine Protease NS3

Urbani¹,

Bianchi²,

Narjes³

et al. 1997

Journal of Biological Chemistry

110

122

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The substrate specificity of a purified protein encompassing the hepatitis C virus NS3 serine protease domain was investigated by introducing systematic modifications, including non-natural amino acids, into substrate peptides derived from the NS4A/NS4B cleavage site. Kinetic parameters were determined in the absence and presence of a peptide mimicking the protease co-factor NS4A (Pep4A). Based on this study we draw the following conclusions: (i) the NS3 protease domain has an absolute requirement for a small residue in the P1 position of substrates, thereby confirming previous modelling predictions. (ii) Optimization of the P1 binding site occupancy primarily influences transition state binding, whereas the occupancy of distal binding sites is a determinant for both ground state and transition state binding. (iii) Optimized contacts at distal binding sites may contribute synergistically to cleavage efficiency.The N-terminal third of the hepatitis C virus (HCV) 1 NS3 protein contains a trypsin-like serine protease that accomplishes four out of the five processing events that take place during maturation of the nonstructural portion of the HCV polyprotein, performing cleavages at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions (1-6). It has been shown that cleavage between NS3 and NS4A is an intramolecular event, whereas all remaining junctions are processed in trans.In vivo, NS3 appears to form a heterodimer whereby the protease domain associates with the viral protein NS4A. The latter is a 54-residue protein that has been shown to bind to the N-terminal region of the protease via a central hydrophobic domain spanning residues 21-34 (7-13). NS4A acts as a cofactor of the protease enhancing cleavage at all sites and being an absolute requirement for processing of the NS4B/NS5A junction ex vivo (7). Several studies have shown that a peptide encompassing the central hydrophobic domain of NS4A is sufficient for eliciting activation of the protease (12, 14 -17).Serine proteases contact the P1 residue of their substrates through characteristic specificity pockets. The residues flanking the specificity pocket are important determinants of substrate recognition. Homology modelling of the S1 specificity pocket of the NS3 protease has predicted the presence of a phenylalanine as a prominent feature, thus rendering the pocket rather small and hydrophobic (18). These characteristics have led to the prediction of the preference for small, hydrophobic residues, ideally cysteine residues, in the P1 position of NS3 substrates. Radiosequencing of the single cleavage products has subsequently confirmed these predictions, yielding the consensus sequence (D/E)XXXXC(A/S) for all trans cleavage sites, with X being any amino acid and the scissile bond being located between Cys and Ala or Ser (2, 18). The homology model has been used to successfully redesign the enzyme's specificity, thereby increasing its validity. Very recentyl the three-dimensional structure of the protease has been solved by two different groups (20, 21...

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Section: Discussionsupporting

confidence: 53%

mentioning

confidence: 94%

mentioning

confidence: 99%

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Substrate Specificity of the Hepatitis C Virus Serine Protease NS3

Urbani¹,

Bianchi²,

Narjes³

et al. 1997

Journal of Biological Chemistry

110

122

View full text Add to dashboard Cite

show abstract

“…NS3 is composed of both a DExH/D-box protein superfamily of the helicase domain and a trypsin/chymotrypsin family of the serine protease domain (Hahm et al 1995, Kim et al 1996, Love et al 1996 and has a molecular weight of 67 kDa. NS3 is responsible for the cleavage of the viral polyprotein during the HCV replication cycle (Penin et al 2004).…”

mentioning

confidence: 99%

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

Zeminian

Padovani

Corvino

et al. 2013

Mem. Inst. Oswaldo Cruz

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The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

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“…This enzyme has a typical chymotrypsin-like fold and is composed of two ␤-barrel domains displaying on their interface the classical active site residues (His, Asp, Ser) that are a hallmark of serine-type proteases (Fig. 1B) (1,5,16,17). Although NS3 possesses intrinsic proteolytic activity, polyprotein cleavage is dramatically enhanced by the NS4A cofactor.…”

mentioning

confidence: 99%

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

Appel

Schaller

Pénin

et al. 2006

Journal of Biological Chemistry

177

120

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With an estimated number of about 180 million affected people worldwide and a limited therapy option, infection with the hepatitis C virus (HCV) 2 is an important medical problem. Initial limitations in propagating HCV in cell culture have been overcome step-by-step in the past few years and culminated in the recent development of a system allowing efficient propagation of infectious HCV in tissue culture. Nevertheless, structural and biochemical studies of viral proteins as well as molecular analysis of viral RNA replication are still major challenges. The recent resolution of the three-dimensional structures of some HCV proteins or domains along with elegant reverse genetic and cell biological studies provided first insights into how HCV amplifies its RNA, exploits the cellular machinery, and eventually overcomes innate immune responses. Organization of the Viral GenomeHCV is a positive strand RNA virus that has been classified as the genus Hepacivirus in the Flaviviridae family. The HCV genome is an uncapped, linear molecule with a length of ϳ9600 nucleotides (nt; Fig. 1A). It carries a long open reading frame that is flanked at the 5Ј-and 3Ј-ends by short highly structured non-translated regions (NTRs). The 5Ј-NTR has a length of about 340 nt and contains an internal ribosome entry site (IRES) required for translation of the HCV genome (1). This RNA element binds the 40 S ribosomal subunit in the absence of other translation initiation factors in a way that the initiation codon is placed in the immediate vicinity of the P site (2). Part of the IRES (domain II) overlaps with RNA signals essential for viral replication (Fig. 1A) arguing for a possible role of domain II in regulating a translation-RNA replication switch. The 3Ј-NTR has a tripartite structure composed of an ϳ40-nt-long variable region downstream of the HCV coding sequence, a poly(U/UC) tract of heterogeneous length, and a highly conserved 98-nt-long sequence designated X-tail (Fig. 1A). Genetic studies have shown that a poly(U/UC) tract of at least ϳ25 nt as well as the complete X-tail are required for RNA replication in cell culture and for infectivity of the viral genome in vivo. An additional cis-acting RNA element (CRE) has been identified in the 3Ј-terminal coding region of NS5B (Fig. 1A). This CRE (designated 5BSL3.2) (3) forms a long distance RNA-RNA interaction with SL2 in the X-tail (4), which is indispensable for RNA replication.Expression of the viral proteins from the monocistronic genome is primarily achieved by production of a polyprotein that is proteolytically cleaved into the structural proteins (core, envelope proteins E1 and E2), the hydrophobic peptide p7, and the non-structural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (Fig. 1A) (1, 5). Processing of the core to p7 region is mediated by host cell signalases, and in the case of the core protein, in addition by signal peptide peptidase. All remaining cleavages are carried out by two viral proteases: the NS2/3 protease mediating cleavage between NS2 and NS3 and the NS3...

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Crystal Structure of the Hepatitis C Virus NS3 Protease Domain Complexed with a Synthetic NS4A Cofactor Peptide

Cited by 699 publications

References 74 publications

Substrate Specificity of the Hepatitis C Virus Serine Protease NS3

Substrate Specificity of the Hepatitis C Virus Serine Protease NS3

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

From Structure to Function: New Insights into Hepatitis C Virus RNA Replication

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