A central hydrophobic domain of the hepatitis C virus NS4A protein is necessary and sufficient for the activation of the NS3 protease

Tomei, Licia; Failla, Cristina Maria; Vitale, Rosa; Bianchi, Elisabetta; Francesco, Raffaele De

doi:10.1099/0022-1317-77-5-1065

Cited by 103 publications

(92 citation statements)

References 20 publications

(4 reference statements)

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“…For activation of the NS3 protease domain, only residues 21 to 34 of NS4A are required (7). Due to its shallow, solvent-exposed substratebinding pocket, the substrate coordination extends over the whole binding region (6,7). This also correlates with the reported reduced ability to cleave substrates smaller than 10 amino acids (8).…”

supporting

confidence: 51%

“…The NS3 protease domain located within the N-terminal region (aa 1-180) of NS3 is a chymotrypsin-like serine protease that forms a heterodimeric co-complex with NS4A, a 54-residue long important cofactor for NS3 proteolytic activity (5,6). For activation of the NS3 protease domain, only residues 21 to 34 of NS4A are required (7). Due to its shallow, solvent-exposed substratebinding pocket, the substrate coordination extends over the whole binding region (6,7).…”

mentioning

confidence: 99%

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High Affinity Peptide Inhibitors of the Hepatitis C Virus NS3-4A Protease Refractory to Common Resistant Mutants

Kügler

Schmelz

Gentzsch

et al. 2012

Journal of Biological Chemistry

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Background: NS3-4A is a validated target for antiviral therapeutics whereby HCV rapidly develops resistant mutants. Results: Peptides coordinating with a unique NS3-4A site and strongly inhibiting common resistance mutants were developed. Conclusion: A unique "finger" structure extends binding of the inhibitory peptides to a novel druggable site. Significance: Novel leads are of interest for the design of inhibitors refractory to known resistance mutants.

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supporting

confidence: 51%

mentioning

confidence: 99%

High Affinity Peptide Inhibitors of the Hepatitis C Virus NS3-4A Protease Refractory to Common Resistant Mutants

Kügler

Schmelz

Gentzsch

et al. 2012

Journal of Biological Chemistry

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“…Transient expression of constructs directing the expression ofN-and C-terminally truncated NS4A proteins mapped a minimum proteinase activation domain from Gly-21 to Ser-32 (Bartenschlager et al, 1995a,b;Tanji et al, 1995a). This result was later confirmed with various in vitro trans-processing assays in which NS4A fragments were supplied as synthetic peptides and identified the central NS4A region as an autonomous proteinase activation domain (Butkiewicz et al, 1996;Shimizu et al, 1996b;Tomei et al, 1996). Although most non-structural proteins, including NS4A, are tightly associated with intracellular membranes, at least in vitro membranes are not required for proteinase activation Koch et al, 1996;Tomei et al, 1996).…”

Section: The Ns3-4a Proteinase Complexsupporting

confidence: 50%

Molecular Targets in Inhibition of Hepatitis C Virus Replication

Bartenschlager

1997

Antivir Chem Chemother

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SummaryHepatitis C virus (HCY) is the major cause of transfusion-associated hepatitis and accounts for a significant proportion of hepatitis cases worldwide. Most, if not all, infections become persistent and about 60% of cases develop chronic liver disease with various outcomes ranging from an asymptomatic carrier state to chronic active hepatitis and liver cirrhosis, which is strongly associated with the development of hepatocellular carcinoma. Since the initial cloning of the viral genome in 1989, our knowledge of the molecular biology of HCY has increased rapidly and led to the identification of several potential targets for antiviral intervention. In contrast, the low replication of the virus in cell culture, the lack of convenient animal models and the high genome variability present major challenges for drug development. This review will describe candidate drug targets and summarize 'classical' and 'novel' approaches currently being pursued to develop efficient HCYspecific therapies.

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“…The amino acid that defines the S1 pocket specificity (Phe 154 ) is conserved between the HCV and GBV-B NS3 molecules (57). However, the reported results were surprising in that the GBV-B NS3 protease did not seem to require a cofactor for activity, a hallmark of flavivirus-like viruses (6,14,15,39,63,64,66). This lack of cofactor dependency could be due to the fact that only a truncated catalytic domain was used in the characterization of the protease activity which might be different from that of the full-length NS3 as demonstrated with HCV (20).…”

Section: Resultsmentioning

confidence: 81%

Virus-Specific Cofactor Requirement and Chimeric Hepatitis C Virus/GB Virus B Nonstructural Protein 3

Butkiewicz

Yao²,

Zhong

et al. 2000

J Virol

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GB virus B (GBV-B) is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species), making it an attractive surrogate virus for in vivo testing of anti-HCV inhibitors in a small monkey model. It has been reported that the nonstructural protein 3 (NS3) serine protease of GBV-B shares similar substrate specificity with its counterpart in HCV. Authentic proteolytic processing of the HCV polyprotein junctions (NS4A/4B, NS4B/5A, and NS5A/5B) can be accomplished by the GBV-B NS3 protease in an HCV NS4A cofactor-independent fashion. We further characterized the protease activity of a full-length GBV-B NS3 protein and its cofactor requirement using in vitro-translated GBV-B substrates. Cleavages at the NS4A/4B and NS5A/5B junctions were readily detectable only in the presence of a cofactor peptide derived from the central region of GBV-B NS4A. Interestingly, the GBV-B substrates could also be cleaved by the HCV NS3 protease in an HCV NS4A cofactor-dependent manner, supporting the notion that HCV and GBV-B share similar NS3 protease specificity while retaining a virus-specific cofactor requirement. This finding of a strict virus-specific cofactor requirement is consistent with the lack of sequence homology in the NS4A cofactor regions of HCV and GBV-B. The minimum cofactor region that supported GBV-B protease activity was mapped to a central region of GBV-B NS4A (between amino acids Phe22 and Val36) which overlapped with the cofactor region of HCV. Alanine substitution analysis demonstrated that two amino acids, Val27 and Trp31, were essential for the cofactor activity, a finding reminiscent of the two critical residues in the HCV NS4A cofactor, Ile25 and Ile29. A model for the GBV-B NS3 protease domain and NS4A cofactor complex revealed that GBV-B might have developed a similar structural strategy in the activation and regulation of its NS3 protease activity. Finally, a chimeric HCV/GBV-B bifunctional NS3, consisting of an N-terminal HCV protease domain and a C-terminal GBV-B RNA helicase domain, was engineered. Both enzymatic activities were retained by the chimeric protein, which could lead to the development of a chimeric GBV-B virus that depends on HCV protease function.GB virus B (GBV-B) is a single-stranded (ss) positive-sense RNA virus associated with GB agent hepatitis (47, 60). It infects tamarins (Saguinus species) and causes acute hepatitis in naive animals (13). Phylogenetic tree analysis and the genome organization of GBV-B suggest that this virus belongs to the Flaviviridae family which at present consists of three genera: Flavivirus, Pestivirus, and Hepacivirus (47, 60). Like other members of this family (27, 52), the genome of GBV-B encodes a single large polyprotein of approximately 2,860 amino acids. The polyprotein is likely processed into several structural (C, E1, E2, and p7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins by either host-or virusencoded proteases (47). Among all animal viruses, GBV-B shares closest nucleotide homology with he...

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A central hydrophobic domain of the hepatitis C virus NS4A protein is necessary and sufficient for the activation of the NS3 protease

Cited by 103 publications

References 20 publications

High Affinity Peptide Inhibitors of the Hepatitis C Virus NS3-4A Protease Refractory to Common Resistant Mutants

High Affinity Peptide Inhibitors of the Hepatitis C Virus NS3-4A Protease Refractory to Common Resistant Mutants

Molecular Targets in Inhibition of Hepatitis C Virus Replication

Virus-Specific Cofactor Requirement and Chimeric Hepatitis C Virus/GB Virus B Nonstructural Protein 3

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