Establishment of an in vitro Assay to Characterize Hepatitis C Virus NS3-4A Protease &lt;i&gt;Trans&lt;/i&gt;-Processing Activity

Hamatake, Robert; Wang, Hwei-Gene Heidi; Butcher, J A; Bifano, Marc; Clark, Gwendelíne; Hernandez, Dennis; Zhang, Sharon; Racela, Jason; Standring, David N.; Colonno, Richard J.

doi:10.1159/000150525

Cited by 9 publications

(4 citation statements)

References 16 publications

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“…This stringent requirement for NS4A in NS4A-NS4B and NS5A-NS5B trans processing in vitro was not detected with the isolated NS3 protease domain in analogous experiments (55), but our finding is in agreement with the study of Hamatake et al (23), who compared the trans-cleavage activity of purified FL NS3 and NS3-NS4A complex in the same type of assay.…”

Section: Expression and Purification Of The Full-length Ns3 Proteinsupporting

confidence: 93%

Multiple Enzymatic Activities Associated with Recombinant NS3 Protein of Hepatitis C Virus

Gallinari

Brennan

Nardi

et al. 1998

J Virol

179

112

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The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full-length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein in Escherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase, and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and C-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.

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Section: Expression and Purification Of The Full-length Ns3 Proteinsupporting

confidence: 93%

Multiple Enzymatic Activities Associated with Recombinant NS3 Protein of Hepatitis C Virus

Gallinari

Brennan

Nardi

et al. 1998

J Virol

179

112

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“…In these transfection experiments, in which a full-length form of the enzyme was produced, NS3 was apparently active only in the presence of the cofactor, at least for the NS4B–5A and NS5A–5B cleavage sites, in contrast to what occurs with the protease domain when tested on HCV substrates (Scarselli et al , 1997 ) and on the predicted GBV-B NS4A–4B cleavage site peptide (A. Sbardellati, unpublished). This is not surprising, since it is known that the HCV NS3 protease domain shows baseline NS4A-independent activity whereas the cofactor is essential for the full-length enzyme (Gallinari et al , 1998 , 1999 ; Hamatake et al , 1996 ; Steinkühler et al , 1996 a ).…”

Section: Full Textmentioning

confidence: 95%

“…Sbardellati, unpublished). This is not surprising, since it is known that the HCV NS3 protease domain shows baseline NS4A-independent activity whereas the cofactor is essential for the full-length enzyme (Gallinari et al, 1998(Gallinari et al, , 1999Hamatake et al, 1996 ;.…”

mentioning

confidence: 97%

Processing of GB virus B non-structural proteins in cultured cells requires both NS3 protease and NS4A cofactor

Sbardellati¹,

Scarselli²,

Amati³

et al. 2000

Journal of General Virology

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The identification of antivirals and vaccines against hepatitis C virus (HCV) infection is hampered by the lack of convenient animal models. The need to develop surrogate models has recently drawn attention to GB virus B (GBV-B), which produces hepatitis in small primates. In a previous study in vitro, it was shown that GBV-B NS3 protease shares substrate specificity with the HCV enzyme, known to be crucial for virus replication. In this report, GBV-B NS3 activity on GBV-B precursor proteins has been analysed in a cell-based system. It is shown that mature protein products are obtained that are compatible with the cleavage sites proposed on the basis of sequence homology with HCV and that GBV-B NS4A protein is required as a cofactor for optimal enzymatic activity. Experiments in vitro supported by a structural model mapped the region of NS4A that interacts with NS3 and showed that the GBV-B cofactor cannot be substituted for by its HCV analogue.GB virus B (GBV-B) is a recently discovered flavivirus that causes hepatitis in small New World monkeys of the genus Saguinus (Schlauder et al., 1995 a, b ;Traboni et al., 1999) and shows striking similarities to hepatitis C virus (HCV) (De Francesco, 1999 ;Rice, 1996), which is responsible for a very severe and widespread form of hepatitis (WHO, 1997 ;NIH, 1997). These observations encouraged us to establish a surrogate animal model of HCV infection using tamarins infected by GBV-B or chimeric GBV-B\HCV viruses. The need for such an indirect animal model arises from the extremely narrow host range of HCV, infecting only humans and chimpanzees (Farci et al., 1992 a, b). In order to investigate the extent to which HCV and GBV-B are similar and whether any of their genes can be shuffled to construct chimeric viruses, several studies have been performed recently on the GBV-B genome and its protein products (Bukh et al., 1999 ;Grace et al., 1999 ;Rijnbrand et al., 2000 ;Sbardellati et al., 1999 ;Scarselli et al., 1997 ;Zhong et al., 1999).A key molecule in the life-cycle of HCV is the NS3 protein, which shows different enzymatic activities organized in two domains. The N-terminal domain is responsible for the proteolytic cleavages that lead to the mature non-structural proteins, whereas the C-terminal domain shows helicase and NTPase activity (De Francesco, 1999). A similar molecule is encoded by the GBV-B genome that shows limited amino acid identity (about 40 %) to the HCV protein . However, a high level of conservation of specific sequence motifs is present in both the protease and helicase Zhong et al., 1999) domains. In a previous study, we described the purification of an active recombinant GBV-B NS3 protease domain and demonstrated that this enzyme is able to cleave HCV substrates in the form of synthetic peptides or in vitro-translated proteins. We also showed that the addition of an HCV NS4A activator peptide, Pep4A #" -$% , did not modify the activity of the GBV-B enzyme .In order to characterize further the substrate specificity and cofactor requirements of GBV-B...

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“…The HCV genome encodes a polypeptide precursor consisting of about 3010 residues [2]. Cleavage of the polypeptide precursor results in production of the individual viral structural (core, envelope) and nonstructural (NS) proteins [3][4][5]. NS3 requires the adjacent NS4A cofactor and is essential for replication of the virus because it directs the proteolytic cleavages at the same junctions as NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B [6].…”

Section: Introductionmentioning

confidence: 99%

Computer-Assisted Analysis of the Interactions of Macrocyclic Inhibitors with wild Type and Mutant D168A Hepatitis C Virus NS3 Serine Protease

Cunha¹,

Ramalho²,

Taft³

et al. 2006

LDDD

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The high frequency of treatment failures suggests the need for more specific, less toxic and more active antiviral therapies for the Hepatitis C virus (HCV). HCV NS3 is currently regarded as a prime target for anti-viral drugs, thus, molecular modeling studies were used to try to understand the interaction of BILN 2061 macrocyclic analogs with the wild-type and the D168A mutant NS3 serine protease, with the aim of rendering them better therapeutic agents of the Hepatitis C virus infection.

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Establishment of an in vitro Assay to Characterize Hepatitis C Virus NS3-4A Protease <i>Trans</i>-Processing Activity

Cited by 9 publications

References 16 publications

Multiple Enzymatic Activities Associated with Recombinant NS3 Protein of Hepatitis C Virus

Multiple Enzymatic Activities Associated with Recombinant NS3 Protein of Hepatitis C Virus

Processing of GB virus B non-structural proteins in cultured cells requires both NS3 protease and NS4A cofactor

Computer-Assisted Analysis of the Interactions of Macrocyclic Inhibitors with wild Type and Mutant D168A Hepatitis C Virus NS3 Serine Protease

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