In vitro cleavage of hepatitis C virus polyprotein substrates by purified recombinant NS3 protease

D'Souza, E. D. A.; Grace, Ken; Sangar, D. V.; Rowlands, David J.; Clarke, Berwyn

doi:10.1099/0022-1317-76-7-1729

Cited by 29 publications

(25 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…it requires NS4A for /ram processing. However, this observation contradicts the in vitro results reported by others [24,26,43].…”

Section: Introductioncontrasting

confidence: 57%

“…Nonionic detergents were not essential for cleavage, but Thesit and Tween 20 did stimulate pro tease activity 4-to 10-fold when present above their crit ical micelle concentration. The zwitterionic detergent CHAPSO only slightly enhanced NS3 activity, as was reported for CHAPS [24], NaCI up to I M strongly stim ulated the ability of NS3 to cleave the 5A/5B substrate. No significant effect of divalent ions.…”

Section: Establishment and Characterization O F An In Vitro Cleavage mentioning

confidence: 49%

“…In this report, we have been able to simplify and refine the published cell-free-based assays [23][24][25][26][27] by accomplishing efficient and authentic processing of the purified 5A/5B and 4A/4B cleavage site substrates using an affinity-puri fied active NS3-4A protease expressed in baculovirusinfected cells. We have also demonstrated clearly that in vitro processing of the 5A/5B and 4A/4B substrates requires the presence of NS4A, whereas in vivo trans-pro cessing does not.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Hamatake¹,

Wang²,

Butcher³

et al. 1996

Intervirology

View full text Add to dashboard Cite

An in vitro cleavage system was established to measure HCV NS3 protease trans-processing activity. This system utilizes purified NS3-4A protein from baculovirus, purified substrates expressed by in vitro transcription and translation and defined buffer components. The 41-residue substrates, 5A/5B and 4A/4B, were processed efficiently in trans by wild-type NS3 but not by a catalytically inactive mutant protease; radiolabel sequencing confirmed that NS3-mediated cleavage occurred at the correct cysteine/serine sites, thereby authenticating this system. Two striking features of this in vitro assay are: (1) analogous 4B/5A and 3/4A substrates cannot be processed in trans under the same conditions, and (2) in vitro cleavage of the 5A/5B and 4A/4B sites is highly dependent on the presence of NS4A, which we show is not the case in vivo.

show abstract

“…it requires NS4A for /ram processing. However, this observation contradicts the in vitro results reported by others [24,26,43].…”

Section: Introductioncontrasting

confidence: 57%

Section: Establishment and Characterization O F An In Vitro Cleavage mentioning

confidence: 49%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Hamatake¹,

Wang²,

Butcher³

et al. 1996

Intervirology

View full text Add to dashboard Cite

show abstract

“…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%

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

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...

show abstract

“…Other aptamers (10G-2 and 10G-8) showed similar inhibition activity (Table 2). We assayed 10G-1 for the inhibition of NS3 activity using a NS5A-NS5B protein containing the amino acid region 2203-2506 [26] which was synthesized in vitro by a coupled transcriptiodtranslation system [35], as a substrate, and observed a similar amount of inhibition (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Selection of RNA Aptamers that Bind Specifically to the NS3 Protease of Hepatitis C Virus

Urvil

Kakiuchi

Zhou

et al. 1997

European Journal of Biochemistry

View full text Add to dashboard Cite

The RNA genome of human hepatitis C virus (HCV) is translated into a large precursor polyprotein. The NS3 protease of HCV has a crucial role in the processing of the polyprotein into functional viral proteins. We have used an in vitro genetic-selection strategy to isolate high-affinity RNA aptamers that bind to the NS3 protein, especially to its protease domain. Starting from a RNA pool that had a random sequence core of 12-18 nucleotides, aptamers that bind specifically to the NS3 protein were selected after 10 rounds of selection and amplification. A single aptamer, 10G-1, was found predominantly (71 %) in the selected pool. This aptamer could bind to the NS3 protein with a binding constant of 650 nM and inhibit the proteolytic activity in vitro. By phosphate-modification-interference analysis we showed that the phosphate residues that are critical for the binding of 10G-1 to NS3 lie within the selected regions of the aptamer and that binding involves electrostatic contacts with the phosphates of regions G28-U34 and A47-A55. The NS3-binding region in 10G-1 can serve as a basis for designing more potential inhibitors of the NS3 protein.Keywords: hepatitis C virus; NS3 protein; in vitro selection; aptamer; RNA.Hepatitis C virus (HCV) of the Flaviviridue family is the major cause of parenterally transmitted and sporadic non-A, non-B-type hepatitis [I-31. HCV infection is estimated to affect around 50 million people worldwide, and more than half of the patients develop chronic hepatitis. The positive-stranded RNA genome of HCV is translated into a large precursor polyprotein of about 3000 amino acid residues. The proteins are translated in the order C-El-E2-(P7)-NS2-NS3-NS4A-NS4B-NSSA-NSSB (Fig. 1 A). The core protein (C) and envelope proteins (El and E2) are structural proteins. The region between NS2 and NS5 contains the non-structural proteins. The processing of the polyprotein is mediated by host-cell signal peptidases The NS3 protein (Fig. 1 A) has a trypsin-like serine-protease motif in its N-terminal region and a putative helicase motif in the C-terminal region [l]. The residues His1083, Aspll07, and Serll65 form the catalytic triad of the protease domain of NS3 [5-71. It was shown that the NS4A peptide enhances the cleav- age activity of NS3 at the NS3-NS4A, NS4B-NSSA and NSSA-NS5B sites [ I I , 121. In addition to its role as a protease, the NS3 protein is associated with the development of hepatocellular carcinoma 1131. The serine-protease domain of NS3 is conserved in HCV isolates and in related pestiviruses and flaviviruses [13]. Furthermore, antigens to the NS3 protein are used in HCV diagnosis [14]. These properties of NS3 protease make it an attractive and challenging target in the development of anti-viral drugs and diagnostic reagents. In view of this, several well-characterized serine-protease inhibitors (such as aprotinin, benzamidine, chymostatin) have been tested for their effects on NS3 in vitro, but it was found that millimolar concentrations of these compounds are required for a moderate in...

show abstract

In vitro cleavage of hepatitis C virus polyprotein substrates by purified recombinant NS3 protease

Cited by 29 publications

References 17 publications

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

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

Substrate Specificity of the Hepatitis C Virus Serine Protease NS3

Selection of RNA Aptamers that Bind Specifically to the NS3 Protease of Hepatitis C Virus

Contact Info

Product

Resources

About