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Hepatitis C viral NS3-4A protease activity is enhanced by the NS3 helicase.

Beran RK, Pyle AM - J. Biol. Chem. (2008)

Bottom Line: Our results indicate that the helicase domain enhances serine protease activity, just as the protease domain enhances helicase activity.This is the first time that such a complete interdependence has been demonstrated for a multifunctional, single chain enzyme.NS3-4A domain interdependence has important implications for function during the viral lifecycle as well as for the design of inhibitor screens that target the NS3-4A protease.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.

ABSTRACT
Non-structural protein 3 (NS3) is a multifunctional enzyme possessing serine protease, NTPase, and RNA unwinding activities that are required for hepatitis C viral (HCV) replication. HCV non-structural protein 4A (NS4A) binds to the N-terminal NS3 protease domain to stimulate NS3 serine protease activity. In addition, the NS3 protease domain enhances the RNA binding, ATPase, and RNA unwinding activities of the C-terminal NS3 helicase domain (NS3hel). To determine whether NS3hel enhances the NS3 serine protease activity, we purified truncated and full-length NS3-4A complexes and examined their serine protease activities under a variety of salt and pH conditions. Our results indicate that the helicase domain enhances serine protease activity, just as the protease domain enhances helicase activity. Thus, the two enzymatic domains of NS3-4A are highly interdependent. This is the first time that such a complete interdependence has been demonstrated for a multifunctional, single chain enzyme. NS3-4A domain interdependence has important implications for function during the viral lifecycle as well as for the design of inhibitor screens that target the NS3-4A protease.

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Related in: MedlinePlus

Steady-state velocity curves for NS3-4A proteolysis of RET-S1 under a range of pH and salt conditions. The steady-state rates of proteolysis were 0.006 ± 0.001 μm product/s at pH 6.5, 30 mm NaCl (solid circles), 0.019 ± 0.001μm product/s at pH 8, 30 mm NaCl (pierced circles), 0.019 ± 0.001 μm product/s at pH 6.5, 75 mm NaCl (solid diamonds), 0.020 ± 0.001 μm product/s at pH 8, 75 mm NaCl (pierced diamonds), 0.010 ± 0.001 μm product/second at pH 6.5, 200 mm NaCl (solid squares), and 0.010 ± 0.001 μm product/s at pH 8, 200 mm NaCl (pierced squares). The active fraction in each case, as determined by intersection with the y intercept, was 68 ± 9% for pH 6.5, 30 mm NaCl, 84 ± 16% for pH 8.0, 30 mm NaCl, 84 ± 15% for pH 6.5, 75 mm NaCl, 70 ± 12% for pH 8.0, 75 mm NaCl, 95 ± 5% for pH 6.5, 200 mm NaCl, and 95 ± 5% for pH 8.0, 200 mm NaCl. The data shown were determined using NS3-4A of the 1b genotype and represent the steady-state data points fit to a line. Similar results were observed with NS3-4A (1a). This data are the average of three experiments and the error values represent standard deviation.
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fig4: Steady-state velocity curves for NS3-4A proteolysis of RET-S1 under a range of pH and salt conditions. The steady-state rates of proteolysis were 0.006 ± 0.001 μm product/s at pH 6.5, 30 mm NaCl (solid circles), 0.019 ± 0.001μm product/s at pH 8, 30 mm NaCl (pierced circles), 0.019 ± 0.001 μm product/s at pH 6.5, 75 mm NaCl (solid diamonds), 0.020 ± 0.001 μm product/s at pH 8, 75 mm NaCl (pierced diamonds), 0.010 ± 0.001 μm product/second at pH 6.5, 200 mm NaCl (solid squares), and 0.010 ± 0.001 μm product/s at pH 8, 200 mm NaCl (pierced squares). The active fraction in each case, as determined by intersection with the y intercept, was 68 ± 9% for pH 6.5, 30 mm NaCl, 84 ± 16% for pH 8.0, 30 mm NaCl, 84 ± 15% for pH 6.5, 75 mm NaCl, 70 ± 12% for pH 8.0, 75 mm NaCl, 95 ± 5% for pH 6.5, 200 mm NaCl, and 95 ± 5% for pH 8.0, 200 mm NaCl. The data shown were determined using NS3-4A of the 1b genotype and represent the steady-state data points fit to a line. Similar results were observed with NS3-4A (1a). This data are the average of three experiments and the error values represent standard deviation.

Mentions: NS3-4A Exhibits Robust Serine Protease Activity Under a Variety of Salt and pH Conditions—It has previously been reported that NS3-4A only functions as a robust serine protease under conditions of high salt (≥150 mm NaCl) and high pH (7.5–8.0) (17, 27). These same conditions are incompatible with those required for NS3 helicase activity (13, 17, 32), and it therefore has been suggested that the two activities (proteolysis and unwinding) might be mutually exclusive (17). We therefore sought to determine whether protease activity of the wild-type NS3-4A complex is restricted to the narrow range of previously reported conditions. To this end, we measured NS3-4A serine protease activity under a variety of pH (6.5 and 8.0) and salt conditions (30, 75, and 200 mm NaCl) (Fig. 4). We did not observe significant differences in the active fraction of protein (i.e. the y intercept of the velocity plots) under these varying pH and salt conditions, particularly when accounting for error (Fig. 4 legend). The steady-state proteolysis velocities in 30 mm NaCl at pH 6.5 and 8.0 were observed to be 0.006 ± 0.001 and 0.019 ± 0.001 μm/s, respectively. In 200 mm NaCl at pH 6.5 and 8.0, the steady-state proteolysis velocities were observed to be 0.010 ± 0.001 μm/s in both cases. Therefore, native, full-length NS3-4A is a robust serine protease under a broad range of salt and pH conditions, including those that are compatible with helicase activity.


Hepatitis C viral NS3-4A protease activity is enhanced by the NS3 helicase.

Beran RK, Pyle AM - J. Biol. Chem. (2008)

Steady-state velocity curves for NS3-4A proteolysis of RET-S1 under a range of pH and salt conditions. The steady-state rates of proteolysis were 0.006 ± 0.001 μm product/s at pH 6.5, 30 mm NaCl (solid circles), 0.019 ± 0.001μm product/s at pH 8, 30 mm NaCl (pierced circles), 0.019 ± 0.001 μm product/s at pH 6.5, 75 mm NaCl (solid diamonds), 0.020 ± 0.001 μm product/s at pH 8, 75 mm NaCl (pierced diamonds), 0.010 ± 0.001 μm product/second at pH 6.5, 200 mm NaCl (solid squares), and 0.010 ± 0.001 μm product/s at pH 8, 200 mm NaCl (pierced squares). The active fraction in each case, as determined by intersection with the y intercept, was 68 ± 9% for pH 6.5, 30 mm NaCl, 84 ± 16% for pH 8.0, 30 mm NaCl, 84 ± 15% for pH 6.5, 75 mm NaCl, 70 ± 12% for pH 8.0, 75 mm NaCl, 95 ± 5% for pH 6.5, 200 mm NaCl, and 95 ± 5% for pH 8.0, 200 mm NaCl. The data shown were determined using NS3-4A of the 1b genotype and represent the steady-state data points fit to a line. Similar results were observed with NS3-4A (1a). This data are the average of three experiments and the error values represent standard deviation.
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fig4: Steady-state velocity curves for NS3-4A proteolysis of RET-S1 under a range of pH and salt conditions. The steady-state rates of proteolysis were 0.006 ± 0.001 μm product/s at pH 6.5, 30 mm NaCl (solid circles), 0.019 ± 0.001μm product/s at pH 8, 30 mm NaCl (pierced circles), 0.019 ± 0.001 μm product/s at pH 6.5, 75 mm NaCl (solid diamonds), 0.020 ± 0.001 μm product/s at pH 8, 75 mm NaCl (pierced diamonds), 0.010 ± 0.001 μm product/second at pH 6.5, 200 mm NaCl (solid squares), and 0.010 ± 0.001 μm product/s at pH 8, 200 mm NaCl (pierced squares). The active fraction in each case, as determined by intersection with the y intercept, was 68 ± 9% for pH 6.5, 30 mm NaCl, 84 ± 16% for pH 8.0, 30 mm NaCl, 84 ± 15% for pH 6.5, 75 mm NaCl, 70 ± 12% for pH 8.0, 75 mm NaCl, 95 ± 5% for pH 6.5, 200 mm NaCl, and 95 ± 5% for pH 8.0, 200 mm NaCl. The data shown were determined using NS3-4A of the 1b genotype and represent the steady-state data points fit to a line. Similar results were observed with NS3-4A (1a). This data are the average of three experiments and the error values represent standard deviation.
Mentions: NS3-4A Exhibits Robust Serine Protease Activity Under a Variety of Salt and pH Conditions—It has previously been reported that NS3-4A only functions as a robust serine protease under conditions of high salt (≥150 mm NaCl) and high pH (7.5–8.0) (17, 27). These same conditions are incompatible with those required for NS3 helicase activity (13, 17, 32), and it therefore has been suggested that the two activities (proteolysis and unwinding) might be mutually exclusive (17). We therefore sought to determine whether protease activity of the wild-type NS3-4A complex is restricted to the narrow range of previously reported conditions. To this end, we measured NS3-4A serine protease activity under a variety of pH (6.5 and 8.0) and salt conditions (30, 75, and 200 mm NaCl) (Fig. 4). We did not observe significant differences in the active fraction of protein (i.e. the y intercept of the velocity plots) under these varying pH and salt conditions, particularly when accounting for error (Fig. 4 legend). The steady-state proteolysis velocities in 30 mm NaCl at pH 6.5 and 8.0 were observed to be 0.006 ± 0.001 and 0.019 ± 0.001 μm/s, respectively. In 200 mm NaCl at pH 6.5 and 8.0, the steady-state proteolysis velocities were observed to be 0.010 ± 0.001 μm/s in both cases. Therefore, native, full-length NS3-4A is a robust serine protease under a broad range of salt and pH conditions, including those that are compatible with helicase activity.

Bottom Line: Our results indicate that the helicase domain enhances serine protease activity, just as the protease domain enhances helicase activity.This is the first time that such a complete interdependence has been demonstrated for a multifunctional, single chain enzyme.NS3-4A domain interdependence has important implications for function during the viral lifecycle as well as for the design of inhibitor screens that target the NS3-4A protease.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.

ABSTRACT
Non-structural protein 3 (NS3) is a multifunctional enzyme possessing serine protease, NTPase, and RNA unwinding activities that are required for hepatitis C viral (HCV) replication. HCV non-structural protein 4A (NS4A) binds to the N-terminal NS3 protease domain to stimulate NS3 serine protease activity. In addition, the NS3 protease domain enhances the RNA binding, ATPase, and RNA unwinding activities of the C-terminal NS3 helicase domain (NS3hel). To determine whether NS3hel enhances the NS3 serine protease activity, we purified truncated and full-length NS3-4A complexes and examined their serine protease activities under a variety of salt and pH conditions. Our results indicate that the helicase domain enhances serine protease activity, just as the protease domain enhances helicase activity. Thus, the two enzymatic domains of NS3-4A are highly interdependent. This is the first time that such a complete interdependence has been demonstrated for a multifunctional, single chain enzyme. NS3-4A domain interdependence has important implications for function during the viral lifecycle as well as for the design of inhibitor screens that target the NS3-4A protease.

Show MeSH
Related in: MedlinePlus