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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 proteolysis of RET-S1 by reconstituted NS3 + NS4A and NS3 protease domain + NS4A. The steady-state velocity for NS3 + NS4A is 0.005 ± 001 μm/s (pierced circles) and for NS3 protease ± NS4A is 0.001 ± 0.001 μm/s (solid squares). The y intercept of the fitted lines show NS3 + NS4A and NS3 protease domain + NS4A to have 75 ± 10% active fraction and 75 ± 12% active fraction, respectively. The data shown are the average of three experiments and the error values represent standard deviation.
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fig5: Steady-state proteolysis of RET-S1 by reconstituted NS3 + NS4A and NS3 protease domain + NS4A. The steady-state velocity for NS3 + NS4A is 0.005 ± 001 μm/s (pierced circles) and for NS3 protease ± NS4A is 0.001 ± 0.001 μm/s (solid squares). The y intercept of the fitted lines show NS3 + NS4A and NS3 protease domain + NS4A to have 75 ± 10% active fraction and 75 ± 12% active fraction, respectively. The data shown are the average of three experiments and the error values represent standard deviation.

Mentions: Protease Assays—Protease assays were performed at 37 °C using the Resonance Energy Transfer-S1 substrate (RET-S1) (Anaspec) designed by Taliani et al. (5). RET-S1 is an NS4A/NS4B junction mimic that fluoresces upon cleavage. All assays, unless otherwise indicated, were performed in 60-μl reaction volumes containing 40 nm NS3-4A and 5 μm RET-S1. The data shown in Fig. 6 and Table 1 were collected using 120 nm protein with the indicated amounts of RET-S1 substrate. In Table 1, the kcat values were calculated based upon the observation that 75% of the protein was active in each case (Fig. 5). The buffer conditions were the same as those normally used for helicase assays (13): 25 mm (pH 6.5), 3 mm MgCl2, 1% glycerol, 2 mm dithiothreitol, 30 mm NaCl, 0.2% (v/v) Triton X-100, unless otherwise indicated. Data were collected using a Cary Eclipse Spectrophotometer (Varian) with a temperature-controlled cuvette holder. The excitation wavelength was 350 nm and the emission wavelength was 440 nm. The initial background value (the value at time 0, just before substrate addition) was subtracted from all subsequent time points.


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

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

Steady-state proteolysis of RET-S1 by reconstituted NS3 + NS4A and NS3 protease domain + NS4A. The steady-state velocity for NS3 + NS4A is 0.005 ± 001 μm/s (pierced circles) and for NS3 protease ± NS4A is 0.001 ± 0.001 μm/s (solid squares). The y intercept of the fitted lines show NS3 + NS4A and NS3 protease domain + NS4A to have 75 ± 10% active fraction and 75 ± 12% active fraction, respectively. The data shown are the average of three experiments and the error values represent standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2573085&req=5

fig5: Steady-state proteolysis of RET-S1 by reconstituted NS3 + NS4A and NS3 protease domain + NS4A. The steady-state velocity for NS3 + NS4A is 0.005 ± 001 μm/s (pierced circles) and for NS3 protease ± NS4A is 0.001 ± 0.001 μm/s (solid squares). The y intercept of the fitted lines show NS3 + NS4A and NS3 protease domain + NS4A to have 75 ± 10% active fraction and 75 ± 12% active fraction, respectively. The data shown are the average of three experiments and the error values represent standard deviation.
Mentions: Protease Assays—Protease assays were performed at 37 °C using the Resonance Energy Transfer-S1 substrate (RET-S1) (Anaspec) designed by Taliani et al. (5). RET-S1 is an NS4A/NS4B junction mimic that fluoresces upon cleavage. All assays, unless otherwise indicated, were performed in 60-μl reaction volumes containing 40 nm NS3-4A and 5 μm RET-S1. The data shown in Fig. 6 and Table 1 were collected using 120 nm protein with the indicated amounts of RET-S1 substrate. In Table 1, the kcat values were calculated based upon the observation that 75% of the protein was active in each case (Fig. 5). The buffer conditions were the same as those normally used for helicase assays (13): 25 mm (pH 6.5), 3 mm MgCl2, 1% glycerol, 2 mm dithiothreitol, 30 mm NaCl, 0.2% (v/v) Triton X-100, unless otherwise indicated. Data were collected using a Cary Eclipse Spectrophotometer (Varian) with a temperature-controlled cuvette holder. The excitation wavelength was 350 nm and the emission wavelength was 440 nm. The initial background value (the value at time 0, just before substrate addition) was subtracted from all subsequent time points.

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