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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 NS3-4A and His-NS3-4A. The y intercept of the line in each case corresponds to the active fraction of protein (see “Experimental Procedures”). For NS3-4A (solid circles), the active fraction was 75 ± 14%. For His-NS3-4A (hatched squares), the active fraction was 25 ± 12%. NS3/4A polyprotein did not display any measurable serine protease activity (solid squares). The data shown are determined using proteins of the 1b genotype. Similar results were observed using NS3-4A(1a) and His-NS3-4A(1a). This data are the average of three experiments and the error values represent standard deviation.
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fig3: Steady-state proteolysis of RET-S1 by NS3-4A and His-NS3-4A. The y intercept of the line in each case corresponds to the active fraction of protein (see “Experimental Procedures”). For NS3-4A (solid circles), the active fraction was 75 ± 14%. For His-NS3-4A (hatched squares), the active fraction was 25 ± 12%. NS3/4A polyprotein did not display any measurable serine protease activity (solid squares). The data shown are determined using proteins of the 1b genotype. Similar results were observed using NS3-4A(1a) and His-NS3-4A(1a). This data are the average of three experiments and the error values represent standard deviation.

Mentions: To quantify the fraction of fully processed, active NS3-4A in the preparation, we analyzed the proteolysis of a depsipeptide substrate known as “RET-S1,” which was originally derived from the NS4A–NS4B cleavage junction (5). As expected (16), both free NS3 and the uncleavable mutant NS3/4A polyprotein are proteolytically inactive, as they fail to react with RET-S1 (see below). However, the wild-type NS3-4A preparation displays efficient protease activity against RET-S1, with a rate constant of 0.020 ± 0.001 μm product/s). This value is significantly faster than values obtained by others for NS3-4A with a non-native N terminus incubated with a 5AB peptide substrate (27). Importantly, burst kinetics experiments reveal that the NS3-4A preparation contains 75 ± 14% active enzyme (Fig. 3), indicating that the majority of the population is properly folded, assembled, and catalytically active. That this value falls short of 100% is consistent with the electrophoretic analyses, which indicated that ∼20% of the wild-type NS3-4A preparation remains uncleaved (Fig. 2). Taken together, the physical and functional information provided by both the electrophoretic and proteolysis assays indicate that we have successfully overexpressed untagged, unmodified NS3-4A in a form that is conformationally homogeneous, appropriately cleaved, and highly reactive. This approach now makes it possible to quantitatively assess the proteolysis and helicase activities of the NS3-4A complex, and to compare these activities with the behavior of other NS3 constructs.


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 NS3-4A and His-NS3-4A. The y intercept of the line in each case corresponds to the active fraction of protein (see “Experimental Procedures”). For NS3-4A (solid circles), the active fraction was 75 ± 14%. For His-NS3-4A (hatched squares), the active fraction was 25 ± 12%. NS3/4A polyprotein did not display any measurable serine protease activity (solid squares). The data shown are determined using proteins of the 1b genotype. Similar results were observed using NS3-4A(1a) and His-NS3-4A(1a). This data 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

fig3: Steady-state proteolysis of RET-S1 by NS3-4A and His-NS3-4A. The y intercept of the line in each case corresponds to the active fraction of protein (see “Experimental Procedures”). For NS3-4A (solid circles), the active fraction was 75 ± 14%. For His-NS3-4A (hatched squares), the active fraction was 25 ± 12%. NS3/4A polyprotein did not display any measurable serine protease activity (solid squares). The data shown are determined using proteins of the 1b genotype. Similar results were observed using NS3-4A(1a) and His-NS3-4A(1a). This data are the average of three experiments and the error values represent standard deviation.
Mentions: To quantify the fraction of fully processed, active NS3-4A in the preparation, we analyzed the proteolysis of a depsipeptide substrate known as “RET-S1,” which was originally derived from the NS4A–NS4B cleavage junction (5). As expected (16), both free NS3 and the uncleavable mutant NS3/4A polyprotein are proteolytically inactive, as they fail to react with RET-S1 (see below). However, the wild-type NS3-4A preparation displays efficient protease activity against RET-S1, with a rate constant of 0.020 ± 0.001 μm product/s). This value is significantly faster than values obtained by others for NS3-4A with a non-native N terminus incubated with a 5AB peptide substrate (27). Importantly, burst kinetics experiments reveal that the NS3-4A preparation contains 75 ± 14% active enzyme (Fig. 3), indicating that the majority of the population is properly folded, assembled, and catalytically active. That this value falls short of 100% is consistent with the electrophoretic analyses, which indicated that ∼20% of the wild-type NS3-4A preparation remains uncleaved (Fig. 2). Taken together, the physical and functional information provided by both the electrophoretic and proteolysis assays indicate that we have successfully overexpressed untagged, unmodified NS3-4A in a form that is conformationally homogeneous, appropriately cleaved, and highly reactive. This approach now makes it possible to quantitatively assess the proteolysis and helicase activities of the NS3-4A complex, and to compare these activities with the behavior of other NS3 constructs.

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