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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

Composition and purification of NS3-4A. The NS3-4A complex organization and construct design are illustrated schematically (A and B). In panel A, pro refers to the serine protease domain and the Roman numerals indicate the respective NS3 helicase subdomains. The regions where ATP, RNA, and the NS4A co-factor bind are indicated as well. The protein construct expressed in E. coli is depicted in panel B. The numbers below the map refer to the HCV polyprotein numbering of the amino acids of NS3-4A. In panel C, His-SUMO-NS3 and His-SUMO-NS4A fusion construct designs are presented. These proteins were purified separately and their His-SUMO tags were removed using the same methods as for NS3-4A. Subsequently, the native NS3 or native NS3 protease domain was reconstituted with native NS4A (see “Experimental Procedures”).
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fig1: Composition and purification of NS3-4A. The NS3-4A complex organization and construct design are illustrated schematically (A and B). In panel A, pro refers to the serine protease domain and the Roman numerals indicate the respective NS3 helicase subdomains. The regions where ATP, RNA, and the NS4A co-factor bind are indicated as well. The protein construct expressed in E. coli is depicted in panel B. The numbers below the map refer to the HCV polyprotein numbering of the amino acids of NS3-4A. In panel C, His-SUMO-NS3 and His-SUMO-NS4A fusion construct designs are presented. These proteins were purified separately and their His-SUMO tags were removed using the same methods as for NS3-4A. Subsequently, the native NS3 or native NS3 protease domain was reconstituted with native NS4A (see “Experimental Procedures”).

Mentions: NS3-4A is a multifunctional enzyme with serine protease and helicase activities that are essential for hepatitis C viral (HCV)3 replication (1–3). The NS3 N-terminal domain adopts a chymotrypsin-like fold, and it displays serine protease activity in the presence of the NS4A co-factor protein (4, 5) (Fig. 1A). The genomic HCV RNA is initially translated into a long polyprotein that must be cleaved into the core, envelope, and nonstructural (NS) proteins, which are required for viral replication and packaging. The NS3-4A protease is essential for this process, as it cleaves several sites within the HCV polyprotein (4). It may also enhance HCV replication by cleaving host proteins such as Cardif (also known as IPS-1, MAVS, or VISA), which are involved in innate immunity (6–9). In this way, NS3-4A may down-regulate the cellular antiviral response.


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

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

Composition and purification of NS3-4A. The NS3-4A complex organization and construct design are illustrated schematically (A and B). In panel A, pro refers to the serine protease domain and the Roman numerals indicate the respective NS3 helicase subdomains. The regions where ATP, RNA, and the NS4A co-factor bind are indicated as well. The protein construct expressed in E. coli is depicted in panel B. The numbers below the map refer to the HCV polyprotein numbering of the amino acids of NS3-4A. In panel C, His-SUMO-NS3 and His-SUMO-NS4A fusion construct designs are presented. These proteins were purified separately and their His-SUMO tags were removed using the same methods as for NS3-4A. Subsequently, the native NS3 or native NS3 protease domain was reconstituted with native NS4A (see “Experimental Procedures”).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Composition and purification of NS3-4A. The NS3-4A complex organization and construct design are illustrated schematically (A and B). In panel A, pro refers to the serine protease domain and the Roman numerals indicate the respective NS3 helicase subdomains. The regions where ATP, RNA, and the NS4A co-factor bind are indicated as well. The protein construct expressed in E. coli is depicted in panel B. The numbers below the map refer to the HCV polyprotein numbering of the amino acids of NS3-4A. In panel C, His-SUMO-NS3 and His-SUMO-NS4A fusion construct designs are presented. These proteins were purified separately and their His-SUMO tags were removed using the same methods as for NS3-4A. Subsequently, the native NS3 or native NS3 protease domain was reconstituted with native NS4A (see “Experimental Procedures”).
Mentions: NS3-4A is a multifunctional enzyme with serine protease and helicase activities that are essential for hepatitis C viral (HCV)3 replication (1–3). The NS3 N-terminal domain adopts a chymotrypsin-like fold, and it displays serine protease activity in the presence of the NS4A co-factor protein (4, 5) (Fig. 1A). The genomic HCV RNA is initially translated into a long polyprotein that must be cleaved into the core, envelope, and nonstructural (NS) proteins, which are required for viral replication and packaging. The NS3-4A protease is essential for this process, as it cleaves several sites within the HCV polyprotein (4). It may also enhance HCV replication by cleaving host proteins such as Cardif (also known as IPS-1, MAVS, or VISA), which are involved in innate immunity (6–9). In this way, NS3-4A may down-regulate the cellular antiviral response.

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