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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

NS3-4A purifies as two separable proteins. A, purified proteins were subjected to denaturing electrophoresis on a 4–12% gradient gel. Purified NS3 (lane 2), NS3-4A (lane 3), and NS3/4A polyprotein (lane 4) were subjected to electrophoresis side by side for comparison of mobilities. The band shown in lane 3 represents the NS3 component of a native, fully cleaved NS3-4A preparation. The band shown in lane 4 represents an NS3/4A polyprotein preparation produced by mutating the Thr/Ser cleavage site between NS3 and NS4A to a non-cleavable sequence (AA). In panels B and C, anti-NS3 and anti-NS4A Western blot analysis confirm the identity of our purified proteins (see “Experimental Procedures”). Panel B depicts an anti-NS3 Western blot. In panel B, lane 1 contains purified NS3, lane 2 contains purified, full-length NS3-4A, and lane 3 contains purified NS3/4A polyprotein. Truncated forms of NS3 are visible below the full-length protein in each lane in the anti-NS3 blot. These truncated forms of NS3 are likely produced during bacterial expression as well as during the multiday purification performed in the absence of protease inhibitors. These truncated forms could represent either N- or C-terminal truncations of NS3 as the monoclonal anti-NS3 antibody binds to the central region of the helicase domain. Panel C depicts an anti-NS4A Western blot. In panel C, lane 1 contains purified NS3, lane 2 contains purified, full-length NS3-4A, and lane 3 contains purified NS3/4A polyprotein. Truncated forms of the NS3/4A polyprotein are visible below the full-length polyprotein in the anti-NS4A blot. These truncated forms of NS3/4A polyprotein likely represent N-terminal degraded NS3/4A as the monoclonal anti-NS4A antibody binds the final 11 C-terminal residues of NS4A.
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fig2: NS3-4A purifies as two separable proteins. A, purified proteins were subjected to denaturing electrophoresis on a 4–12% gradient gel. Purified NS3 (lane 2), NS3-4A (lane 3), and NS3/4A polyprotein (lane 4) were subjected to electrophoresis side by side for comparison of mobilities. The band shown in lane 3 represents the NS3 component of a native, fully cleaved NS3-4A preparation. The band shown in lane 4 represents an NS3/4A polyprotein preparation produced by mutating the Thr/Ser cleavage site between NS3 and NS4A to a non-cleavable sequence (AA). In panels B and C, anti-NS3 and anti-NS4A Western blot analysis confirm the identity of our purified proteins (see “Experimental Procedures”). Panel B depicts an anti-NS3 Western blot. In panel B, lane 1 contains purified NS3, lane 2 contains purified, full-length NS3-4A, and lane 3 contains purified NS3/4A polyprotein. Truncated forms of NS3 are visible below the full-length protein in each lane in the anti-NS3 blot. These truncated forms of NS3 are likely produced during bacterial expression as well as during the multiday purification performed in the absence of protease inhibitors. These truncated forms could represent either N- or C-terminal truncations of NS3 as the monoclonal anti-NS3 antibody binds to the central region of the helicase domain. Panel C depicts an anti-NS4A Western blot. In panel C, lane 1 contains purified NS3, lane 2 contains purified, full-length NS3-4A, and lane 3 contains purified NS3/4A polyprotein. Truncated forms of the NS3/4A polyprotein are visible below the full-length polyprotein in the anti-NS4A blot. These truncated forms of NS3/4A polyprotein likely represent N-terminal degraded NS3/4A as the monoclonal anti-NS4A antibody binds the final 11 C-terminal residues of NS4A.

Mentions: The NS3-4A complex eluted between fractions 45 and 55, presumably in a detergent micelle (17). Fractions containing NS3-4A were identified using the RET-S1 protease assay (the RET-S1 protease assay details are described later in this section) as well as through SDS-PAGE analysis. Moreover, anti-NS3 and anti-NS4A (monoclonal antibodies in both cases) Western blotting were used to confirm the identity of the purified protein complex, which was then used for experimentation (see Fig. 2, B and C, and the Western blotting procedure described later in this section).


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

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

NS3-4A purifies as two separable proteins. A, purified proteins were subjected to denaturing electrophoresis on a 4–12% gradient gel. Purified NS3 (lane 2), NS3-4A (lane 3), and NS3/4A polyprotein (lane 4) were subjected to electrophoresis side by side for comparison of mobilities. The band shown in lane 3 represents the NS3 component of a native, fully cleaved NS3-4A preparation. The band shown in lane 4 represents an NS3/4A polyprotein preparation produced by mutating the Thr/Ser cleavage site between NS3 and NS4A to a non-cleavable sequence (AA). In panels B and C, anti-NS3 and anti-NS4A Western blot analysis confirm the identity of our purified proteins (see “Experimental Procedures”). Panel B depicts an anti-NS3 Western blot. In panel B, lane 1 contains purified NS3, lane 2 contains purified, full-length NS3-4A, and lane 3 contains purified NS3/4A polyprotein. Truncated forms of NS3 are visible below the full-length protein in each lane in the anti-NS3 blot. These truncated forms of NS3 are likely produced during bacterial expression as well as during the multiday purification performed in the absence of protease inhibitors. These truncated forms could represent either N- or C-terminal truncations of NS3 as the monoclonal anti-NS3 antibody binds to the central region of the helicase domain. Panel C depicts an anti-NS4A Western blot. In panel C, lane 1 contains purified NS3, lane 2 contains purified, full-length NS3-4A, and lane 3 contains purified NS3/4A polyprotein. Truncated forms of the NS3/4A polyprotein are visible below the full-length polyprotein in the anti-NS4A blot. These truncated forms of NS3/4A polyprotein likely represent N-terminal degraded NS3/4A as the monoclonal anti-NS4A antibody binds the final 11 C-terminal residues of NS4A.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2573085&req=5

fig2: NS3-4A purifies as two separable proteins. A, purified proteins were subjected to denaturing electrophoresis on a 4–12% gradient gel. Purified NS3 (lane 2), NS3-4A (lane 3), and NS3/4A polyprotein (lane 4) were subjected to electrophoresis side by side for comparison of mobilities. The band shown in lane 3 represents the NS3 component of a native, fully cleaved NS3-4A preparation. The band shown in lane 4 represents an NS3/4A polyprotein preparation produced by mutating the Thr/Ser cleavage site between NS3 and NS4A to a non-cleavable sequence (AA). In panels B and C, anti-NS3 and anti-NS4A Western blot analysis confirm the identity of our purified proteins (see “Experimental Procedures”). Panel B depicts an anti-NS3 Western blot. In panel B, lane 1 contains purified NS3, lane 2 contains purified, full-length NS3-4A, and lane 3 contains purified NS3/4A polyprotein. Truncated forms of NS3 are visible below the full-length protein in each lane in the anti-NS3 blot. These truncated forms of NS3 are likely produced during bacterial expression as well as during the multiday purification performed in the absence of protease inhibitors. These truncated forms could represent either N- or C-terminal truncations of NS3 as the monoclonal anti-NS3 antibody binds to the central region of the helicase domain. Panel C depicts an anti-NS4A Western blot. In panel C, lane 1 contains purified NS3, lane 2 contains purified, full-length NS3-4A, and lane 3 contains purified NS3/4A polyprotein. Truncated forms of the NS3/4A polyprotein are visible below the full-length polyprotein in the anti-NS4A blot. These truncated forms of NS3/4A polyprotein likely represent N-terminal degraded NS3/4A as the monoclonal anti-NS4A antibody binds the final 11 C-terminal residues of NS4A.
Mentions: The NS3-4A complex eluted between fractions 45 and 55, presumably in a detergent micelle (17). Fractions containing NS3-4A were identified using the RET-S1 protease assay (the RET-S1 protease assay details are described later in this section) as well as through SDS-PAGE analysis. Moreover, anti-NS3 and anti-NS4A (monoclonal antibodies in both cases) Western blotting were used to confirm the identity of the purified protein complex, which was then used for experimentation (see Fig. 2, B and C, and the Western blotting procedure described later in this section).

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