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Characterization of the Mechanism of Translocation and Unwinding of Hepatitis C Virus NS3 Helicase

by
Vaishnavi Rajagopal
Indian Institute of Technology
MS, 2003
Bombay, India

Thesis Advisor: Smita S. Patel, PhD
Graduate Program in Biochemistry & Molecular Biology

Research Building-School of Public Health
2nd floor Conference Room

Friday, December 12, 2008
10:30 am


Abstract

The hepatitis C virus (HCV) non-structural protein 3 (NS3) is a multi-domain, multi-functional enzyme carrying an N-terminal serine-protease activity and a C-terminal NTPase/Helicase activity. The two domains connected by a flexible linker can be expressed independently, with each retaining its individual function. The NS3 protease forms a non-covalent complex with the non-structural protein 4A (NS4A) and the heterodimeric NS3/4A complex, which constitutes the active protease, is involved in the viral poly-protein processing. Whilst the exact role of NS3 protease in viral replication has been clearly established, the role of its NTPase/helicase is still unclear. Thus, understanding the mechanism of the NTPase/helicase and its interaction with the protease domain can give insights into role of NS3 helicase in viral replication. In this study, two different constructs of the NS3 protein – the NS3 helicase domain and a single chain NS3-4A construct with its protease co-factor NS4A covalently attached to NS3 were characterized using thermodynamic, kinetic and structure-function approaches for their nucleic acid binding, ATPase, duplex unwinding and single-stranded translocation activities.

My study has characterized the enzymatic activities of the two constructs: (i) The dsDNA unwinding processivity of both is improved by the addition of single-strand binding protein; (ii) Increased stability of the duplex nucleic acid to be unwound compromises the enzymes’ unwinding rate and processivity; (iii) The ATPase activity of both is stimulated to a greater extent by ssRNA than by ssDNA; And (iv) Both translocate faster on ssRNA than on ssDNA. The biggest difference between the two constructs is that NS3-4A is capable of processive dsRNA unwinding while no significant dsRNA unwinding activity was detected for NS3h. NS3-4A unwinds dsDNA at a 10-fold slower rate than NS3h. NS3-4A also possesses a 5-fold lower kcat for polyU stimulated ATPase as compared to NS3h. However, NS3-4A possesses a 10-fold lower kcat for intrinsic hydrolysis than NS3h, indicating that NS3-4A undergoes lesser cycles of futile ATP hydrolysis. ATP hydrolysis is tightly coupled to translocation of NS3-4A on ssDNA and ssRNA substrates. While NS3-4A translocates 2 bases for every ATP hydrolyzed on ssRNA, NS3h hydrolyzes 2ATPs to translocate a single base on ssRNA and ~4 ATP to translocate a single base on ssDNA.

The presence of the protease domain not only enhances the nucleic acid binding and the unwinding processivity, but also tightly couples ATP hydrolysis to ss translocation, making NS3-4A an efficient motor. The fact that NS3-4A displays robust RNA translocation and unwinding activities could directly implicate the protein’s role as a potential replicative helicase involved in viral genome replication.


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