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Journal of Virology, April 2009, p. 3268-3275, Vol. 83, No. 7
0022-538X/09/$08.00+0     doi:10.1128/JVI.01849-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The NS4A Protein of Hepatitis C Virus Promotes RNA-Coupled ATP Hydrolysis by the NS3 Helicase{triangledown}

Rudolf K. F. Beran,1 Brett D. Lindenbach,2 and Anna Marie Pyle1,3*

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520,1 Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut 06536,2 Howard Hughes Medical Institute, Chevy Chase, Maryland 208153

Received 3 September 2008/ Accepted 9 January 2009

Nonstructural protein 3 (NS3) is an essential replicative component of the hepatitis C virus (HCV) and a member of the DExH/D-box family of proteins. The C-terminal region of NS3 (NS3hel) exhibits RNA-stimulated NTPase and helicase activity, while the N-terminal serine protease domain of NS3 enhances RNA binding and unwinding by NS3hel. The nonstructural protein 4A (NS4A) binds to the NS3 protease domain and serves as an obligate cofactor for NS3 serine protease activity. Given its role in stimulating protease activity, we sought to determine whether NS4A also influences the activity of NS3hel. Here we show that NS4A enhances the ability of NS3hel to bind RNA in the presence of ATP, thereby acting as a cofactor for helicase activity. This effect is mediated by amino acids in the C-terminal acidic domain of NS4A. When these residues are mutated, one observes drastic reductions in ATP-coupled RNA binding and duplex unwinding by NS3. These same mutations are lethal in HCV replicons, thereby establishing in vitro and in vivo that NS4A plays an important role in the helicase mechanism of NS3 and its function in replication.

* Corresponding author. Mailing address: Department of Molecular Biophysics and Biochemistry, 266 Whitney Ave., Bass Bldg., Rm. 334, Yale University, New Haven, CT 06520. Phone: (203) 432-5633. Fax: (203) 432-5316. E-mail: anna.pyle{at}yale.edu

{triangledown} Published ahead of print on 19 January 2009.

Journal of Virology, April 2009, p. 3268-3275, Vol. 83, No. 7
0022-538X/09/$08.00+0     doi:10.1128/JVI.01849-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Phan, T., Beran, R. K. F., Peters, C., Lorenz, I. C., Lindenbach, B. D. (2009). Hepatitis C Virus NS2 Protein Contributes to Virus Particle Assembly via Opposing Epistatic Interactions with the E1-E2 Glycoprotein and NS3-NS4A Enzyme Complexes. J. Virol. 83: 8379-8395 [Abstract] [Full Text]  


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