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Journal of Virology, September 2007, p. 8905-8918, Vol. 81, No. 17
0022-538X/07/$08.00+0     doi:10.1128/JVI.00937-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The C Terminus of Hepatitis C Virus NS4A Encodes an Electrostatic Switch That Regulates NS5A Hyperphosphorylation and Viral Replication

Brett D. Lindenbach,^1,* Béla M. Prágai,^1, Roland Montserret,² Rudolf K. F. Beran,³ Anna M. Pyle,³ François Penin,² and Charles M. Rice^1*

Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Ave., New York, New York 10021,¹ Institut de Biologie et Chimie des Protéines, CNRS-UMR 5086, IFR128 BioSciences Gerland-Lyon Sud, University of Lyon, 7 Passage de Vercors, Lyon F-69367, Cedex 07, France,² Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520³

Received 1 May 2007/ Accepted 14 June 2007

Hepatitis C virus (HCV) nonstructural protein 4A (NS4A) is only 54 amino acids (aa) in length, yet it is a key regulator of the essential serine protease and RNA helicase activities of the NS3-4A complex, as well as a determinant of NS5A phosphorylation. Here we examine the structure and function of the C-terminal acidic region of NS4A through site-directed mutagenesis of a Con1 subgenomic replicon and through biophysical characterization of a synthetic peptide corresponding to this region. Our genetic studies revealed that in 8 of the 15 C-terminal residues of NS4A, individual Ala substitutions or charge reversal substitutions led to severe replication phenotypes, as well as decreased NS5A hyperphosphorylation. By selecting for replication-competent mutants, several second-site changes in NS3 were identified and shown to suppress these defects in replication and NS5A hyperphosphorylation. Circular-dichroism spectroscopy and nuclear magnetic resonance spectroscopy on a peptide corresponding to the C-terminal 19 aa of NS4A revealed that this region can adopt an alpha-helical conformation, but that this folding requires neutralization of a cluster of acidic residues. Taken together, these data suggest that the C terminus of NS4A acts as a dynamic regulator of NS3-4A interaction, NS5A hyperphosphorylation, and HCV replicase activity.

Published ahead of print on 20 June 2007.

Present address: Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06536.

Permanent address: Department of Microbiology, Albert Szent-Györgyi Medical University, Szeged, Hungary.

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