HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Paolini, C. Articles by Gallinari, P. Search for Related Content PubMed PubMed Citation Articles by Paolini, C. Articles by Gallinari, P. Agricola Articles by Paolini, C. Articles by Gallinari, P.

Mutational analysis of hepatitis C virus NS3-associated helicase

Istituto di Ricerche di Biologia Molecolare ‘P. Angeletti’ (IRBM), Via Pontina Km 30.600, 00040 Pomezia (Rome), Italy¹

Author for correspondence: Paola Gallinari. Fax +39 06 91093225. e-mail Gallinari{at}IRBM.it

Nonstructural protein 3 (NS3) of hepatitis C virus contains a bipartite structure consisting of an N-terminal serine protease and a C-terminal DEXH box helicase. To investigate the roles of individual amino acid residues in the overall mechanism of unwinding, a mutational–functional analysis was performed based on a molecular model of the NS3 helicase domain bound to ssDNA, which has largely been confirmed by a recently published crystal structure of the NS3 helicase–ssDNA complex. Three full-length mutated NS3 proteins containing Tyr(392)Ala, Val(432)Gly and Trp(501)Ala single substitutions, respectively, together with a Tyr(392)Ala/Trp(501)Ala double-substituted protein were expressed in Escherichia coli and purified to homogeneity. All individually mutated forms showed a reduction in duplex unwinding activity, single-stranded polynucleotide binding capacity and polynucleotide-stimulated ATPase activity compared to wild-type, though to different extents. Simultaneous replacement of both Tyr₃₉₂ and Trp₅₀₁ with Ala completely abolished all these enzymatic functions. On the other hand, the introduced amino acid substitutions had no influence on NS3 intrinsic ATPase activity and proteolytic efficiency. The results obtained with Trp(501)Ala and Val(432)Gly single-substituted enzymes are in agreement with a recently proposed model for NS3 unwinding activity. The mutant phenotype of the Tyr(392)Ala and Tyr(392)Ala/Trp(501)Ala enzymes, however, represents a completely novel finding.

This article has been cited by other articles:

				A. M. I. Lam and D. N. Frick Hepatitis C Virus Subgenomic Replicon Requires an Active NS3 RNA Helicase J. Virol., January 1, 2006; 80(1): 404 - 411. [Abstract] [Full Text] [PDF]

				D. N. Frick, R. S. Rypma, A. M. I. Lam, and C. M. Frenz Electrostatic analysis of the hepatitis C virus NS3 helicase reveals both active and allosteric site locations Nucleic Acids Res., October 12, 2004; 32(18): 5519 - 5528. [Abstract] [Full Text] [PDF]

				A. M. I. Lam, D. Keeney, and D. N. Frick Two Novel Conserved Motifs in the Hepatitis C Virus NS3 Protein Critical for Helicase Action J. Biol. Chem., November 7, 2003; 278(45): 44514 - 44524. [Abstract] [Full Text] [PDF]

				A. M. I. Lam, D. Keeney, P. Q. Eckert, and D. N. Frick Hepatitis C Virus NS3 ATPases/Helicases from Different Genotypes Exhibit Variations in Enzymatic Properties J. Virol., April 1, 2003; 77(7): 3950 - 3961. [Abstract] [Full Text] [PDF]

				J. W. Kim, M. Y. Seo, A. Shelat, C. S. Kim, T. W. Kwon, H.-h. Lu, D. T. Moustakas, J. Sun, and J. H. Han Structurally Conserved Amino Acid W501 Is Required for RNA Helicase Activity but Is Not Essential for DNA Helicase Activity of Hepatitis C Virus NS3 Protein J. Virol., December 6, 2002; 77(1): 571 - 582. [Abstract] [Full Text] [PDF]

				A. E. Matusan, M. J. Pryor, A. D. Davidson, and P. J. Wright Mutagenesis of the Dengue Virus Type 2 NS3 Protein within and outside Helicase Motifs: Effects on Enzyme Activity and Virus Replication J. Virol., October 15, 2001; 75(20): 9633 - 9643. [Abstract] [Full Text] [PDF]

				C.-L. Tai, W.-C. Pan, S.-H. Liaw, U.-C. Yang, L.-H. Hwang, and D.-S. Chen Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase J. Virol., September 1, 2001; 75(17): 8289 - 8297. [Abstract] [Full Text] [PDF]

				M. S. Dillingham, P. Soultanas, P. Wiley, M. R. Webb, and D. B. Wigley Defining the roles of individual residues in the single-stranded DNA binding site of PcrA helicase PNAS, July 17, 2001; 98(15): 8381 - 8387. [Abstract] [Full Text] [PDF]