Domain 3 of non-structural protein 5A from hepatitis C virus is natively unfolded

https://doi.org/10.1016/j.bbrc.2009.02.108Get rights and content

Abstract

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is involved both in the viral replication and particle production. Its third domain (NS5A-D3), although not absolutely required for replication, is a key determinant for the production and assembly of novel HCV particles. As a prerequisite to elucidate the precise functions of this domain, we report here the first molecular characterization of purified recombinant HCV NS5A-D3. Sequence analysis indicates that NS5A-D3 is mostly unstructured but that short structural elements may exist at its N-terminus. Gel filtration chromatography, circular dichroism and finally NMR spectroscopy all point out the natively unfolded nature of purified recombinant NS5A-D3. This lack of stable folding is thought to be essential for primary interactions of NS5A-D3 domain with other viral or host proteins, which could stabilize some specific conformations conferring new functional features.

Introduction

Hepatitis C virus (HCV) is classified in the Hepacivirus genus within the Flaviviridae family. HCV infection often lead to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma [1]. With 120–180 million people persistently infected worldwide, the absence of a vaccine and limited efficacy of current drug treatments turn HCV into a serious health challenge. HCV is a small (+)RNA enveloped virus. The HCV viral genome (∼9.6 kb) is translated in a unique polyprotein of ∼3000 amino acids [2]. Its processing by viral encoded or host proteases, in a co- and post-translational way, leads to at least 10 different proteins. These viral proteins are classified into structural (Core, E1 and E2) and non-structural (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins. Non-structural HCV proteins are involved in the processing of the polyprotein precursor and in the viral replication. The minimal set of proteins required to achieve viral replication is NS3, NS4A, NS4B, NS5A and NS5B [3]. NS5A is a large (49 kDa) phospho-protein absolutely required for HCV replication and particle assembly [4], [5], [6], [7] but for which the precise function(s) remains to be elucidated. Up to date, no enzymatic activity has been detected for this viral protein. Recently, de Chassey et al. have reported a protein interaction network during HCV infection [8]. NS5A is the viral protein, following NS3, displaying the highest number of interactions with human proteins. NS5A is anchored to the ER membrane on its cytoplasmic side via an amphipathic N-terminal α-helix [5], [9]. Its cytoplasmic part is constituted by three domains, NS5A-D1, -D2 and -D3, that are connected by low-complexity sequences [10]. NS5A-D1, for which a X-ray structure has been solved [11], is a zinc-binding domain with RNA binding activity [12]. Sequences of NS5A-D2 and -D3 are significantly less conserved among the HCV genotypes than for -D1. Domain 2 of NS5A is required for HCV replication [7], [13] and was shown to be natively unfolded [14]. NS5A-D3 (residues 356–447) is dispensable for HCV RNA replication, but has an essential role for viral particle production and assembly [4], [15]. It has been proposed to be equally natively unfolded [6], [10], but despite its key role in HCV infection, no biochemical and structural data have been presented to underscore this feature. This study combines bioinformatics with experimental biochemical and biophysical tools to characterize, for the very first time, HCV NS5A-D3 at a molecular level. We report direct experimental evidence showing the disordered nature of isolated NS5A-D3.

Section snippets

Materials and methods

Sequence analysis. Sequence analyses were performed using tools available at the Institut de Biologie et Chimie des Protéines (IBCP) Network Protein Sequence Analysis (NPSA) website (http://npsa-pbil.ibcp.fr) [16]. HCV NS5A sequences were retrieved from the European HCV Database (http://euhcvdb.ibcp.fr/) [17]. Multiple-sequence alignments were performed with Clustal W. The repertoire of residues at each aa position and their frequencies observed in natural sequence variants were computed by the

Sequence analyses of NS5A domain 3

Sequence analyses and structure predictions were performed to assess the degree of conservation of the NS5A-D3 domain and to identify potential essential amino acids (aa) and motifs. The aa repertoire deduced from the analysis of 363 HCV isolates of genotype 1b revealed some aa strictly conserved in 50% of sequence positions (denoted by asterisks in Fig. 1), mainly in the 359–380 and 408–439 regions. The apparent variability of the central 381–407 region is however not that important at many

Acknowledgments

This work was supported by the French Centre National de la Recherche Scientifique and Universities of Lille and Lyon and a grant from the French National Agency for Research on AIDS and viral Hepatitis (ANRS). X. Hanoulle was supported by a fellowship from the ANRS. The NMR facility used in this study was funded by the Région Nord-Pas-de-Calais (France), the CNRS, the Universities of Lille1 and Lille2 and the Institut Pasteur de Lille.

The authors gratefully acknowledge RD-Biotech (Besançon,

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