Biochemical and Biophysical Research Communications
Domain 3 of non-structural protein 5A from hepatitis C virus is natively unfolded
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,
References (29)
- et al.
Structure and function of the membrane anchor domain of hepatitis C virus nonstructural protein 5A
J. Biol. Chem.
(2004) - et al.
An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A
J. Biol. Chem.
(2002) - et al.
The NS5A protein of hepatitis C virus is a zinc metalloprotein
J. Biol. Chem.
(2004) - et al.
Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein
J. Biol. Chem.
(2005) - et al.
NPS@: network protein sequence analysis
Trends Biochem. Sci.
(2000) - et al.
In vitro asymmetric binding of the pleiotropic regulatory protein, FruR, to the ace operator controlling glyoxylate shunt enzyme synthesis
J. Biol. Chem.
(1994) Intrinsically unstructured proteins
Trends Biochem. Sci.
(2002)- NIH, Consensus Development Conference statement. Management of hepatitis C, Hepatology 36 (2002)...
- et al.
Replication of hepatitis C virus
Nat. Rev. Microbiol.
(2007) - et al.
Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line
Science
(1999)
Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly
PLoS Pathog.
Structural biology of hepatitis C virus
Hepatology
Identification of residues required for RNA replication in domains II and III of the hepatitis C virus NS5A protein
J. Virol.
Hepatitis C virus infection protein network
Mol. Syst. Biol.
Cited by (75)
Intrinsically disordered proteins of viruses: Involvement in the mechanism of cell regulation and pathogenesis
2020, Progress in Molecular Biology and Translational ScienceCitation Excerpt :NS5A is a membrane-associated protein that has both disordered and ordered regions, an anchor attaches its N-terminal region to the membrane, but its cytoplasmic regions are mostly disordered and contain three domains. Among these three domains, domain I (D1) is highly conserved and has ordered sequence,252 while domain II (D2) and III (D3) are highly disordered and less conserved.253,254 Promiscuity of NS5A is well studied, and some of the interactions that involve its disordered domain have been identified.255
Cyclophilin A allows the allosteric regulation of a structural motif in the disordered domain 2 of NS5A and thereby fine-tunes HCV RNA replication
2019, Journal of Biological ChemistryNMR reveals the intrinsically disordered domain 2 of NS5A protein as an allosteric regulator of the hepatitis C virus RNA polymerase NS5
2017, Journal of Biological ChemistryVirology and Pathogenesis of Hepatitis C
2017, Zakim and Boyer's Hepatology: A Textbook of Liver DiseaseThe disordered region of the HCV protein NS5A: Conformational dynamics, SH3 binding, and phosphorylation
2015, Biophysical JournalCitation Excerpt :Together with biochemical data on RNA binding to NS5A (22), this led to the hypothesis that one function of NS5A is the translocation of the viral RNA from the site of replication to the site of particle assembly (23). The remainder of the NS5A protein, made up of domains 2 (D2) and 3 (D3), is intrinsically disordered as demonstrated both experimentally (24,25) and by computational analysis of the intrinsic disorder in the HCV genome (11). In addition, it has been shown that D2 is required for RNA replication (26) while domain 3 is indispensable for particle assembly (27).
A proline-tryptophan turn in the intrinsically disordered domain 2 of NS5A protein is essential for hepatitis C Virus RNA Replication
2015, Journal of Biological Chemistry