Domain 2 of Nonstructural Protein 5A (NS5A) of Hepatitis C Virus Is Natively Unfolded

Abstract

Nonstructural protein 5A protein (NS5A) of hepatitis C virus (HCV) plays an important role in the regulation of viral replication, interferon resistance, and apoptosis. HCV NS5A comprises three domains. Recently the structure of domain 1 has been determined, revealing a structural scaffold with a novel zinc-binding motif and a disulfide bond. At present, the structures of domains 2 and 3 remain undefined. Domain 2 of HCV NS5A (NS5A-D2) is important for functions of NS5A and involved in molecular interactions with its own NS5B and PKR, a cellular interferon-inducible serine/threonine specific protein kinase. In this study we performed structural analysis of domain 2 by multinuclear nuclear magnetic resonance (NMR) spectroscopy. The analysis of the backbone 1H, 13C, and 15N resonances, 3JHNalpha coupling constants ,and 3D NOE data indicates that NS5A-D2 lacks secondary structural elements and reveals characteristics of unfolded proteins. NMR relaxation parameters confirmed the lack of rigid structure in the domain. The absence of an ordered conformation and the observation of a highly dynamic behavior of NS5A-D2 may provide an underlying molecular basis on its physiological function to allow NS5A-D2 to interact with a variety of biological partners.

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

“…(D1), two additional domains (D2 and D3) that are mainly intrinsically disordered (23)(24)(25) and that we previously characterized by NMR spectroscopy (27,28). IDPs/IDRs exist as highly dynamic and interconverting conformers and thus escape to the rule "one three-dimensional structure, one function."…”

“…Crystallographic studies revealed that this domain could adopt at least three different homodimeric conformations (19 -21), underscoring the multifunctionality of the protein. By using several biophysical techniques such as NMR spectroscopy, circular dichroism, and gel filtration chromatography, NS5A-D2, which is essential for viral RNA replication (12), and NS5A-D3, which is involved in production and assembly of new virions (13,22), were shown to be mainly disordered (23)(24)(25). In this respect, NS5A is the HCV protein with the highest percentage of its primary sequence predicted to be disordered (26).…”

Hepatitis C Virus NS5A Protein Is a Substrate for the Peptidyl-prolyl cis/trans Isomerase Activity of Cyclophilins A and B

“…2A). As these anomalous chemical shifts were present equally in the genotype 1a NS5A-D2 domain (37), and as this Trp 312 -Ala 313 segment, as well as Pro 310 and Pro 315 , is fully conserved in all genotypes (Fig. 1A), we have synthesized a peptide centered on this motif and are currently pursuing a detailed NMR analysis to interpret the anomalous chemical shift values in structural terms.…”

“…NS5A-D2 and -D3, for which sequence conservation among HCV genotypes is significantly lower than for D1, have been proposed to be natively unfolded domains (28,32). Molecular and structural characterization of NS5A-D2 from HCV genotype 1a has confirmed the disordered nature of this domain (36,37).…”

A Synthetic Codon-Optimized Hepatitis C Virus Nonstructural 5A DNA Vaccine Primes Polyfunctional CD8+ T Cell Responses in Wild-Type and NS5A-Transgenic Mice

“…Domain I is essential for RNA repli-cation and has a zinc-binding motif, and the domain is a dimer as determined by crystallography (27). Domains II and III are less conserved and natively unfolded (28)(29)(30). Domain III is required for proper assembly of viral particles (31) but is not needed for RNA replication (32).…”