Molecular and structural characterization of the domain 2 of hepatitis C virus non-structural protein 5A (original) (raw)
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Crystal Structure of a Novel Dimeric Form of NS5A Domain I Protein from Hepatitis C Virus
Journal of Virology, 2009
A new protein expression vector design utilizing an N-terminal six-histidine tag and tobacco etch virus protease cleavage site upstream of the hepatitis C virus NS5A sequence has resulted in a more straightforward purification method and improved yields of purified NS5A domain I protein. High-resolution diffracting crystals of NS5A domain I (amino acids 33 to 202) [NS5A(33-202)] were obtained by using detergent additive crystallization screens, leading to the structure of a homodimer which is organized differently from that published previously (T. L. Tellinghuisen, J. Marcotrigiano, and C. M. Rice, Nature 435:374-379, 2005) yet is consistent with a membrane association model for NS5A. The monomer-monomer interface of NS5A(33-202) features an extensive buried surface area involving the most-highly conserved face of each monomer. The two alternate structural forms of domain I now available may be indicative of the multiple roles emerging for NS5A in viral RNA replication and viral particle assembly.
Domain 2 of Nonstructural Protein 5A (NS5A) of Hepatitis C Virus Is Natively Unfolded †
Biochemistry, 2007
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 1 H, 13 C, and 15 N resonances, 3 J HNR 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
Biomol NMR Assign, 2011
Non-structural protein 5A (NS5A) plays an important role in the life cycle of hepatitis C virus. This proline-rich phosphoprotein is organized into three domains. Besides its role in virus replication and virus assembly, NS5A is involved in a variety of cellular regulation processes. Recent studies on domain 2 and 3 revealed that both belong to the class of intrinsically disordered proteins as they adopt a natively unfolded state. In particular, domain 2 together with its vicinal regions is responsible for NS5A's multiple interactions with other proteins necessary for virus persistence. The low chemical shift dispersion observed for instrinsically disordered proteins presents a challenge for NMR spectroscopy. Here we report sequential resonance assignment of a 179-residue fragment of NS5A, comprising the entire domain 2, using a set of sensitivity and resolution optimized 3D correlation experiments, as well as amino-acid-type editing in 1 H-15 N correlation spectra. Our assignment reveals the presence of several segments with high propensity to form a-helical structure that may be of importance to the function of this protein fragment as a versatile interaction platform.
The non-structural 5A protein of hepatitis C virus
Journal of Viral Hepatitis, 1999
The non-structural (NS)5A protein of hepatitis C virus (HCV) is cleaved, after translation, by the NS3-encoded zinc-dependent serine proteinase, from the NS4B protein upstream and the NS5B protein downstream. The released, mature NS5A protein is a 56 000 MW phosphoprotein (p56), which also exists within infected cells in a hyperphosphorylated form (p58). The NS5A gene has a quasispecies distribution, meaning that various NS5A sequences co-exist, in various proportions, in infected individuals. HCV NS5A appears to be located in cytoplasmic membranes surrounding the nucleus. Its precise functions are not known. HCV non-structural proteins, including NS5A, form a large multiprotein replication complex, which probably directs the replication of the HCV genome. HCV NS5A lacking the 146 N-terminal amino acids is a potent transcriptional activator in vitro. NS5A can also bind to single-strand RNA-dependent protein kinase (PKR) and inhibit its antiviral function. An 'interferon (IFN) sensitivity-determining region' has recently been postulated in the NS5A protein central region in hepatitis C virus (HCV) genotype 1b, but strongly conflicting evidence has been published. In fact, there would seem to be no such region in the NS5A protein, even though NS5A plays an important and complex role in HCV resistance to IFN. Structure-function studies are required to identify precisely how NS5A and IFN interact.
Pharmacological disruption of hepatitis C NS5A protein intra- and intermolecular conformations
Journal of General Virology, 2014
Non-structural 5A protein (NS5A) has emerged as an important pharmacological target for hepatitis C virus (HCV). However, little is known about the conformation of NS5A intracellularly or how NS5A inhibitors achieve the picomolar (pM) inhibition of virus replication. Here, we have presented two structurally related small molecules, one that potently inhibits HCV replication and selects for resistance in NS5A, and another that is inactive. Resistance to this antiviral was greater in genotype 1a than in genotype 1b replicons and mapped to domain 1 of NS5A. Using a novel cell-based assay that measures the intracellular proximity of fluorescent tags covalently attached to NS5A, we showed that only the active antiviral specifically disrupted the close proximity of inter-and intramolecular positions of NS5A. The active antiviral, termed compound 1, caused a repositioning of both the N and C termini of NS5A, including disruption of the close approximation of the N termini of two different NS5A molecules in a multimolecular complex. These data provide the first study of how antivirals that select resistance in domain 1 of NS5A alter the cellular conformation of NS5A. This class of antiviral disrupts the close proximity of the N termini of domain 1 in a NS5A complex but also alters the conformation of domain 3, and leads to large aggregates of NS5A. Current models predict that a multicomponent cocktail of antivirals is needed to treat HCV infection, so a mechanistic understanding of what each component does to the viral machinery will be important.
2013
25 26 NS5A has emerged as an important pharmacologic target for Hepatitis C Virus (HCV). Little is 27 known though about the conformation of NS5A intracellularly or how NS5A inhibitors achieve 28 the picomolar (pM) inhibition of viral replication (Gao 2010). 29 Here we present two structurally related small molecules, one that potently inhibits HCV 30 replication and selects for resistance in NS5A and the other that is inactive. Resistance to this 31 antiviral is greater in genotype 1a replicons than genotype 1b and maps to domain 1 of NS5A. 32 Using a novel cell based assay that measures the intracellular proximity of fluorescent tags 33 covalently attached to NS5A we show only the active antiviral specifically disrupts the close 34 proximity of interand intra-molecular positions of NS5A. The active antiviral compound 1 35 causes a repositioning of both the amino and carboxy termini of NS5A including a disruption of 36 the close approximation of the amino termini of two different N...
An Integrative In Silico Model of Hepatitis C Virus Non-structural 5a Protein
A model for the non-structural 5a (NS5A) protein of Hepatitis-C virus is proposed. It is a hub promiscuous multifunctional protein with roles in virus replication and host interactions. The 3D-structure for domainII was predicted based on, the Homo Sapiens Replication factor-A protein-1 (RPA1), as a template using consensus meta-servers results. DomainIII is an intrinsically unstructured domain (61% intrinsically-disorder regions) that lacks a unique 3Dstructure and participates in diverse protein interactions in different situations. It also has a single-stranded DNAbinding protein motif (SSDP) signature for pyrimidine binding during viral replication. Two protein binding motifs with high sequence conservation and disordered regions are proposed; the first corresponds to an Interleukin-8B receptor signature (IL-8R-B), while the second has a lymphotoxin beta receptor (LTβR) high local similarity. A mechanism is proposed to their contribution to NS5A Interferon signaling pathway interception. Lastly, the overlapping between LTβR and SSDP is considered as a sign for NS5A date hubs.
An integrative in silico model of Hepatitis C Virus nonstructural 5a protein.pdf
A model for the non-structural 5a (NS5A) protein of Hepatitis-C virus is proposed. It is a hub promiscuous multifunctional protein with roles in virus replication and host interactions. The 3D-structure for domainII was predicted based on, the Homo Sapiens Replication factor-A protein-1 (RPA1), as a template using consensus meta-servers results. DomainIII is an intrinsically unstructured domain (61% intrinsically-disorder regions) that lacks a unique 3Dstructure and participates in diverse protein interactions in different situations. It also has a single-stranded DNAbinding protein motif (SSDP) signature for pyrimidine binding during viral replication. Two protein binding motifs with high sequence conservation and disordered regions are proposed; the first corresponds to an Interleukin-8B receptor signature (IL-8R-B), while the second has a lymphotoxin beta receptor (LTβR) high local similarity. A mechanism is proposed to their contribution to NS5A Interferon signaling pathway interception. Lastly, the overlapping between LTβR and SSDP is considered as a sign for NS5A date hubs.
PLoS Pathogens, 2008
Persistent infection with the hepatitis C virus (HCV) is a major risk factor for the development of liver cirrhosis and hepatocellular carcinoma. With an estimated about 3% of the world population infected with this virus, the lack of a prophylactic vaccine and a selective therapy, chronic hepatitis C currently is a main indication for liver transplantation. The establishment of cell-based replication and virus production systems has led to first insights into the functions of HCV proteins. However, the role of nonstructural protein 5A (NS5A) in the viral replication cycle is so far not known. NS5A is a membrane-associated RNA-binding protein assumed to be involved in HCV RNA replication. Its numerous interactions with the host cell suggest that NS5A is also an important determinant for pathogenesis and persistence. In this study we show that NS5A is a key factor for the assembly of infectious HCV particles. We specifically identify the C-terminal domain III as the primary determinant in NS5A for particle formation. We show that both core and NS5A colocalize on the surface of lipid droplets, a proposed site for HCV particle assembly. Deletions in domain III of NS5A disrupting this colocalization abrogate infectious particle formation and lead to an enhanced accumulation of core protein on the surface of lipid droplets. Finally, we show that mutations in NS5A causing an assembly defect can be rescued by trans-complementation. These data provide novel insights into the production of infectious HCV and identify NS5A as a major determinant for HCV assembly. Since domain III of NS5A is one of the most variable regions in the HCV genome, the results suggest that viral isolates may differ in their level of virion production and thus in their level of fitness and pathogenesis.