Dataset Information

C-Terminal Auto-Regulatory Motif of Hepatitis C Virus NS5B Interacts with Human VAPB-MSP to Form a Dynamic Replication Complex.

ABSTRACT: Hepatitis C virus (HCV) is a pathogen of global importance and nearly 200 million people are chronically infected with HCV. HCV is an enveloped single-stranded RNA virus, which is characteristic of the formation of the host membrane associated replication complex. Previous functional studies have already established that the human ER-anchored VAPB protein acts as a host factor to form a complex with HCV NS5A and NS5B, which may be established as a drug target. However, there is lacking of biophysical characterization of the structures and interfaces of the complex, partly due to the dynamic nature of the complex formation and dissociation, which is extensively involved in intrinsically-disordered domains. Here by an integrated use of domain dissection and NMR spectroscopy, for the first time we have successfully deciphered that the HCV NS5B utilizes its auto-regulatory C-linker to bind the VAPB-MSP domain to form a dynamic complex. This finding implies that the NS5B C-linker is capable of playing dual roles by a switch between the folded and disordered states. Interestingly, our previous and present studies together reveal that both HCV NS5A and NS5B bind to the MSP domains of the dimeric VAP with significantly overlapped interfaces and similar affinities. The identification that EphA2 and EphA5 bind to the MSP domain with higher affinity than EphA4 provides a biophysical basis for further exploring whether other than inducing ALS-like syndrome, the HCV infection might also trigger pathogenesis associated with signalling pathways mediated by EphA2 and EphA5.

SUBMITTER: Gupta G

PROVIDER: S-EPMC4718513 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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C-Terminal Auto-Regulatory Motif of Hepatitis C Virus NS5B Interacts with Human VAPB-MSP to Form a Dynamic Replication Complex.

Gupta Garvita G Song Jianxing J

PloS one 20160119 1

Hepatitis C virus (HCV) is a pathogen of global importance and nearly 200 million people are chronically infected with HCV. HCV is an enveloped single-stranded RNA virus, which is characteristic of the formation of the host membrane associated replication complex. Previous functional studies have already established that the human ER-anchored VAPB protein acts as a host factor to form a complex with HCV NS5A and NS5B, which may be established as a drug target. However, there is lacking of biophy ...[more]

PMID: 26784321

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Project description:Hepatitis C virus (HCV) affects nearly 200 million people worldwide and is a leading factor for serious chronic liver diseases. For replicating HCV genome, the membrane-associated replication machinery needs to be formed by both HCV non-structural proteins including NS5A and human host factors. Recently NS5A has been identified to bind ER-anchored human VAP proteins and consequently this interaction may serve as a novel target for design of anti-HCV drugs. So far no biophysical characterization of this interaction has been reported. Here, we dissected the 243-residue VAPB into 4 and 447-residue NS5A into 10 fragments, followed by CD and NMR characterization of their structural properties. Subsequently, binding interactions between these fragments have been extensively assessed by NMR HSQC titration which is very powerful in detecting even very weak binding. The studies lead to three important findings: 1). a "fuzzy complex" is formed between the intrinsically-unstructured third domain (D3) of NS5A and the well-structured MSP domain of VAPB, with an average dissociation constant (Kd) of ~5 µM. 2). The binding-important residues on both NS5A-D3 and VAPB-MSP have been successfully mapped out, which provided experimental constraints for constructing the complex structure. In the complex, unstructured D3 binds to three surface pockets on one side of the MSP structure. Interestingly, two ALS-causing mutations T46I and P56S are also located on the D3-MSP interface. Moreover, NS5A-D3, FFAT-containing proteins and EphA4 appear to have overlapped binding interfaces on the MSP domain. 3). NS5A-D3 has been experimentally confirmed to competes with EphA4 in binding to the MSP domain, and T46I mutation of MSP dramatically abolishes its binding ability to D3. Our study not only provides essential foundation for further deciphering structure and function of the HCV replication machinery, but may also shed light on rationalizing a recent observation that a chronic HCV patient surprisingly developed ALS-like syndrome.

Dataset Information

C-Terminal Auto-Regulatory Motif of Hepatitis C Virus NS5B Interacts with Human VAPB-MSP to Form a Dynamic Replication Complex.

Publications

C-Terminal Auto-Regulatory Motif of Hepatitis C Virus NS5B Interacts with Human VAPB-MSP to Form a Dynamic Replication Complex.

Similar Datasets

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