Abstract

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.

Highlights

  • Hepatitis C virus (HCV), first discovered in 1989, is a member of the Flaviviridae family of enveloped, positive-strand RNA viruses [1,2]

  • Formation of the host membrane associated replication complexes appears to be a common property for RNA viruses such as HCV [74,75]

  • Currently little is known about the structure of the complexes, it is anticipated that studies of RNA virus replication machineries will have a critical impact on antiviral drug development due to their specific roles in virus replication [74,75]

Read more

Summary

Introduction

Hepatitis C virus (HCV), first discovered in 1989, is a member of the Flaviviridae family of enveloped, positive-strand RNA viruses [1,2] It is the major causative agent of non-A, non-B hepatitis and about 200 million people are infected with HCV worldwide [3]. Replication of HCV initiates immediately after translation and processing of the viral protein and all of HCV gene products remain associated with intracellular membranes [11,12,13,14,15,16,17]. The membrane-associated replication machinery copies the genome RNA into a negativestrand intermediate, which is used to generate additional positive-strand RNAs for subsequent rounds of translation and packaging into virus particles. HCV nonstructural proteins including NS3, NS4A, NS4B, NS5A, and NS5B appear to be the key components of the RNA replication machinery but the exact details are poorly understood, such as the identities of the host factors and detailed interactions among them

Methods
Results
Conclusion
Full Text
Paper version not known

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.