Characterization and applications of a Crimean-Congo hemorrhagic fever virus nucleoprotein-specific Affimer: Inhibitory effects in viral replication and development of colorimetric diagnostic tests

PLoS Negl Trop Dis. 2020 Jun 3;14(6):e0008364. doi: 10.1371/journal.pntd.0008364. eCollection 2020 Jun.

Abstract

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is one of the most widespread medically important arboviruses, causing human infections that result in mortality rates of up to 60%. We describe the selection of a high-affinity small protein (Affimer-NP) that binds specifically to the nucleoprotein (NP) of CCHFV. We demonstrate the interference of Affimer-NP in the RNA-binding function of CCHFV NP using fluorescence anisotropy, and its inhibitory effects on CCHFV gene expression in mammalian cells using a mini-genome system. Solution of the crystallographic structure of the complex formed by these two molecules at 2.84 Å resolution revealed the structural basis for this interference, with the Affimer-NP binding site positioned at the critical NP oligomerization interface. Finally, we validate the in vitro application of Affimer-NP for the development of enzyme-linked immunosorbent and lateral flow assays, presenting the first published point-of-care format test able to detect recombinant CCHFV NP in spiked human and animal sera.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Viral / blood
  • Antigens, Viral / genetics
  • Colorimetry / methods*
  • Diagnostic Tests, Routine / methods*
  • Enzyme-Linked Immunosorbent Assay
  • Gene Expression
  • Hemorrhagic Fever Virus, Crimean-Congo / genetics
  • Hemorrhagic Fever Virus, Crimean-Congo / physiology*
  • Hemorrhagic Fever, Crimean / diagnosis*
  • Hemorrhagic Fever, Crimean / virology*
  • Humans
  • Immunoglobulin G / blood
  • Models, Molecular
  • Nucleoproteins / chemistry
  • Nucleoproteins / genetics
  • Protein Conformation
  • Virus Replication*

Substances

  • Antibodies, Viral
  • Antigens, Viral
  • Immunoglobulin G
  • Nucleoproteins

Grants and funding

This work was supported by an EU Marie Skłodowska-Curie Actions (MSCA, ec.europa.eu) Innovative Training Network (ITN): H2020- MSCA-ITN-2016, under grant No 721367 (to B. Álvarez-Rodríguez). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.