Application of error-prone PCR to functionally probe the morbillivirus Haemagglutinin protein.

Giulia Gallo,Brian Willett,Christina Tsirigoti,Carina Conceicao,Dalan Bailey,Stephen C Graham

doi:10.1099/jgv.0.001580

Abstract

The enveloped morbilliviruses utilise conserved proteinaceous receptors to enter host cells: SLAMF1 or Nectin-4. Receptor binding is initiated by the viral attachment protein Haemagglutinin (H), with the viral Fusion protein (F) driving membrane fusion. Crystal structures of the prototypic morbillivirus measles virus H with either SLAMF1 or Nectin-4 are available and have served as the basis for improved understanding of this interaction. However, whether these interactions remain conserved throughout the morbillivirus genus requires further characterisation. Using a random mutagenesis approach, based on error-prone PCR, we targeted the putative receptor binding site for SLAMF1 interaction on peste des petits ruminants virus (PPRV) H, identifying mutations that inhibited virus-induced cell-cell fusion. These data, combined with structural modelling of the PPRV H and ovine SLAMF1 interaction, indicate this region is functionally conserved across all morbilliviruses. Error-prone PCR provides a powerful tool for functionally characterising functional domains within viral proteins.

Highlights

The enveloped morbilliviruses utilise conserved proteinaceous receptors to enter host cells: SLAMF1 or Nectin-4
A characteristic feature of these viruses is their conserved usage of proteinaceous receptors, SLAMF1 or Nectin-4, to enter cells, respectively
Recent work by our lab and others in the field has highlighted how the specificity of this receptor usage may influence the host-range of individual viruses. This has been led by experimental identification of mutations that confer additional receptor tropisms (D540G in CDV H [7]) as well as structure-g uided approaches to identify similar restrictions (R191P in peste des petits ruminants virus (PPRV) H [8]), mutations which are both within the receptor binding domain (RBD) of morbillivirus H

Summary

Introduction

The enveloped morbilliviruses utilise conserved proteinaceous receptors to enter host cells: SLAMF1 or Nectin-4. Our own work mutating key residues in the RBD ( position 191 within the intermolecular β-sheet interaction of site 3 [8, 9]), together with modelling of this interaction, indicates that the PPRV and ovine (sheep) SLAMF1 RBD is likely to be structurally similar, and important in determining host-range ([8], Fig. 1a).

Results

Conclusion