Work

Analysis of mechanisms of the rabies virus P protein-nucleocapsid interaction using engineered N-protein peptides and potential applications in antivirals design

Year: 2024

Type: article

Abstract: The Phosphoprotein (P protein) of the rabies virus has multiple roles in virus replication. A critical function is to act as a cofactor in genome replication and mRNA production through binding via its N-terminal region to the L protein, the essential enzyme for mRNA and genome synthesis/processing, and via its C-terminal domain (P CTD ) to the N protein and viral RNA (N-RNA) ribonucleoprotein complex. The binding site of the P CTD on the N protein is a disordered loop that is expected to be phosphorylated at Ser389. This interface may provide novel targets for antiviral approaches. Following an alanine scan of the peptide we selected two single site mutations that showed improved affinity and combined these mutations with a phosphomimetic (S389E) to produce double and triple mutants in the context of linear and cyclic peptides of the disordered loop, with the goal of generating a competitive peptide against the N-RNA complex. To assess the binding properties of the peptides we characterized their thermodynamics identifying complex properties of improved enthalpy but with compensating entropy for mutants and cyclized peptides. Nevertheless, a triple mutant shows 3.5-fold stronger affinity for P CTD than the full-length S389E N protein. Structural characterization of the triple mutant suggests the improved affinity may be due to trapping a favoured β-strand structure for binding to the P CTD . This novel peptide may serve as a template for the future design of antivirals. • Development of peptides based on the P protein-binding loop of the RABV N protein. • Engineered peptides based on the N protein loop can bind more strongly to the P protein than to full-length N protein. • Cyclic and mutant peptides reveal mechanistic details of the P protein-nucleocapsid interface, a potential antiviral target. • The improved binding properties of the engineered peptides provide templates and tools for design of antivirals, against RABV.

Source: Antiviral Research

DOI: 10.1016/j.antiviral.2024.106075