Nucleoprotein-phosphoprotein interactions in the assembly of respiratory syncytial virus nucleocapsids: deciphering the mechanisms and exploring antivirals development. – PneumoPEPS

Human respiratory syncytial virus (hRSV) infections primarily affect infants, the elderly, and immunocompromised patients. They are a major cause of morbidity and mortality worldwide and represent a significant social and economic burden. However, we still lack a complete understanding of the key molecular mechanisms of its replication. Although a new generation of monoclonal antibodies preventing severe hRSV infections in infants is now available there are still no treatment options for the vast majority of the population including vulnerable patients at risk of severe infections. The development of novel antivirals to treat hRSV infections, whether prophylactic and/or curative, remains a strong societal and medical need.
In PneumoPEPS, we focus on the encapsidation of viral RNA by the nucleoprotein (N) to form new nucleocapsids (NC), an essential step in the virus replication cycle. In this process, the production of unassembled RNA-free N (N0) requires interaction with the phosphoprotein (P), followed by its transfer and polymerization onto a newly synthesized RNA.
NC assembly involves protein-protein interactions involving two Short Linear Motifs (SliMs) that bind to overlapping surfaces of the N protein. One is located at the N-terminus of the N protein, called the N-terminal arm (NNT-ARM), and stabilizes the polymeric assembly of N by binding to the surface of the neighboring N protein. The other is located at the N-terminus of the P protein and acts as a molecular chaperone (PCHAP) for N0. During viral replication, PCHAP dissociates from N, the latter is transferred to a neosynthesized RNA where it assembles to the growing NC notably by the attachment of NNT-ARM on the last protein N already incorporated.
In this project, we formulate several hypotheses: (1) the assembly process of hRSV NCs is controlled by competition between PCHAP and NNT-ARM for binding N0; (2) interfering with either of these interactions blocks virus replication, (3) peptides with sequences corresponding to these SLiMs are natural inhibitors of these interactions; (4) The amino acid sequence of SLiMs involved in these interactions (in particular PCHAP) has not been naturally selected through virus evolution to confer the highest possible binding affinity for their targets. Instead, these sequences were optimised to support viral transcription and replication, which requires balancing the relative affinities of PCHAP and NNT-ARM for NCORE. The sequence of the interfering peptides or miniproteins (potential drug candidates) can thus be redesigned to increase this affinity.
Pneumopeps propose to:
(1) Use an integrative approach combining computational design, directed evolution and cell-based assays to identify high affinity peptides and miniproteins targeting the N protein (WP1).
(2) Select peptides with varying affinities for N and characterize their interactions with biophysical and structural methods (WP2).
(3) Evaluate the antiviral capabilities of the best candidates in cellular and mice infection assays (WP3).
We will thus test the hypothesis that the strength and dynamics of interactions between the SLiMs and N control NC assembly and generate a quantitative model of this process. We will demonstrate that interference with NC assembly blocks viral replication, building up on the initial hits identified in preliminary work. This research will pave the way for the development of antiviral peptides and miniproteins to treat hRSV infections.