Investigating the Structure and Function of the Menangle Virus Replication Machinery

Abstract

The Paramyxoviruses infect many species including mammals, birds, reptiles, and fish and pose a significant health and economic burden worldwide. The well-known human diseases measles and mumps are caused by members of this large viral family, which also includes newly emergent zoonotic pathogens like Nipah virus. Despite the prevalence of these viruses, the molecular details of their replication remain unclear. Initiation of viral replication requires formation of the RNA-dependent RNA-polymerase complex (RdRp) and subsequent attachment of the RdRp to the replication template. This template is a helical protein-RNA complex, termed the nucleocapsid which is formed when the viral nucleocapsid protein (N protein) encapsidates the single-stranded RNA genome. The protein-protein interactions required for RNA synthesis are coordinated by the viral phosphoprotein (P protein) – the non-catalytic subunit of the RdRp. The P protein binds both the catalytic subunit of the polymerase, and the N protein, facilitating attachment of the RdRp to the nucleocapsid. This research probes the interaction between the N and P proteins of Menangle virus (MenV), a bat borne pathogen closely related to human mumps virus, aiming to increase understanding of the structural basis for polymerase attachment and translocation. The MenV N and P proteins were individually isolated and characterised, using a range of structural and biophysical techniques. Expression of the N protein in bacteria results in formation of nucleocapsid-like-particles (NLPs). Methods were developed to isolate rings that model a single turn of the helical nucleocapsid. Each ring is comprised of thirteen N protein subunits, with each subunit binding six nucleotides of RNA. A high-resolution image reconstruction, derived using Cryo-electron microscopy, allowed an atomic model of the MenV nucleocapsid protein to be developed. Analysis of the P protein demonstrated the presence of structurally disordered and highly dynamic sequences connecting the known binding elements. This flexibility is likely required for polymerase translocation along the nucleocapsid. An improved structural model for the nucleocapsid-binding domains (NBDs), located at the C-terminus of the P protein, was determined using X-ray crystallography. The attachment of the NBDs to NLPs was characterised spectroscopically. The modest affinity determined for the interaction between P and the nucleocapsid likely facilitates the repeated cycles of binding and release that are required for polymerase translocation.