Structural Characterisation of Proteins from the Replication Machinery of Paramyxoviruses

Abstract

The paramyxoviruses are responsible for a myriad of animal diseases, including the well-known measles and mumps viruses, and the new dangerous Hendra and Nipah viruses. The risk posed by these dangerous viruses, as well as the therapeutic potential of other paramyxoviruses, necessitates a better understanding of their replication. These non-segmented single-stranded RNA viruses encoded their own RNA polymerase to direct transcription and genome replication. The polymerase is a complex of a large catalytic protein (L) and an accessory protein (P). The polymerase cannot access the genome directly, as it is encased by the viral N protein in a helical complex termed the nucleocapsid. P provides the “legs and feet” that allow the polymerase to traverse along the nucleocapsid. Our understanding of polymerase functions is hampered by the lack of structural information on L, and is complicated by subtle variations in the interaction between the polymerase and the nucleocapsid among paramyxoviruses. This project investigated the role of disorder in the polymerase foot (Pfoot) domain from mumps virus and its closest relatives, the rubulaviruses. The Pfoot domains were expressed in bacteria, purified, and subjected to structural investigation. Circular dichroism and NMR spectroscopy, along with X-ray crystallography, were used to demonstrate that the Pfoot domains span the structural continuum. Some are folded and globular, while others have only residual amounts of secondary structure. Despite this diverse structural behaviour, the rubulavirus Pfoot domains possess a conserved binding function, as determined by GST pulldown assays. A generic chemical stabiliser was able to force fold the partially ordered domains, suggesting that all Pfoot domains form a common structure upon binding to the nucleocapsid. Fundamentally, the Pfoot domains present a unique system for studying protein disorder and folding. Additionally we attempted to produce the measles and mumps virus polymerase proteins in Pichia pastoris to enable more detailed structural studies. The measles virus L protein was expressed in P. pastoris, representing the first time a paramyxoviranae L protein has been expressed in yeast. However, the system requires further development to confirm the identity of expressed proteins.