Paramyxoviruses infect many animals including mammals, birds, reptiles and fish. Well-known human pathogens such as measles virus, mumps virus, the parainfluenza viruses and respiratory syncytial virus, all belong to this large family. Despite their prominence, the molecular replication machinery used by these viruses to synthesize RNA is poorly understood. Here we report experimental methods to produce and characterize one element of the replication machinery from Menangle virus, a zoonotic bat-borne Paramyxovirus, which is closely related to human mumps virus.

Viruses within the Paramyxovirus family possess a non-segmented, single-stranded RNA genome of negative polarity. The viral N protein packages the genome into a helical complex termed the nucleocapsid, which acts as the template for all virally directed RNA synthesis. Our interest is in understanding how the viral RNA polymerase engages and moves along the nucleocapsid during transcription and genome replication. This requires the production and characterization of nucleocapsid-like particles (NLPs) in order to facilitate structural, biochemical and biophysical analysis. NLPs are readily generated by heterologous expression of the viral N protein in bacteria.­­­­

Novel procedures to purify bacterially produced NLPs from Menangle virus are reported. We demonstrate that the NLPs are homogenous in size, and package RNA. Preliminary structural characterization of Menangle virus NLPs is presented, showing the NLPs have a ring-like organization and correspond to a single turn of the helical nucleocapsid.