Drosophila melanogaster Nora virus virus-like particles: In vitro assembly

Nora virus is a recently discovered RNA picorna-like virus that produces a persistent infection in Drosophila melanogaster. This virus is of interest because it is similar to the human picornaviruses that are responsible for human diseases, such as polio, hepatitis A, foot and mouth disease, and the common cold. The Nora virus RNA genome is approximately 12,000 bases long and is split into four open reading frames (ORF 1, -2, -3 and -4). ORF 4 is most likely expressed as a polyprotein that is cleaved into three polypeptides by a viral-encoded protease, and these are designated as viral protein (VP) 4a, VP4b, and VP4c. These three viral proteins are thought to be the major capsid components of the virus, making ORF4 of particular interest in how this virus assembles. Virus-like particles (VLPs) are non-infectious virions, which contain only empty capsids with no enclosed packaged genetic material. Assembly of VLPs, for diseases caused by known viruses that lack efficient treatment and prevention, could be essential in the production of vaccines. For this study, VLPs of the Nora virus ORF 1, -3, -4a, -4b, and -4c proteins were placed together, in vitro, to determine the gene(s) that are essential in assembling the structural capsid. Proteins were separated through cesium chloride gradients and detected by Western blot analysis. Further visual composition and aggregation of protein was confirmed by electron microscopy. Electron microscopy revealed a size distribution similar to that of wild type virus when viral protein 4A is present with an additional protein, but the lack of VP4A, or VP4A alone, results in scattered size distribution, potentially indicating VP4A as the scaffold protein. The Nora virus assembly pathway is not yet known, but consideration of this could lead to a deeper understanding of how picornaviruses in general undergo assembly.