Structural characterization of a nematode-infecting virus - Orsay
Guo, Yusong R
Tao, Yizhi Jane; Zhong, Weiwei
Doctor of Philosophy
Orsay virus is the first and so far the only virus known to naturally infects the model organism Caenorhabditis elegans. It has a bipartite, positive-sense RNA genome, encoding the viral polymerase, the capsid protein (CP), and a protein named Delta whose functions are currently unknown. Based upon sequence analysis, the Orsay virus is most closely related to members of the Nodaviridae family, but may define a novel family given its unique features, such as the non-AUG initiation of CP translation and the expression of a CP-Delta fusion protein. To better understand the molecular mechanisms of Orsay virus replication, I performed structural and functional studies of Orsay viral proteins. Recombinant Orsay CP expressed in E. coli spontaneously forms virus-like particle (VLP). Its crystal structure has been determined to 3.25-Å resolution by averaging and phase extension using a 9-Å resolution cryo-EM reconstruction as a phasing model. The Orsay VLP shows T=3 icosahedral symmetry with 60 trimeric spikes. Each CP can be divided into three regions: an N-terminal peptide, a S domain forming the continuous capsid shell, and a P domain forming surface protrusions. The jelly-roll β-barrel in the S domain closely resembles those from small plant viruses, while the P domain is remotely related to betanodaviruses. The N-terminal peptide forms an extensive internal network and may regulate capsid stability and genome packaging. Recombinant CP could also be purified as stable dimers, indicating that Orsay capsid assembly may start from dimer formation. The Delta protein, which has no homology with any other known protein, is rich in β-sheets with high valine content. Under electron microscope, recombinant Delta appeared as a rod-shaped, fibrous protein. The crystal structure of the N-terminal 66 residues at 2.1Å resolution shows a pentameric helical bundle with interesting biological implications. Considering the unique advantage of the C. elegans-Orsay system for studying viral pathogenesis and virus-host interactions and also its promising application in anti-viral drug screening, my studies of the Orsay virus will not only provide important insights into the fundamental biology of RNA viruses in general, but will also facilitate development of the C. elegans-Orsay as a powerful virological model.
Orsay virus; structure, crystallography