Structural and Functional Studies on the Infectious Salmon Anemia Virus Nucleoprotein
Tao, Yizhi Jane
Doctor of Philosophy
Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses which are able to cause infectious disease, and even worldwide pandemics. The genome of Orthomyxovirus consists of 6-8 negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes which perform multiple essential functions throughout the virus life cycle. To better understand the structural features of orthomyxovirus RNPs that allow them to be specifically packaged, we performed structural/functional studies of the nucleoprotein (NP), the major protein component of the RNPs, from the infectious salmon anemia virus (ISAV). The crystal structure of the ISAV-NP was determined to 2.7Å resolution. The ISAV-NP possesses a 112-aa N-terminal domain and a bi-lobular core structure that strongly resembles the structure of the influenza virus NP. Because the ISAV-NP forms homogenous dimers that are stable in solution, I was able to study the NP:RNA binding affinity as well as stoichiometry with fluorescence polarization, using recombinant proteins and synthetic oligos. Surprisingly, the RNA binding analysis revealed that each NP binds ~12 nts of RNA, shorter than the 24-28 nts originally estimated for the influenza A virus NP. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. These results suggest that free RNA exists in the orthomyxovirus RNPs, and selective RNP packaging is likely accomplished through direct RNA-RNA interactions.