Hu T, et al., 26(5):1037-1048, Protein Sci, 2017
Coronaviruses (CoVs) are positive-strand RNA viruses with an ability to infect animals as well as humans. CoVs produce nonstructural proteins (nsps) that play a key role in assembling the RNA replicase complex, which is essential for viral replication and transcription. Nsp9 forms a homodimer, and its dimeric form is critical for RNA binding. An investigation directed towards the exploration of the structure and oligomerization state of a gamma-CoV, reported herein is an avian infectious bronchitis virus (IBV) nsp9. The crystal structure is solved for IBV nsp9. According to the crystal structure, a hydrophobic region and two parallel α-helices are vital for nsp9 dimerization. The dimeric state was further studied using various biochemical techniques. Binding interactions of wild-type and mutant nsp9 with RNA were investigated by using Bio-Layer Interferometry (BLI). A Pall ForteBio Octet RED96 system equipped with Streptavidin (SA) Biosensor probes were used to perform BLI assays. Biotinylated 20-mer RNA was loaded onto SA Biosensor tips. Sensor tips were immersed in various concentrations of nsp9 to determine association and dissociation rates. KD values were calculated for the binding interactions. Data obtained by BLI was confirmed also by Surface Plasmon Resonance (SPR) method. Overall results of this study suggest that the dimeric form of nsp9 is essential for its binding to RNA. Furthermore, three conserved residues within the dimeric interface of IBV nsp9 were revealed pivotal for both dimerization and RNA interactions.