Abstract
The potential for infection by coronaviruses (CoVs) has become a serious concern with the recent emergence of Middle East respiratory syndrome and severe acute respiratory syndrome (SARS) in the human population. CoVs encode two large polyproteins, which are then processed into 15-16 nonstructural proteins (nsps) that make significant contributions to viral replication and transcription by assembling the RNA replicase complex. Among them, nsp9 plays an essential role in viral replication by forming a homodimer that binds single-stranded RNA. Thus, disrupting nsp9 dimerization is a potential anti-CoV therapy. However, different nsp9 dimer forms have been reported for alpha- and beta-CoVs, and no structural information is available for gamma-CoVs. Here we determined the crystal structure of nsp9 from the avian infectious bronchitis virus (IBV), a representative gamma-CoV that affects the economy of the poultry industry because it can infect domestic fowl. IBV nsp9 forms a homodimer via interactions across a hydrophobic interface, which consists of two parallel alpha helices near the carboxy terminus of the protein. The IBV nsp9 dimer resembles that of SARS-CoV nsp9, indicating that this type of dimerization is conserved among all CoVs. This makes disruption of the dimeric interface an excellent strategy for developing anti-CoV therapies. To facilitate this effort, we characterized the roles of six conserved residues on this interface using site-directed mutagenesis and a multitude of biochemical and biophysical methods. We found that three residues are critical for nsp9 dimerization and its abitlity to bind RNA.