Structural basis for dimerization and RNA binding of avian infectious bronchitis virus nsp9

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.

Dimerization of Coronavirus nsp9 with Diverse Modes Enhances Its Nucleic Acid Binding Affinity

“…CoV nsp9 has diverse dimerization, while its function of enhancement nuclear binding affinity is conservative (22)(23)(24)(25). We propose that most nsp9 proteins originated from ancestral coronavirus, which have N-finger motifs and GXXXG motifs that both play critical roles in dimerization.…”

“…Nsp9 is able to bind ssDNA or ssRNA (22,25), although binding of ssRNA is expected to be the native function. In the infected cell, the coupling/compartmentation of the viral RNA synthesis with the RNA-binding function of nsp9 might render RNA versus DNA specificity unnecessary (22).…”

“…We propose that most nsp9 proteins originated from ancestral coronavirus, which have N-finger motifs and GXXXG motifs that both play critical roles in dimerization. The N-terminal loops and GXXXG motifs of PDCoV, IBV, and SARS-CoV nsp9 are also involved in dimerization, while the extra amino acids (the residues of fusion tag) in the N-finger may block the fully function of the N-finger (22,24,25). Interestingly, PEDV and HCoV-229E evolve different disulfide bonds to enhance the dimerization (23).…”

“…Meanwhile, the dimerization of nsp9 is critical for viral replication (21). For nsp9, there are five crystallographic structures that showed a variety of dimeric interfaces (22)(23)(24)(25). However, the dimerization mechanism of all CoV nsp9s, whether from emerging viruses or not, is not so clear yet.…”

Structural Basis for the Inhibition of Host Gene Expression by Porcine Epidemic Diarrhea Virus nsp1

“…After these proteins are processed by papain-like and 3C-like proteases, pp1a and pp1ab yield a total of 15 or 16 mature nonstructural proteins (NSPs) (11,12). Many of the NSPs perform essential functions in viral RNA synthesis (13)(14)(15)(16)(17), and nsp1, nsp3, nsp5, nsp14, nsp15, and nsp16 play important roles in the host immune system . Although the CoV genome is highly conserved within genera, nsp1, which is located at the N terminus of the replicase polyprotein pp1a, is responsible for the majority of variation (40).…”