Coronaviruses pose serious health threats to humans and other animals. Understanding the mechanisms of their replication has important implications for global health and economic stability. Nonstructural protein 9 (nsp9) is an essential RNA binding protein for coronavirus replication. However, the mechanisms of the dimerization and nucleic acid binding of nsp9 remain elusive. Here, we report four crystal structures, including wild-type porcine delta coronavirus (PDCoV) nsp9, PDCoV nsp9-ΔN7 (N-terminal 7 amino acids deleted), wild-type porcine epidemic diarrhea virus (PEDV) nsp9, and PEDV nsp9-C59A mutant. These structures reveal the diverse dimerization forms of coronavirus nsp9. We first found that the N-finger of nsp9 from PDCoV plays a critical role in dimerization. Meanwhile, PEDV nsp9 is distinguished by the presence of a disulfide bond in the dimer interface. Interestingly, size exclusion chromatography and analytical ultracentrifugation analyses indicate that the PDCoV nsp9-ΔN7 and PEDV nsp9-C59A mutants are monomeric in solution. In addition, electrophoretic mobility shift assays and microscale thermophoresis analysis indicate that the monomeric forms of PDCoV nsp9 and PEDV nsp9 still have nucleic acid binding affinity, although it is lower than that of the wild type. Our results show that the diverse dimerization forms of coronavirus nsp9 proteins enhance their nucleic acid binding affinity.
Bibliographical noteFunding Information:
This study was supported by the National Natural Science Foundation of China (grant 31722056), the National Key R&D Plan of China (program no. 2016YFD0500103), and the Huazhong Agricultural University Scientific and Technological Self-Innovation Foundation (program no. 2662015JQ003 and 2662017PY028). We thank the staff at the SSRF BL17U1 beamline for assistance with X-ray data collection. Moreover, we also thank research associates at the Center for Protein Research (CPR), x, for technical support.
© 2018 American Society for Microbiology.
- Coronavirus nsp9
- Disulfide bond
- Nucleic acid binding