3EE7

Crystal Structure of SARS-CoV nsp9 G104E

Summary for 3EE7

• Entry DOI: 10.2210/pdb3ee7/pdb
• Related: 1QZ8 1UW7 2J97 2J98
• Descriptor: Replicase polyprotein 1a, PHOSPHATE ION, GLYCEROL, ... (4 entities in total)
• Functional Keywords: gxxxg, dimerization, sars-cov, helix-helix, hydrolase, membrane, metal-binding, protease, rna-binding, thiol protease, transmembrane, zinc-finger, viral protein
• Biological source: SARS coronavirus (SARS-CoV)
• Cellular location: Non-structural protein 3: Host membrane ; Multi-pass membrane protein . Non-structural protein 4: Host membrane ; Multi-pass membrane protein . Non-structural protein 6: Host membrane ; Multi-pass membrane protein . Non-structural protein 7: Host cytoplasm, host perinuclear region . Non-structural protein 8: Host cytoplasm, host perinuclear region . Non-structural protein 9: Host cytoplasm, host perinuclear region . Non-structural protein 10: Host cytoplasm, host perinuclear region : P0C6U8
• Total number of polymer chains: 4
• Total formula weight: 56466.66

Authors

Miknis, Z.J.,Donaldson, E.F.,Umland, T.C.,Rimmer, R.,Baric, R.S.,Schultz, L.W. (deposition date: 2008-09-04, release date: 2009-03-24, Last modification date: 2023-08-30)

Primary citation

Miknis, Z.J.,Donaldson, E.F.,Umland, T.C.,Rimmer, R.A.,Baric, R.S.,Schultz, L.W.
Severe acute respiratory syndrome coronavirus nsp9 dimerization is essential for efficient viral growth
J.Virol., 83:3007-3018, 2009

PubMed Abstract: The severe acute respiratory syndrome coronavirus (SARS-CoV) devotes a significant portion of its genome to producing nonstructural proteins required for viral replication. SARS-CoV nonstructural protein 9 (nsp9) was identified as an essential protein with RNA/DNA-binding activity, and yet its biological function within the replication complex remains unknown. Nsp9 forms a dimer through the interaction of parallel alpha-helices containing the protein-protein interaction motif GXXXG. In order to study the role of the nsp9 dimer in viral reproduction, residues G100 and G104 at the helix interface were targeted for mutation. Multi-angle light scattering measurements indicated that G100E, G104E, and G104V mutants are monomeric in solution, thereby disrupting the dimer. However, electrophoretic mobility assays revealed that the mutants bound RNA with similar affinity. Further experiments using fluorescence anisotropy showed a 10-fold reduction in RNA binding in the G100E and G104E mutants, whereas the G104V mutant had only a 4-fold reduction. The structure of G104E nsp9 was determined to 2.6-A resolution, revealing significant changes at the dimer interface. The nsp9 mutations were introduced into SARS-CoV using a reverse genetics approach, and the G100E and G104E mutations were found to be lethal to the virus. The G104V mutant produced highly debilitated virus and eventually reverted back to the wild-type protein sequence through a codon transversion. Together, these data indicate that dimerization of SARS-CoV nsp9 at the GXXXG motif is not critical for RNA binding but is necessary for viral replication.

PubMed: 19153232
DOI: 10.1128/JVI.01505-08

Experimental method

X-RAY DIFFRACTION (2.6 Å)