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 (nsp 9) was identified as an essential protein with RNA/DNA-binding activity, and yet its biological function within the replication complex remains unknown. Nsp 9 forms a dimer through the interaction of parallel ฮฑ-helices containing the protein-protein interaction motif GXXXG. In order to study the role of the nsp 9 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 nsp 9 was determined to 2.6-ร… resolution, revealing significant changes at the dimer interface. The nsp 9 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 nsp 9 at the GXXXG motif is not critical for RNA binding but is necessary for viral replication. Copyright ยฉ 2009, American Society for Microbiology.