Severe Acute Respiratory Syndrome Coronavirus nsp9 Dimerization Is Essential for Efficient Viral Growth

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 ␣-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-Å 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.

Continuous and Discontinuous RNA Synthesis in Coronaviruses

“…Also interacting with nsp8 is nsp9, a small protein that binds ssRNA without sequence specificity (130–132). Dimerization of nsp9 is critical for virus replication (133), and its structural and functional features strongly suggest that it could be a component of the RTC catalytic core, stabilizing viral RNAs during RNA synthesis and processing.…”

Insights into RNA synthesis, capping, and proofreading mechanisms of SARS-coronavirus

“…Nsp9 is a small (13 kDa in SARS-CoV) protein unique to coronavirus (Snijder et al, 2003). The two available crystal structures of the protein (Egloff et al, 2004;Sutton et al, 2004) reveal a novel fold made of an open six-stranded ␤-barrel and an additional ␣-helix that contains a GXXXG protein/protein interaction motif (Miknis et al, 2009) involved in dimer formation. Noteworthy, this dimerization is critical for virus replication (Miknis et al, 2009).…”

“…The two available crystal structures of the protein (Egloff et al, 2004;Sutton et al, 2004) reveal a novel fold made of an open six-stranded ␤-barrel and an additional ␣-helix that contains a GXXXG protein/protein interaction motif (Miknis et al, 2009) involved in dimer formation. Noteworthy, this dimerization is critical for virus replication (Miknis et al, 2009). In addition, SARS-CoV nsp9 binds ssRNA without sequence specificity (Egloff et al, 2004), and has been shown to interact with nsp8 (Sutton et al, 2004).…”

Atlas of coronavirus replicase structure

“…Subunits nsp3-6 contain all the viral factors that are necessary to form viral replicative organelles (Angelini et al, 2013), as well as two proteinases that are responsible for processing all of the viral replicase proteins (Ziebuhr et al, 2000). The small subunits nsp7-11 comprise the viral primer-making activities and provide other essential support for replication (Donaldson et al, 2007b;Imbert et al, 2006;Miknis et al, 2009). The final part of the replicase from nsp12-16 contains the remaining RNA-modifying enzymes needed for replication, RNA capping and proofreading.…”

“…Nsp9 colocalizes in the perinuclear region along with other components of the replication complex (Bost et al, 2000). While the precise role of nsp9 in viral replication is not yet clear, Minkis and co-workers investigated the role of the dimer interface demonstrated that SARS-CoV nsp9 is essential for efficient viral growth (Miknis et al, 2009).…”