Crystal structure of the SARS-CoV-2 non-structural protein 9, Nsp9

Dene Littler; Benjamin Gully; Rhys N Colson; Jamie Rossjohn

During infection of human cells, SARS-CoV Non-structural protein 9 (Nsp9SARS) was found to be important for replication (Frieman et al., 2012) . Homologs of the Nsp9 protein have been identified in numerous coronaviruses including SARS-Cov-2 (Nsp9COV19), human coronavirus 229E (Nsp9HcoV), avian infectious bronchitis virus (Nsp9IBV), porcine epidemic diarrhea virus (Nsp9PEDV) and porcine delta virus (Nsp9PDCoV). Nsp9SARS has been shown to have modest affinity for long oligonucleotides with binding thought to be dependent on oligomerisation state (Egloff et al., 2004) (Sutton et al., 2004) . Nsp9SARS dimerises in solution via a conserved a-helical 'GxxxG' motif. Disruption of key residues within this motif reduces both RNA-binding (Sutton et al., 2004) and SARS-CoV viral proliferation (Frieman et al., 2012) . The mechanism of RNA binding within the Nsp9 protein family is not understood as these proteins have an unusual structural fold not previously seen in RNA binding proteins (Egloff et al., 2004) (Sutton et al., 2004) . The fold's Greek-key motif exhibits topological similarities with OB-fold proteins and small protein B but such vestiges have proven insufficient to provide clear insight into Nsp9 function (Egloff et al., 2004) . As a consequence of the weak affinity of Nsp9SARS for long oligonucleotide stretches it was suggested that the natural RNA substrate may instead be conserved features at the 3' end of the viral-genome (the stem-loop II RNA-motif) (Ponnusamy et al., 2008) . Furthermore, potential direct interactions with the co-factors of the RNA polymerase have been reported . However, it remains to be determined how the oligonucleotide-binding activity of Nsp9 proteins promote viral replication during infection.