The recently identified etiological agent of the severe acute respiratory syndrome (SARS) belongs to Coronaviridae (CoV), a family of viruses replicating by a poorly understood mechanism. Here, we report the crystal structure at 2.7-Å resolution of nsp9, a hitherto uncharacterized subunit of the SARS-CoV replicative polyproteins. We show that SARS-CoV nsp9 is a single-stranded RNA-binding protein displaying a previously unreported, oligosaccharide͞oligo-nucleotide fold-like fold. The presence of this type of protein has not been detected in the replicative complexes of RNA viruses, and its presence may reflect the unique and complex CoV viral replication͞transcription machinery.I n 2003, a human coronavirus (CoV) was identified as the causative agent of a form of atypical pneumonia: severe acute respiratory syndrome-CoV (SARS-CoV) (1-5). Coronaviridae have the longest known single-stranded (ss)RNA genome (27-31.5 kb), with a complex genetic organization and sophisticated replication͞transcription cycle (6, 7). Twenty-eight proteins are predicted to be encoded by the SARS-CoV genome (8, 9). The nonstructural (nsp) or ''replicase'' proteins of CoVs are derived from an unusually large replicase gene of Ͼ20 kb that consists of two large ORFs (ORFs 1a and 1b). Translation of this replicase gene from the incoming genomic RNA is the first step in CoV genome expression and includes a Ϫ1 ribosomal frameshift to express the ORF1b-encoded polypeptide. Translation products are the pp1a polyprotein (Ͼ4,000 amino acids) and the C-terminally extended pp1ab polyprotein (Ͼ7,000 amino acids), which are both cleaved by two or three ORF1a-encoded viral proteinases (10). Most of these replicase cleavage products assemble into a membrane-associated viral replication͞ transcription complex. Among other components, this complex includes a set of relatively small polypeptides (nsp6 to nsp11) encoded by the 3Ј region of ORF1a, for which no predicted nor proven function has been assigned. For the mouse hepatitis CoV, several of these cleavage products were reported to colocalize with other components of the viral replication complex in the perinuclear region of the infected cell (11), suggesting their involvement (directly or indirectly) in viral RNA metabolism.As part of a viral structural genomics program (12), we have cloned the 28 gene products of SARS-CoV and expressed them either as full-length proteins or as (predicted) functional domains. The determination of the three-dimensional structures of these gene products is expected to facilitate and accelerate discovery of drugs against this emerging and life-threatening pathogen. Furthermore, structural homology search is becoming a powerful method to infer biochemical and͞or biological function of previously uncharacterized proteins. We report here the crystal structure of nsp9, one the SARS-CoV uncharacterized nonstructural protein, as well as evidence for its function as an ssDNA͞RNA-binding protein.
Materials and MethodsCrystallization, Structure Determination, and Refinement. SARS...