Background: SARS-CoV nsp10 binds and stimulates both the nsp14 and nsp16 activities. Results: We mapped the nsp10 surface interacting with nsp14 and demonstrated that this surface plays a critical role in SARS-CoV replication. Conclusion:The core interaction domain of nsp10 is essential for SARS-CoV replication. Significance: This nsp10 core represents an attractive target for antiviral drug development against various pathogenic coronaviruses.

Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between nonstructural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several "hot spots," such as Val 42 Coronaviruses (CoVs),7 classified into the family Coronaviridae in the order Nidovirales, possess a viral RNA genome that is among the largest known (2). They include important pathogens of livestock, wild and companion animals, and humans, such as the severe acute respiratory syndrome CoV (SARSCoV) (3-5). They are mainly etiological agents of respiratory and enteric diseases, exemplified by the worldwide pandemic of SARS-CoV spreading in 2003 from Asia, with a final number of cases around 8,000 and a 10% mortality.The genome of SARS-CoV contains a single-stranded plussense RNA of ϳ29.7 kb (2). At the molecular level, CoVs employ a variety of unusual strategies to accomplish a complex program of gene expression (5). Coronavirus replication requires the synthesis of both genomic and multiple subgenomic RNA species and the assembly of progeny virions by a pathway that is unique among enveloped RNA viruses (5-7). Fourteen open reading frames (ORFs) have been identified, of which 12 are located in the 3Ј-end of the genome. The other two ORFs (1a and 1b), which are located in the 5Ј-proximal twothirds of the genome, encode two large polyproteins translated directly from genomic RNA. ORF 1b is expressed by a Ϫ1 ribosomal frameshifting at the end of pp1a, extending its coding sequence and thus generating the pp1ab polyprotein (6). These two polyproteins are cleaved into 16 functional viral replicase proteins called nsp1 to -16 (for non-structural proteins 1-16). Those nsps form the membrane-bound replication-transcription complex, which is localized to a network of endoplasmic reticulum-derived membranes in the infected cell (8, 9). Bioinformatics, structural biology, (reverse) genetics, and biochemical studies have contributed to the characterization of CoV