Molecular Mapping of the RNA Cap 2′-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16*

Abstract

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

Biogenesis and Dynamics of the Coronavirus Replicative Structures

“…Although the enzyme that subsequently adds the guanine to the terminal phosphates (guanylyl transferase) has not been identified yet, nsp14 has been shown to exert S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) activity [31]. Finally, the cap-1 structure is formed by the AdoMet-dependent (nucleoside-2'O)-methyltransferase (2'O-MTase) activity that is present in nsp16 [32], and for which the latter needs to form a complex with nsp10 [33,34]. …”

Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase

“…CoVs replicate in the host cytoplasm and encode their own capping enzymes. Among the four capping enzymes involved in coronavirus m7GpppAm-cap formation, guanine-N7-methyltransferase (N7-MTase) was identified as nsp14 in our previous work using a yeast genetic system (Chen et al, 2009), and 2 0 -O-methyltransferase (2 0 -O-MTase) is formed by nsp16 with nsp10 as a cofactor (Chang et al, 2011;Chen et al, 2011;Decroly et al, 2011;Lugari et al, 2010). SARS-coronavirus nsp14 was previously characterized as a 3 0 -to 5 0 -exoribonuclease (ExoN) (Chen et al, 2007;Minskaia et al, 2006), and the N7-MTase domain was mapped to the carboxy-terminal part of the protein (Chen et al, 2009).…”

Short peptides derived from the interaction domain of SARS coronavirus nonstructural protein nsp10 can suppress the 2′-O-methyltransferase activity of nsp10/nsp16 complex

“…Such knowledge would be useful for designing and developing small molecules as protein-protein interaction inhibitors that could specially inhibit the enzymatic activity of nsp16 and the replication of SARS-CoV. Lugari and colleagues identified a number of key nsp10 residues involved in the interaction with nsp16 and in regulating nsp16 RNA cap 2 -O-MTase activity by site-directed mutagenesis (Lugari et al, 2010). In this study we mapped the linear domain consisting of aa 65-107 of nsp10 as the core area for the interaction with nsp16, which is located inside the interface of the nsp10/nsp16 complex as revealed from the crystal structure (Chen et al, 2011;Decroly et al, 2011).…”