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Abstract
Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural 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 Val42, Met44, Ala71, Lys93, Gly94, and Tyr96, forming a continuous protein-protein surface of about 830 Å2, bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2′-O-methyltransferase (2′O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2′O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2′O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.
Highlights
Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between nonstructural proteins nsp10 and nsp16
Delineation of the nsp10 Surface Involved in Its Interaction with nsp16—We used RY2H with nsp10 and nsp16 to isolate interaction-defective alleles (IDAs) and thereby delineate their surface of interaction [25, 26]
IDAs are alleles that contain mutations affecting their ability to interact with their wild type binding partners, leading to the identification of specific amino acid residues involved in the interaction between nsp10 and nsp16 [39]. nsp16 was used as a bait to screen a library of potential nsp10 IDAs generated by PCR-mutagenesis and selected to express full-length proteins
Summary
CoV, coronavirus; SARS, severe acute respiratory syndrome; AdoMet, S-adenosyl-L-methionine; EYFP, enhanced yellow fluorescent protein; 2ЈO-MTase, 2Ј-O-methyltransferase; MHV, mouse hepatitis virus; RY2H, reverse yeast two-hybrid; BRET, bioluminescence resonance energy transfer; IDA, interaction-defective allele; RLuc, Renilla luciferase; N7-MTase, (guanine-N7)-methyltransferase. The involvement of nsp N7-MTase and of nsp16 2ЈO-MTase in the capping pathway was recently demonstrated biochemically [1, 18, 20] Both nsp and -16 play crucial roles for efficient RNA synthesis within the SARS-CoV replicon and for transcription/replication of MHV-CoV [13, 24]. We have identified a continuous specific surface of ϳ830 A2 on nsp involved in its interaction with nsp
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