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Accueil > Bibliographie > Molecular mapping of the RNA Cap 2’-O-methyltransferase activation interface (...)

Molecular mapping of the RNA Cap 2’-O-methyltransferase (...)

J Biol Chem. 2010 Aug ; [Epub ahead of print]
Molecular mapping of the RNA Cap 2’-O-methyltransferase activation interface between SARS coronavirus nsp10 and nsp16.
Lugari A, Betzi S, Decroly E, Bonnaud E, Hermant A, Guillemot JC, Debarnot C, Borg JP, Bouvet M, Canard B, Morelli X, Lecine P.

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) was identified in 2003 as a previously unknown coronavirus responsible of a sudden outbreak spreading rapidly from Asia, with a final number of cases around 8,000 and a 10% mortality. Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being strong and reciprocal between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction using Reverse Yeast two-Hybrid (RY2H). Alanine-scanning mutagenesis, bioinformatics and molecular modelling studies were used to identify several ’hot spots’ such as V42, M44, L45, A71, K93, G94 and Y96, forming a continuous protein-protein surface of about 830 A(2), bearing very conserved amino acids within coronaviruses. As nsp16 carries RNA cap 2[prime]O-MTase activity only in the presence of its interacting partner nsp10 (1), functional consequences of mutations on this surface were evaluated biochemically. Most changes which disrupted the nsp10/nsp16 interaction without structural perturbations, as judged by heteronuclear NMR experiments, were shown to abrogate stimulation of nsp16 cap 2’O-MTase activity. More strikingly, we show that Y96 plays a central role in the interaction and nsp16 stimulation : the Y96A mutation abrogates stimulation of nsp16 2’O-MTase activity while Y96F over stimulates it. Together with nsp16 2’O-MTase activity, the nsp10/nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.

PubMed

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