Coronavirus Nsp10, a Critical Co-factor for Activation of Multiple Replicative Enzymes

Authors:
Mickaël Bouvet,‡§,1,2 Adrien Lugari,¶,1,3 Clara C. Posthuma, Jessika C. Zevenhoven,? Stéphanie Bernard,¶,4 Stéphane Betzi,¶ Isabelle Imbert,‡§ Bruno Canard,‡§ Jean-Claude Guillemot,‡§ Patrick Lécine,** Susanne Pfefferle,‡‡,5 Christian Drosten,‡‡ Eric J. Snijder,? Etienne Decroly,‡§,6,7 and Xavier Morelli¶,6,8
In:
Source: Biochemistry and Molecular Biology
Publication Date: (2014)
Issue: 289: 37
Cells used in publication:
Vero
Species: monkey
Tissue Origin: kidney
Platform:
Nucleofector™ I/II/2b
Experiment

SARS-CoV Reverse Genetics

Using “en passant recombineering” (recombineering by mutagenesis) (54), mutations in the nsp10-, nsp14-, and nsp16-coding regions of SARS-CoV isolate Frankfurt-1 were engineered in prSCV, a pBeloBac11 derivative containing a full-length cDNA copy of the viral genome (55). The DNA of such BAC clones was linearized with NotI, extracted with phenol-chloroform, and transcribed with the mMessage-mMachine® T7 (Ambion) using 2 µg of DNA template in a 20-µl reaction. Full-length viral RNA was precipitated with LiCl according to the manufacturer's protocol, and 6 µg was electroporated into 5 × 106 BHK-Tet-SARS-N cells, which express the SARS-CoV N protein after> 4 h of induction with 2 µm doxycycline (53). Electroporation was done using the Amaxa Nucleofector (Lonza), Nucleofector Kit T, and program T-020 according to the manufacturer's instructions. Cells were mixed in a 1:1 ratio with Vero-E6 cells and seeded on coverslips for immunofluorescence microscopy and for analysis of virus production. Each mutant was launched twice from independently generated BAC clones. All work with live SARS-CoV was performed inside biosafety cabinets in a biosafety level 3 facility at Leiden University Medical Center.

Abstract

The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1–16) encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3'-5' exoribonuclease and 2'-O-methyltransferase activities. Using alanine-scanning mutagenesis, in cellulo bioluminescence resonance energy transfer experiments, and in vitro pulldown assays, we have now identified the key residues on the nsp10 surface that interact with nsp14. The functional consequences of mutations introduced at these positions were first evaluated biochemically by monitoring nsp14 exoribonuclease activity. Disruption of the nsp10-nsp14 interaction abrogated the nsp10-driven activation of the nsp14 exoribonuclease. We further showed that the nsp10 surface interacting with nsp14 overlaps with the surface involved in the nsp10-mediated activation of nsp16 2'-O-methyltransferase activity, suggesting that nsp10 is a major regulator of SARS-CoV replicase function. In line with this notion, reverse genetics experiments supported an essential role of the nsp10 surface that interacts with nsp14 in SARS-CoV replication, as several mutations that abolished the interaction in vitro yielded a replication-negative viral phenotype. In contrast, mutants in which the nsp10-nsp16 interaction was disturbed proved to be crippled but viable. These experiments imply that the nsp10 surface that interacts with nsp14 and nsp16 and possibly other subunits of the viral replication complex may be a target for the development of antiviral compounds against pathogenic coronaviruses.