Structural basis and functional analysis of the SARS coronavirus nsp14–nsp10 complex

Abstract

Nonstructural protein 14 (nsp14) of coronaviruses (CoV) is important for viral replication and transcription. The N-terminal exoribonuclease (ExoN) domain plays a proofreading role for prevention of lethal mutagenesis, and the C-terminal domain functions as a (guanine-N7) methyl transferase (N7-MTase) for mRNA capping. The molecular basis of both these functions is unknown. Here, we describe crystal structures of severe acute respiratory syndrome (SARS)-CoV nsp14 in complex with its activator nonstructural protein10 (nsp10) and functional ligands. One molecule of nsp10 interacts with ExoN of nsp14 to stabilize it and stimulate its activity. Although the catalytic core of nsp14 ExoN is reminiscent of proofreading exonucleases, the presence of two zinc fingers sets it apart from homologs. Mutagenesis studies indicate that both these zinc fingers are essential for the function of nsp14. We show that a DEEDh (the five catalytic amino acids) motif drives nucleotide excision. The N7-MTase domain exhibits a noncanonical MTase fold with a rare β-sheet insertion and a peripheral zinc finger. The cap-precursor guanosine-P3-adenosine-5′,5′-triphosphate and S-adenosyl methionine bind in proximity in a highly constricted pocket between two β-sheets to accomplish methyl transfer. Our studies provide the first glimpses, to our knowledge, into the architecture of the nsp14-nsp10 complex involved in RNA viral proofreading.CoV | nsp14 | proofreading | exoribonuclease | methyltransferase

Nidovirus RNA polymerases: Complex enzymes handling exceptional RNA genomes

“…The recently solved crystal structure of the SARS-CoV nsp10+14 complex revealed that nsp14 folds into two separate domains, with residues 1-287 and 288-527 forming the N-terminal ExoN and Cterminal N7-methyl transferase domains, respectively (Ma et al, 2015). A single nsp10 molecule interacts with the ExoN domain of nsp14, which supports the available biochemical data that nsp10 only activates ribonuclease activity (Bouvet et al, 2010;Bouvet et al, 2012).…”

“…(Brockway et al, 2003;Imbert et al, 2008;Subissi et al, 2014b;Sutton et al, 2004;von Brunn et al, 2007)) and co-crystallization studies (e.g. (Decroly et al, 2011;Ma et al, 2015;Zhai et al, 2005)) have implicated several additional CoV nsps and the nucleocapsid (N) protein as potential viral cofactors of the RNA polymerase. For the AV PRRSV, using different technical approaches, multiple host proteins that appear to interact with nsp9 were identified, although it remains to be studied whether they directly bind to the RdRp domain of the protein and affect its function(s) in RNA synthesis Li et al, 2014a;Liu et al, 2016).…”

“…Despite the presence of two unusual zinc fingers on the surface of the ExoN domain, the core structure of the enzyme is similar to that of members of the DEDD exonuclease superfamily. CoV ExoN contains five catalytic residues (D90, E92, E191, H268 and D273 in SARS-CoV), and the enzyme was classified into the subgroup of the so-called DEDDh exonucleases (Ma et al, 2015), with E191 replacing a D residue that is conserved in other members of the superfamily. A direct interaction of SARS-CoV nsp14 with nsp12 was observed in pull-down assays (Imbert et al, 2008) and the protein could also be co-purified with the nsp7+8+12 tripartite complex (Subissi et al, 2014b).…”

Avoiding Regions Symptomatic of Conformational and Functional Flexibility to Identify Antiviral Targets in Current and Future Coronaviruses

“…( B ) NSP7, based on PDB id 5F22 (unpublished). ( C ) NSP14, based on PDB id 5C8T (Ma et al 2015). Protein structure visualized with Bioviva Discovery Studio .…”

Toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay

“…The viral MTases (SARS-CoV nsp14, MERS-CoV nsp14, DENV-3 NS5-MTase, West Nile virus NS5-MTase, DENV-2 NS5-RdRp and human RNA N7-methyltransferase (RNMT) coding sequences were cloned in fusion with a N-terminus hexa-histidine tag in Gateway ® plasmids (pDest14 or pDest17, Life technologies). The proteins were expressed in E. coli cells and purified following previously described protocols (Aouadi et al, 2017;Bouvet et al, 2010;Ma et al, 2015;Milhas et al, 2016;Peyrane et al, 2007;Selisko et al, 2006). MERS-CoV nsp14 was produced and purified as follows: the protein were expressed in Arctic Express E. coli strain (Agilent) at 13 C during 24 h after addition of 50 mM IPTG.…”