Structure–function analysis of the nsp14 N7–guanine methyltransferase reveals an essential role inBetacoronavirusreplication

Abstract

As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5′ exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3′-to-5′ ExoN domain and a C-terminal (N7-guanine)–methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14’s enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)–CoV nsp14 to perform an in silico comparative analysis between betacoronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV and Middle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum.

Potent Inhibition of SARS-CoV-2 nsp14 N7-Methyltransferase by Sulfonamide-Based Bisubstrate Analogues

“…Indeed, based on docking studies, our particular scaffold interacts with two key conserved residues—Arg310 and Phe426 in SARS-CoV nsp14—of the catalytic pocket that have been identified as critical for N 7-MTase nsp14 activity and consequently for SARS-CoV-2 replication. 9 Our results strengthen the emerging status of this enzyme as a valid target for antiviral rational-designed inhibitors. Further optimizations are underway to increase the cellular permeability of this series of potent nsp14 inhibitors with physicochemical properties tailored for cellular activity, which will enable the characterization of their mode of action.…”

“… 7 , 8 Nsp14 is also considered as an antiviral target because the replication of N 7-MTase catalytic mutants is strongly impaired. 9 Therefore, this crucial yet uncommon and under-explored enzyme seemed an enticing target to us for the development of antiviral therapies. 9 − 11 …”

“… 9 Therefore, this crucial yet uncommon and under-explored enzyme seemed an enticing target to us for the development of antiviral therapies. 9 − 11 …”

N7-Methylation of the Coronavirus RNA Cap Is Required for Maximal Virulence by Preventing Innate Immune Recognition

“…During the process of revision and resubmission, a paper characterizing N7-MTase structurally, biochemically, and virologically was published ( 51 ). That article complements our study.…”

Synthesis, Structure–Activity Relationships, and Antiviral Profiling of 1-Heteroaryl-2-Alkoxyphenyl Analogs as Inhibitors of SARS-CoV-2 Replication

“…These are highly conserved among various coronaviruses and many of them are excellent candidates as targets for the discovery of antiviral agents [ 9 ]. Examples include the cap guanine N7-methyltransferase and 3′–5′ exonuclease activity of nsp14 [ 10 ], the nsp15 endoribonuclease [ 11 ], and the papain-like protease (PLpro) activity of nsp3 [ 12 ]. Targets that are most intensively studied are nsp5, also known as the chymotrypsin-like protease (3CLpro) or the viral main protease (Mpro) [ 13 ] and the RNA-dependent RNA polymerase (RdRp, encoded by nsp12) [ 14 , 15 ].…”