Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA

Abstract

Coronaviruses (CoVs) stand out among RNA viruses because of their unusually large genomes (∼30 kb) associated with low mutation rates. CoVs code for nsp14, a bifunctional enzyme carrying RNA cap guanine N7-methyltransferase (MTase) and 3'-5' exoribonuclease (ExoN) activities. ExoN excises nucleotide mismatches at the RNA 3'-end in vitro, and its inactivation in vivo jeopardizes viral genetic stability. Here, we demonstrate for severe acute respiratory syndrome (SARS)-CoV an RNA synthesis and proofreading pathway through association of nsp14 with the low-fidelity nsp12 viral RNA polymerase. Through this pathway, the antiviral compound ribavirin 5'-monophosphate is significantly incorporated but also readily excised from RNA, which may explain its limited efficacy in vivo. The crystal structure at 3.38 Å resolution of SARS-CoV nsp14 in complex with its cofactor nsp10 adds to the uniqueness of CoVs among RNA viruses: The MTase domain presents a new fold that differs sharply from the canonical Rossmann fold.

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“…An open question that warrants further investigation is the role of the 3Ј-5Ј exonuclease (nsp14) in susceptibility to RDV. It has previously been reported that nucleotide analog inhibitors can be excised by the viral exonuclease (14). A murine hepatitis virus mutant lacking the 3Ј-5Ј exonuclease activity was shown to be more sensitive to RDV (5).…”

“…Progress has also been made in characterizing the SARS-CoV RdRp complex (13)(14)(15). Biochemical data suggest that the active complex is composed of at least three viral nonstructural proteins nsp7, nsp8, and nsp12.…”

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“…ATAi sa na nionic polymer shown to bind to a variety of protein targets, including gp120o fH IV-1 and HIV-2, that has been demonstrated to prevent SARS-CoV replication (IC 50 = 0.2 mg mL À1 ). [32,34] In order to develop nucleoside analogues to effectively inhibit viral RNA replication, the nucleoside must either evade detection by the exonuclease or must outcompete exonuclease activity.R emdesivir (GS-5734) is an excellent example of the latter.A na denosine analogue prodrug with a1 '-nitrile, it displayed potent efficacy against SARS and MERS in human airway epithelial (HAE) cell models and in mice (IC 50 = 0.069 and 0.074 mm for SARS-CoV and MERS-CoV,r espectively,i nH AE). [28] Beyond this exception,t he remainingR dRp inhibitors have been nucleoside analogues,a nd these provide the most promisinga venue towards disrupting viral RNA replication.…”

Nucleoside analogues for the treatment of coronavirus infections

“…Development of nucleoside analogue inhibitors of CoVs is further hampered by the novel RNA-dependent RNA proofreading activity of CoV nonstructural protein 14, a 3 0 to 5 0 exoribonuclease (nsp14-ExoN), which confers up to 20-fold increase in replication fidelity compared with other RNA viruses. The nsp14-ExoN activity is responsible for native CoV high resistance to many nucleoside analogues including ribavirin and 5-fluorouracil [11][12][13]. Nevertheless, the recent development of nucleotide and nucleoside analogue inhibitors with a high barrier for resistance, broad-spectrum activity against multiple CoVs, and the ability to inhibit WT CoVs in the presence of nsp14-ExoN holds promise for the treatment of CoV disease.…”