Biology subjects

Zevenhoven-Dobbe, J.

Publications and source records attributed to Zevenhoven-Dobbe, J..

2 recordsLinked to original sources

bioRxiv2021

6',6'-Difluoro-aristeromycin is a potent inhibitor of MERS-coronavirus replication

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the lack of treatments to combat infections with human or (potentially) zoonotic CoVs. Thus, it is critical to develop and evaluate antiviral compounds that either directly target CoV functions or modulate host functions involved in viral replication. Here, we demonstrate that low-micromolar concentrations of 6',6'-difluoro-aristeromycin (DFA), an adenosine nucleoside analogue, strongly inhibit the replication of Middle East respiratory syndrome coronavirus (MERS-CoV) in a cell-based infection assay. DFA was designed to target S-adenosylhomocysteine (SAH) hydrolase and, consequently, may affect intracellular levels of the methyl donor S-adenosylmethionine, which is used by two CoV methyltransferases involved in the capping of the 5 end of the viral mRNAs. Passaging of wild-type MERS-CoV in the presence of DFA selected a virus population with a [~]100-fold decreased DFA sensitivity, which carried various amino acid substitutions in viral nonstructural proteins (nsps). Specifically, mutations were present in the RNA polymerase subunit (nsp12) and in nsp13, the helicase subunit containing a nucleoside triphosphate hydrolase activity that has been implicated in CoV capping. We hypothesize that DFA directly or indirectly affects viral cap methylation, either by inhibiting the viral enzymes involved or by binding to SAH hydrolase. We also evaluated the antiviral activity of DFA against other betacoronaviruses, but found it to have limited impact on their replication, while being quite cytotoxic to the Calu-3 cells used for this comparison. Nevertheless, our results justify the further characterization of DFA derivatives as an inhibitor of MERS-CoV replication. ImportanceCurrently, there is a lack of antiviral drugs with proven efficacy against human CoV infections including the MERS-CoV that is endemic in the Middle East, the pandemic SARS-CoV-2 and potential future zoonotic CoV. This highlights the importance to investigate new drug targets and identify compounds that can be used to inhibit CoV replication. In this study, we characterize the inhibitory effect of DFA on MERS-CoV replication by phenotypic studies, time-of-addition studies, and the generation and genotyping of a DFA-resistant virus population. Our results revealed that DFA needs further improvement to reduce its cytotoxic side-effects and potentially enhance its broad-spectrum activity. Despite this observation, we think that DFA can be used to understand the function and metabolic interactions of the CoV RNA-synthesizing machinery, or as a starting point for the design of new compounds of the same class.

microbiology↗

bioRxiv2020

The enzymatic activity of the nsp14 exoribonuclease is critical for replication of Middle East respiratory syndrome-coronavirus

AO_SCPLOWBSTRACTC_SCPLOWCoronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains an N-terminal 3-to-5 exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase) domain. While the latter presumably operates during viral mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN-knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously found to have a crippled but viable hypermutation phenotype. Remarkably, using an identical reverse genetics approach, an extensive mutagenesis study revealed the corresponding ExoN-knockout mutants of another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), to be non-viable. This is in agreement with observations previously made for alpha- and gammacoronaviruses. Only a single MERS-CoV ExoN active site mutant could be recovered, likely because the introduced D191E substitution is highly conservative in nature. For 11 other MERS-CoV ExoN active site mutants, not a trace of RNA synthesis could be detected, unless - in some cases - reversion had first occurred. Subsequently, we expressed and purified recombinant MERS-CoV nsp14 and established in vitro assays for both its ExoN and N7-MTase activities. All ExoN knockout mutations that were lethal when tested via reverse genetics were found to severely decrease ExoN activity, while not affecting N7-MTase activity. Our study thus reveals an additional function for MERS-CoV nsp14 ExoN, which apparently is critical for primary viral RNA synthesis, thus differentiating it from the proofreading activity thought to boost long-term replication fidelity in MHV and SARS-CoV. IO_SCPLOWMPORTANCEC_SCPLOWThe bifunctional nsp14 subunit of the coronavirus replicase contains 3-to-5 exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) domains. For the betacoronaviruses MHV and SARS-CoV, the ExoN domain was reported to promote the fidelity of genome replication, presumably by mediating some form of proofreading. For these viruses, ExoN knockout mutants are alive while displaying an increased mutation frequency. Strikingly, we now established that the equivalent knockout mutants of MERS-CoV ExoN are non-viable and completely deficient in RNA synthesis, thus revealing an additional and more critical function of ExoN in coronavirus replication. Both enzymatic activities of (recombinant) MERS-CoV nsp14 were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.

microbiology↗