Abstract:20 Coronaviruses (CoVs) emerge as zoonoses and cause severe disease in humans, 21 demonstrated by the SARS-CoV-2 (COVID-19) pandemic. RNA recombination is 22 required during normal CoV replication for subgenomic mRNA (sgmRNA) synthesis and 23 35
“…Nsp14 is a 60 kDa bifunctional enzyme with an N-terminal exonuclease (ExoN) domain implicated in replication fidelity and a C-terminal N7-methyltransferase (N7-MTase) domain involved in mRNA capping ( Chen et al., 2009 ) ( Figure 4 A). In addition, nsp14 is also involved in several other processes of the virus life cycle and pathogenicity, including innate immune responses and viral genome recombination ( Becares et al., 2016 ; Gribble et al., 2020 ). Figure 4 The Overall Structure of Nonstructural Protein 14 (A) Cartoon representation of the structure of the nsp14.…”
Section: Introductionmentioning
confidence: 99%
“…A viable catalytically inactivated nsp14-ExoN in MHV-CoV generated significantly altered patterns of recombination and decreased recombination frequency when compared to MHV-CoV wild type. Decreased sgmRNA populations and increased defective viral genomes (DVGs) were also observed in MHV-ExoN(–), further suggesting the importance of ExoN in viral RNA synthesis and viral fitness ( Gribble et al., 2020 ).…”
The coronavirus disease 2019 (COVID-19) that is wreaking havoc on worldwide public health and economies has heightened awareness about the lack of effective antiviral treatments for human coronaviruses (CoVs). Many current antivirals, notably nucleoside analogs (NAs), exert their effect by incorporation into viral genomes and subsequent disruption of viral replication and fidelity. The development of anti-CoV drugs has long been hindered by the capacity of CoVs to proofread and remove mismatched nucleotides during genome replication and transcription. Here, we review the molecular basis of the CoV proofreading complex and evaluate its potential as a drug target. We also consider existing nucleoside analogs and novel genomic techniques as potential anti-CoV therapeutics that could be used individually or in combination to target the proofreading mechanism.
“…Nsp14 is a 60 kDa bifunctional enzyme with an N-terminal exonuclease (ExoN) domain implicated in replication fidelity and a C-terminal N7-methyltransferase (N7-MTase) domain involved in mRNA capping ( Chen et al., 2009 ) ( Figure 4 A). In addition, nsp14 is also involved in several other processes of the virus life cycle and pathogenicity, including innate immune responses and viral genome recombination ( Becares et al., 2016 ; Gribble et al., 2020 ). Figure 4 The Overall Structure of Nonstructural Protein 14 (A) Cartoon representation of the structure of the nsp14.…”
Section: Introductionmentioning
confidence: 99%
“…A viable catalytically inactivated nsp14-ExoN in MHV-CoV generated significantly altered patterns of recombination and decreased recombination frequency when compared to MHV-CoV wild type. Decreased sgmRNA populations and increased defective viral genomes (DVGs) were also observed in MHV-ExoN(–), further suggesting the importance of ExoN in viral RNA synthesis and viral fitness ( Gribble et al., 2020 ).…”
The coronavirus disease 2019 (COVID-19) that is wreaking havoc on worldwide public health and economies has heightened awareness about the lack of effective antiviral treatments for human coronaviruses (CoVs). Many current antivirals, notably nucleoside analogs (NAs), exert their effect by incorporation into viral genomes and subsequent disruption of viral replication and fidelity. The development of anti-CoV drugs has long been hindered by the capacity of CoVs to proofread and remove mismatched nucleotides during genome replication and transcription. Here, we review the molecular basis of the CoV proofreading complex and evaluate its potential as a drug target. We also consider existing nucleoside analogs and novel genomic techniques as potential anti-CoV therapeutics that could be used individually or in combination to target the proofreading mechanism.
“…Mutational reversion and recombination driven processes can pose significant challenges to the use of live-attenuated vaccines (120) emphasizing the need to engineer recombination-resistant strains (124). A recent study suggests the involvement of nsp14-ExoN in mediating recombination frequency and junction site selection in several coronaviruses (125), opening up an exciting avenue of exploration for nsp14 in vaccine development.…”
Section: Mechanisms Underlying High Fidelity Replicationmentioning
Few human pathogens have been the focus of as much concentrated worldwide attention as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), the cause of COVID-19. Its emergence into the human population and ensuing pandemic came on the heels of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), two other highly pathogenic coronavirus spillovers, which collectively have reshaped our view of a virus family previously associated primarily with the common cold. It has placed intense pressure on the collective scientific community to develop therapeutics and vaccines, whose engineering relies on a detailed understanding of coronavirus biology. Here, we present the molecular virology of coronavirus infection, including its entry into cells, its remarkably sophisticated gene expression and replication mechanisms, its extensive remodeling of the intracellular environment and its multi-faceted immune evasion strategies. We highlight aspects of the viral lifecycle that may be amenable to antiviral targeting as well as key features of its biology that await discovery.
“…In cell culture, MHV and SARS-CoV mutants lacking ExoN activity exhibit increased sensitivity to mutagenic agents like 5-fluoracil (5-FU), compounds to which the wildtype virus is relatively resistant (28,29). Recently, ExoN activity was also implicated in CoV RNA recombination, as an MHV ExoN knockout mutant exhibited altered recombination patterns, possibly reflecting its involvement in other activities than error correction during CoV replication and subgenomic mRNA synthesis (30).…”
Coronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) domains. While the latter presumably supports 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 reported to have crippled but viable hypermutation phenotypes. Remarkably, using reverse genetics, a large set of corresponding ExoN knockout mutations was now found to be lethal for another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). For 13 mutants, viral progeny could not be recovered, unless – occasionally – reversion had first occurred. Only a single mutant was viable, likely because its D191E substitution is highly conservative. Remarkably, also a SARS-CoV-2 ExoN knockout mutant was found unable to replicate, resembling observations previously made for alpha- and gammacoronaviruses, but starkly contrasting with the documented phenotype of ExoN knockout mutants of the closely related SARS-CoV. Subsequently, we established in vitro assays with purified recombinant MERS-CoV nsp14 to monitor its ExoN and N7-MTase activities. All ExoN knockout mutations that proved lethal in reverse genetics were found to severely decrease ExoN activity, while not affecting N7-MTase activity. Our study strongly suggests CoV nsp14 ExoN to have an additional function, which apparently is critical for primary viral RNA synthesis and thus differs from the proofreading function that – based on previous MHV and SARS-CoV studies – was proposed to boost longer-term replication fidelity.
IMPORTANCE The bifunctional nsp14 subunit of the coronavirus replicase contains 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase domains. For the betacoronaviruses MHV and SARS-CoV, ExoN was reported to promote the fidelity of genome replication, presumably by mediating a form of proofreading. For these viruses, ExoN knockout mutants are viable while displaying an increased mutation frequency. Strikingly, we now established that the equivalent ExoN knockout mutants of two other betacoronaviruses, MERS-CoV and SARS-CoV-2, are non-viable, suggesting an additional and critical ExoN function in their replication. This is remarkable in light of the very limited genetic distance between SARS-CoV and SARS-CoV-2, which is highlighted, for example, by 95% amino acid sequence identity in their nsp14 sequences. For (recombinant) MERS-CoV nsp14, both its enzymatic activities 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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
