COVID-19: Researchers have discovered what may be the Achilles’ heel of the coronavirus

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Researchers at Vanderbilt University Medical Center (VUMC) and the University of Texas Medical Branch (UTMB) at Galveston have discovered what may be the Achilles’ heel of the coronavirus, a finding that may help close the door on COVID-19 and possibly head off future pandemics.

The coronavirus is an RNA virus that has, in its enzymatic toolkit, a ‘proofreading’ exoribonuclease, called nsp14-ExoN, which can correct errors in the RNA sequence that occur during replication, when copies of the virus are generated.

Using cutting-edge technologies and novel bioinformatics approaches, the researchers discovered that this ExoN also regulates the rate of recombination, the ability of the coronavirus to shuffle parts of its genome and even pull in genetic material from other viral strains while it replicates in order to gain evolutionary advantage.

These patterns of recombination, the researchers reported last week in the journal PLOS Pathogens, are conserved across multiple coronaviruses, including SARS-CoV-2, which causes COVID-19, and MERS-CoV, which causes a similar illness, Middle Eastern respiratory syndrome.

“The coronavirus exoribonuclease is therefore a conserved, important target for inhibition and attenuation in the ongoing pandemic of SARS-CoV-2, and in preventing future outbreaks of novel coronaviruses,” concluded the paper’s first author, Jennifer Gribble, a VUMC graduate student in the laboratory of Mark Denison, MD.

“If you can find a drug that prevents RNA recombination, you really shut down the virus,” added Andrew Routh, Ph.D., assistant professor of Biochemistry and Molecular Biology at UTMB and, with Denison, the paper’s co-corresponding author. “It’s really intriguing in terms of what we understand about virus adaptation and evolution.”

Previous studies have shown that coronaviruses are resistant to many nucleoside antiviral drugs, which work by introducing errors in the viral genetic code to block replication. The coronavirus proofreader corrects the errors so replication can proceed.

Only a few drugs are capable of circumventing the proofreader. They include an approved drug, remdesivir, and EIDD-2801 (molnupiravir), an investigational drug now in clinical trials. Both were developed with the help of VUMC scientists.

“Finding that the viral ExoN plays a key role in recombination is exciting,” said Denison, director of the Division of Pediatric Infectious Diseases at VUMC who has studied coronaviruses for more than 30 years.

“Knocking out this function (in laboratory studies) leads to decreased recombination and a weaker virus,” Denison said. “So we think it may be possible to block this process with drugs as well (and) that it may make other drugs like remdesivir and molnupiravir work even better and last longer.”

In 2007 Denison and his colleagues discovered the coronaviruses proofreader. They also found that blocking the enzyme accelerated the rate of uncorrected errors – mutations – and crippled its ability to cause disease in animals.

Several years later they discovered that remdesivir, an investigational antiviral drug, had highly potent activity against a wide range of coronaviruses, both in laboratory and animal tests. In October 2020 remdesivir was approved for emergency use in patients hospitalized with COVID-19.

For the past two years, Gribble and Routh have collaborated in an effort to understand the role of recombination in the replication of RNA viruses, which include influenza, polio, measles, hepatitis C, HIV and Ebola, as well as the coronaviruses.

Using computational software Routh had developed, which can scour virus-sequencing datasets for evidence of “recombination events,” Gribble was studying recombination in model experimental viruses, such as coronaviruses that infect mice.

Once the pandemic hit, Routh, Gribble and their colleagues were quickly able to apply this approach to SARS-CoV-2 and other coronaviruses that cause disease in humans. Other VUMC co-authors were Laura Stevens, MS, Maria Agostini, Ph.D., Jordan Anderson-Daniels, Ph.D., James Chappell, MD, Ph.D., Xiaotao Lu, MS, and Andrea Pruijssers, Ph.D.

Recombination does not always result in a “fitter,” potentially more virulent virus, Routh noted. If during recombination, for example, some of the genome is deleted, the result is a “defective” viral genome that can mix with, and disable, the more virulent strain.

Coronaviruses frequently produce defective genomes, the researchers found. “That could be useful,” Routh said. “You might be able to exploit defective genomes as a way of making new vaccines … or to perturb replication (of a more virulent strain) … in the patient.”

Much remains to be learned about recombination and the role that plays in the continued spread of evolving variants of SARS-CoV-2 around the world and the ability of anti-viral drugs and vaccines to stop it.

That’s why basic science is so important, said Denison, who holds the Edward Claiborne Stahlman Chair in Pediatric Physiology and Cell Metabolism in the Vanderbilt University School of Medicine.

“We need to understand the capacity of all kinds of viruses to move between species and the mechanisms by which they cause disease,” he said. “We need to make sure that there are fundamental things that we know about all identified viruses—their genomic sequences, for example, and some basics about their biology.”

That takes a lot of creativity, determination—and money. Funding for this study was provided by National Institutes of Health grants AI108197, GM065086 and AI133952, the Dolly Parton COVID-19 Research Fund and the Elizabeth B. Lamb Center for Pediatric Research at Vanderbilt.


RNA viruses commonly exhibit high mutation rates, a feature attributed to the relatively poor fidelity of their RNA-dependent RNA polymerase (RdRp) and the fact that nucleotide incorporation errors go uncorrected. This lack of proofreading contributes to the generation of “quasispecies” populations, clouds of genome sequence variants that are subject to continuous natural selection (1,–3).

On the one hand, their genetic heterogeneity allows RNA viruses to rapidly adapt to changing circumstances, in order to overcome environmental challenges such as host switching, antiviral drug treatment, or host immune responses (4, 5). On the other hand, the accumulation of an excessive number of deleterious mutations can result in “error catastrophe” and, consequently, in the extinction of a viral species (6,–8).

In order to balance these opposing principles, RNA viruses are thought to operate close to their so-called error threshold, while balancing the interdependent parameters of replication fidelity, genome size, and genome complexity (9, 10). This interplay is thought to have restricted the expansion of RNA virus genome sizes, which are below 15 kb for most RNA virus families (10,–12).

The largest RNA virus genomes currently known are found in the order Nidovirales, which includes the family Coronaviridae and also the recently discovered planarian secretory cell nidovirus (PSCNV) (12, 13), which has the largest RNA genome identified thus far (41.1 kb).

One of the molecular mechanisms potentially driving the unprecedented expansion of nidovirus genomes was discovered about 17 years ago, during the in-depth bioinformatics analysis of the genome and proteome of the severe acute respiratory syndrome coronavirus (SARS-CoV).

During this analysis, Alexander Gorbalenya and colleagues identified a putative 3′-to-5′ exoribonuclease (ExoN) signature sequence in the N-terminal domain of nonstructural protein 14 (nsp14), a subunit of the large replicase polyprotein encoded by CoVs and related large-genome nidoviruses. Strikingly, this ExoN domain was found to be lacking in the replicases of nidoviruses with small(er) genomes (specifically, arteriviruses), and therefore, it was proposed that the enzyme may provide a form of proofreading activity that could have promoted the expansion of large nidoviral genomes to their current size (10,–12, 14).

Comparative sequence analysis with cellular homologs classified the nidoviral/CoV ExoN domain as a member of the superfamily of DEDDh exonucleases, which also includes the proofreading domains of many DNA polymerases as well as other eukaryotic and prokaryotic exonucleases (15). These enzymes catalyze the excision of nucleoside monophosphates from nucleic acids in the 3′-to-5′ direction, using a mechanism that depends on two divalent metal ions and a reactive water molecule (16,–18). Five conserved active-site residues arranged in three canonical motifs (I, II, and III) (Fig. 1) orchestrate ExoN activity (14, 19,–21). Additionally, the domain incorporates two zinc finger (ZF) motifs (10), ZF1 and ZF2 (Fig. 1), that were hypothesized to contribute to the structural stability and catalytic activity, respectively, of ExoN (20).

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FIG 1
Alignment of nsp14 amino acid sequences from selected coronaviruses. Sequences of the ExoN and N7-MTase domains in MERS-CoV (NC-019843), SARS-CoV (NC_004718), SARS-CoV-2 (NC_045512.2), MHV (NP_045298), HCoV-229E (NC_002645), TGEV (AJ271965), and IBV (NP_040829) were used for the analysis. The domains indicated at the top are based on the SARS-CoV-nsp14 secondary structure (PDB 5NFY) (21). Fully conserved residues are shown with white letters on dark gray (above 70% conservation), whereas partially conserved residues are displayed with lighter shades of gray. Catalytic residues and residues involved in formation of zinc fingers are marked with asterisks and circles, respectively. Filled circles indicate zinc fingers targeted by mutagenesis (Fig. 2A), while arrowheads identify the two N7-MTase domain residues mutated to generate the MTase negative control used in biochemical assays. The alignment was generated using Clustal Omega (104) and edited using Jalview version 2.11 (105).

The predicted 3′-to-5′ exoribonuclease activity of the CoV ExoN domain was first confirmed in vitro, in biochemical assays using recombinant SARS-CoV nsp14 and different synthetic RNA substrates (19). Originally, residues D90/E92 (motif I), D243 (motif II), and D273 (motif III) were identified as putative active-site residues of SARS-CoV ExoN (14, 19).

However, the SARS-CoV nsp14 crystal structure revealed E191 rather than D243 to be the acidic active residue in motif II, demonstrating that ExoN is in fact a DEEDh enzyme (20). By using reverse genetics for the alphacoronavirus human coronavirus 229E (HCoV-229E), Minskaia et al. demonstrated that inactivation of the ExoN active site results in failure to recover infectious viral progeny (19).

Interestingly, a quite different phenotype was described for the corresponding ExoN knockout mutants of two betacoronaviruses, mouse hepatitis virus (MHV) and SARS-CoV. While ExoN inactivation decreased replication fidelity in these viruses, conferring a ‘mutator phenotype’, the mutants were viable, both in cell culture (22, 23) and in animal models (24).

These findings suggested that ExoN may indeed be part of an error correction mechanism. Subsequently, the ability of ExoN to excise 3′-terminal mismatched nucleotides from a double-stranded RNA (dsRNA) substrate was demonstrated in vitro using recombinant SARS-CoV nsp14 (25). Furthermore, this activity was shown to be strongly enhanced (up to 35-fold) by the addition of nsp10, a small upstream subunit of the CoV replicase (26).

The two subunits were proposed to operate, together with the nsp12 RdRp, in repairing misincorporations that may occur during CoV RNA synthesis (21, 27). In cell culture, MHV and SARS-CoV mutants lacking ExoN activity exhibit increased sensitivity to mutagenic agents like 5-fluorouracil (5-FU), compounds to which the wild-type 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).

Outside the order Nidovirales, arenaviruses are the only other RNA viruses known to employ an ExoN domain, which is part of the arenavirus nucleoprotein and has been implicated in fidelity control (31) and/or immune evasion, the latter by possibly degrading viral dsRNA (32, 33). Based on results obtained with transmissible gastroenteritis virus (TGEV) and MHV ExoN knockout mutants, the CoV ExoN activity was also suggested to counteract innate responses (34, 35).

In the meantime, CoV nsp14 had been proven to be a bifunctional protein by the discovery of a guanine-N7-methyltransferase (N7-MTase) activity in its C-terminal domain (36) (Fig. 1). This enzymatic activity was further corroborated in vitro, using biochemical assays with purified recombinant SARS-CoV nsp14.

The enzyme was found to be capable of methylating cap analogues or GTP substrates, in the presence of S-adenosylmethionine (SAM) as methyl donor (36, 37). The N7-MTase was postulated to be a key factor for equipping CoV mRNAs with a functional 5′-terminal cap structure, as guanine-N7-methylation is essential for cap recognition by the cellular translation machinery (25). Although the characterization of the nsp14 N7-MTase active site and reaction mechanism was not completed, alanine scanning mutagenesis and in vitro assays with nsp14 highlighted several key residues (Fig. 1) (36, 38, 39).

Moreover, crystal structures of SARS-CoV nsp14 in complex with its nsp10 cofactor (PDB entries 5C8U and 5NFY) revealed several unique structural and functional features (20, 21). These combined structural and biochemical studies confirmed that the two enzymatic domains of nsp14 are functionally distinct (36) and physically independent (20, 21).

Still, the two activities are structurally intertwined, as it seems that the N7-MTase activity depends on the integrity of the N-terminal ExoN domain, whereas the flexibility of the protein is modulated by a hinge region connecting the two domains (21).

Coronaviruses are abundantly present in mammalian reservoir species, including bats, and pose a continuous zoonotic threat (40,–43). To date, seven CoVs that can infect humans have been identified, and among these, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently causing an unprecedented pandemic outbreak. The previous zoonotic CoV to emerge, in 2012, was the Middle East respiratory syndrome coronavirus (MERS-CoV) (44).

Due to its transmission from dromedary camels and subsequent nosocomial transmission, MERS-CoV continues to circulate and cause serious human disease, primarily in the Arabian Peninsula (45). Occasional spread to other countries has also occurred, including an outbreak with 186 confirmed cases in South Korea in 2015 (46,–48).

Like SARS-CoV, SARS-CoV-2, and MHV, MERS-CoV is classified as a member of the genus Betacoronavirus, although it belongs to a different lineage (subgenus) of that cluster (49, 50). The current lack of approved therapeutics and vaccines to prevent or treat CoV infections, as well as the general threat posed by emerging CoVs, necessitates the further in-depth characterization of CoV replication and replicative enzymes.

In this context, the quite different phenotypes described for ExoN knockout mutants of other CoVs (see above) prompted us to study the importance of this enzyme for MERS-CoV replication. To this end, using both reverse genetics and biochemical assays with recombinant nsp14, we engaged in an extensive site-directed mutagenesis study, targeting all active-site motifs of the MERS-CoV ExoN domain.

Strikingly, in contrast to what was observed for two other betacoronaviruses, MHV and SARS-CoV, our studies revealed that ExoN inactivation severely impacts MERS-CoV replication, resulting in failure to recover viable progeny. While completing our MERS-CoV nsp14 studies, given the developing pandemic, we also evaluated the impact of ExoN inactivation (using a D90A/E92A ExoN motif I double mutant) on SARS-CoV-2 replication and viability.

Given the close phylogenetic relationship between SARS-CoV and SARS-CoV-2, reflected for example in 95% nsp14 amino acid identity (51), we were highly surprised to find that—as for our MERS-CoV ExoN knockout mutants—it was not possible to rescue viable progeny for this SARS-CoV-2 mutant in which two key residues of the ExoN active site were mutated.

Our biochemical evaluation of MERS-CoV nsp14 mutants suggested that this phenotype is not caused by inadvertent side effects of ExoN inactivation on N7-MTase activity. Our combined data suggest that CoV ExoN and/or nsp14 plays a more direct and fundamental role in CoV RNA synthesis than merely safeguarding the long-term fidelity of replication and can thus be considered a prominent target for the development of antiviral drugs.

reference link : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654266/


More information: Jennifer Gribble et al, The coronavirus proofreading exoribonuclease mediates extensive viral recombination, PLOS Pathogens (2021). DOI: 10.1371/journal.ppat.1009226

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