Yale Study Reveals That Enzyme Present In SARS-Cov-2 Known As Coronavirus 3′-5′ Exoribonuclease (ExoN) Is Responsible For Antiviral Drug Resistance
: A new research led by scientists from Yale University along with other experts from University of Minnesota and Iowa State University has found that a crucial enzyme known as the Coronavirus 3′–5′ exoribonuclease (ExoN) promotes antiviral resistance hence making many identified drugs such as remdesivir, ivermectin and even identified antiviral phytochemicals redundant after a while.
The Coronavirus 3′–5′ exoribonuclease (ExoN)
enzyme, residing in the nonstructural protein (nsp) 10-nsp14 complex, boosts replication fidelity by proofreading RNA synthesis and is critical for the virus life cycle.
ExoN also recognizes and excises nucleotide analog inhibitors incorporated into the nascent RNA, undermining the effectiveness of nucleotide analog-based antivirals.
The study team revealed via cryo-electron microscopy structures of both wild-type and mutant SARS-CoV-2 nsp10-nsp14 in complex with an RNA substrate bearing a 3′-end mismatch at resolutions ranging from 2.5 Å to 3.9 Å. The structures reveal the molecular determinants of ExoN substrate specificity and give insight into the molecular mechanisms of mismatch correction during coronavirus RNA synthesis.
The study findings provide guidance for rational design of improved anti-coronavirus therapies.
The study findings were published in the peer reviewed journal: Science https://science.sciencemag.org/content/early/2021/07/26/science.abi9310
The Coronavirus 3′–5′ exoribonuclease (ExoN) is a proofreading enzyme that removes the wrong nucleosides from the ribonucleic acid (RNA), which is the genetic material of SARS-CoV-2, thus ensuring faithful replication of the SARS-CoV-2 viral genome in the human host.
The study findings describe the structure of a crucial enzyme present in the SARS-CoV-2, which is the coronavirus responsible for COVID-19 disease.
It should be noted that the introduction of nucleosides to inhibit the RNA-dependent RNA polymerase (RdRp) enzyme is one important mechanism of antiviral drugs such as remdesivir; however, the presence of ExoN renders these drugs useless.
Hence understating the structure of ExoN could help identify or assist in the development of inhibitory molecules to ultimately accelerate drug discovery processes.
So far there are no effective antivirals against SARS-CoV-2. However, its replication/transcription complex (RTC) is a promising target, along with its core of RdRp and other non-structural proteins (nsps). Nucleotide analogs like remdesivir insert the wrong nucleotide into the SARS-CoV-2 genome in order to arrest replication; however, this event can be rescued by the proofreading activity of ExoN.
The ExoN enzyme, which is found as the N-terminal ExoN domain of the viral non-structural protein 14 (nsp14), is stimulated by nsp10, which also stabilizes its active site structure. ExoN also breaks down viral double-stranded RNA (dsRNA), which would otherwise activate ho
st-pathogen recognition receptors (PRRs).
Importantly this contributes to the immune escape capabilities of SARS-CoV-2.
The study findings discusses the molecular binding of substrates by ExoN, as well as how this enzyme recognizes and removes wrongly inserted nucleotides or analogs in the newly forming RNA strand.
The study team used a hairpin RNA substrate known as TSP31 to explore the catalytic activity of ExoN. This allowed binding to RNA but not RNA cleavage by the enzyme.
Utilizing a combination of size-exclusion chromatography (SEC), single-particle cryo-electron microscopy (EM), and in silico methods, the researchers found that the nsp10-nsp14-RNA complex binds also to nsp8, though in a weak and dynamic manner. This binding promotes stable RNA substrate binding that allows for RNA cleavage by ExoN. It also increases RNA degradation by the nsp10-nsp14 complex.
Interestingly as a common factor to both RdRp and ExoN enzyme complexes, nsp8 appears to play a crucial role in transferring the RNA substrate between these molecules. However, more work will need to be conducted to determine how nsp8 corrects mismatched nucleotides.
Also when a mutant form of the ExoN was studied, part of the sample contained the tetramerized form of the nsp10-nsp14-RNA complex, which appears to prevent nsp8 binding.
The study team found that the binding of the RNA substrate to the ExoN active site of the SARS-CoV-2 wildtype (WT) nsp10-nsp14-RNA complex causes two metal ions to bind to the catalytic center. This triggers further reactions that complete the active site.
Most significantly, the detailed description of the interactions with the dsRNA suggests that the enzyme maintains its specificity for the substrate.
Additionally, these study findings provide information on the structure of the substrate relative to other RNA viral and proofreading ExoNs, as well as the predicted SARS-CoV ExoN.
It should be noted that all coronavirus ExoNs share the same RNA-contacting residues in nsp14. Thus, they have the same substrate recognition mechanism.
Additionally, the study findings shed light on the development of ExoN-resistant nucleotide analog inhibitors. In particular, the study showed that a free 3′-OH of the RNA substrate is critical for exonucleolytic degradation by ExoN. It has been shown that 3′-deoxy ribonucleotides can be efficiently incorporated into nascent RNA by RdRp from other positive-strand RNA viruses, such as HCV and poliovirus, and subsequently block RNA extension. Therefore, 3′-deoxy nucleotide analogs can potentially act as effective coronavirus RdRp chain terminators that also resist ExoN excision. Nonetheless, modifications at other positions on the ribose ring are also worth further exploration.
Just as important to note, the SARS-CoV-2 ExoN was also found to accept both single-stranded RNA (ssRNA) and dsRNA substrates, thereby indicating that mismatch correction in vivo may operate in at least two different ways.
The study team also observed that RNA containing remdesivir monophosphate (RMP), following the incorporation of the inhibitor, could still be bound by the viral ExoN. This agrees with the finding that this RMP-terminated RNA remains vulnerable to the enzyme, while the absence of the ExonN increases its susceptibility. Other modifications should also be explored.
The study findings highlight also how mismatch correction occurs during RNA synthesis by the SARS-CoV-2.
The study team also elucidates which parts of the enzyme architecture are required for its recognition of the RNA substrate and catalytic action.
In conclusion, the study team offers several suggestions on possible alterations that could be made on the ribose structure to prevent ExoN-mediated resistance to nucleotide analog RdRp inhibitors. This new but critical data could be particularly useful during the design of powerful ExoN inhibitors that can be combined with the drugs for more effective action against SARS-CoV-2.
The team concluded, “Understanding this structure and the molecular details of how ExoN works can help guide further development of antivirals.”
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