Newly identified helicase might hold the key to a novel family of anti-COVID-19 drugs

By Hugo Francisco de SouzaReviewed by Lily Ramsey, LLMAug 10 2023

In a recent study published in the PNAS Journal, researchers used a multidisciplinary approach to investigate a nonstructural protein essential in SARS-CoV-2 replication.

They characterized the protein and discovered how it contributes to RNA replication. They also discovered the inhibitory effects of the stable nitroxide TEMPOL against the protein, paving the road for a new class of anti-viral drugs against the disease.

Study: An iron–sulfur cluster in the zinc-binding domain of the SARS-CoV-2 helicase modulates its RNA-binding and -unwinding activities. Image Credit: MiniStocker/Shutterstock.com

SARS-CoV-2 replication and RdRp

The severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) is a strain of coronavirus (SARSr‑CoV) that caused the coronavirus disease 2019 (COVID-19) pandemic.

It is a positive-sense single-stranded RNA virus (+ssRNA) from the same family as the SARS virus (SARS‑CoV‑1) and Hepatitis C. These viruses lack DNA, with their RNA serving as messenger RNA and template genome.

In SARS‑CoV‑2, the RNA genome is 30 kb in size and comprises structural- (sp) and nonstructural proteins (nsp), which perform both viral transcription and replication.

Previous studies have identified 16 nsp, three of which collectively make up the RNA-dependent RNA polymerase (RdRp) – nsp12 (catalytic subunit), nsp7, and nsp8 (auxiliary factors). While a growing body of literature characterizing the structure and function of these three for their potential in anti-viral drug therapy exists, the remaining 13 nsp have been largely ignored.

Recent research has begun exploring the functional properties of helicases (enzymes mainly responsible for DNA or RNA unwinding) belonging to the helicase superfamily 1B (SF1B).

One of these, nsp13, is interesting in that its independent helicase activity is poor and inefficient, but when paired with the RdRp complex, shows a significant augmentation in its RNA unwinding action, thereby playing a vital role in the replication of SARS‑CoV‑2. Research has additionally suggested nsp13 as essential in 5' mRNA cap formation.

The 5' mRNA cap is critical in protecting the RNA from environmental RNA exonucleases (enzymes that damage RNA) and promoting viral translation by the infected hosts' ribosomes.

Electron microscopy and X-ray crystallography of the nsp13 structure have shown it to be one of the most structurally conserved parts of the SARSr‑CoV genome. Genetic analyses have confirmed this by elucidating that nsp13s in SARS‑CoV‑2 and SARS‑CoV‑1 differ by a conservative change in only one amino acid (V570I).

One of the main difficulties in developing vaccines and anti-viral drugs against COVID-19 is its high mutation rate. Vaccines and medications developed against one strain usually show poor efficiency against a different strain.

Focusing efforts on genomic regions that remain largely conserved across strains has the potential for developing drugs effective against a broad range of strains. Research thus suggests that compounds effective at inhibiting the action of SARS-CoV-2 nsp13 would have similar effects on SARS-CoV-1 and future SARS‑CoV variants.

About the study

In the present study, researchers added to their previous work on nsp characterization and functional evaluation. They investigated nsp13 using a multidisciplinary approach incorporating inductively coupled plasma mass spectrometry (ICP-MS), UV-visible absorption, electron paramagnetic resonance (EPR), and Mössbauer spectroscopies to characterize the structure.

These spectrometric techniques were conducted on recombinant strains of SARS‑CoV‑2 expressed in Expi293F mammalian cells (a human in vitro cell line). Strains were designed to overexpress nsp13, and site-directed mutagenesis was used to create multiple Expi293F clones will point mutations in the nsp13 region.

This was done so that downstream comparisons between wild-type (WT) and clone nsp13 inhibitions could be made. Proteins were extracted, isolated, and purified using subcellular fractionation and immunoprecipitation (IP).

Researchers began characterization by using mass spectrometry to evaluate the chemical peptide composition of nsp13 and quantify its expression amount by different Expi293F clones.

ICP-MS was then employed to evaluate the proteins' total zinc and iron content. Ferrozine-based colorimetric assays were used to quantify the amount of Fe²⁺ and Fe³⁺  in each nsp13 sample.

UV-vis absorption spectroscopy was used to quantify the densities of Expi293F transfected colonies. Amino acid analysis (AAA) was used to quantify the nsp13 protein contents of the colonies. EPR and Mössbauer spectroscopies were used to investigate the type and stoichiometry of iron-sulfur cluster(s) in nsp13.

Researchers finally used helicase unwinding activity assays to evaluate the efficiency of SARS‑CoV‑2 unwinding by WT and mutant nsp13.

Since their results indicated that the iron content of nsp13 played a vital role in its helicase property, metal-free (apo-) nsp13 was obtained by treating the purified protein with ethylenediaminetetraacetic acid (EDTA) and passing the mixture through a sample cleanup gravity column.

Zinc was reconstituted into the protein at values matching those from ICP-MS analyses to reduce the confounding effects of zinc ion removal.

Study findings

In their previous work on nsp12 (the main catalytic subunit of RdRp), the present research group identified two cubane [Fe₄S₄] iron-sulfur (Fe-S) clusters in the protein. It established the critical role that Fe-S clusters play in CoV replication and the interaction of nsp12 with nsp13.

In the present work, proteomic analysis from mass spectrometry data identified a leucine-tyrosine-lysine (LYK) motif in nsp13, which was indicated to function as the binding site for nsp12 Fe-S clusters.

This suggests the mechanism behind Fe-S transfer from nsp12 to nsp13. WT nsp13 was seen to have zinc and iron ions. Nsp13 variants lacking the LYK motif were observed to retain zinc but lose iron ions, implying replacement by alanines (α-amino acids with the chemical formula C₃H₇NO₂).

(UV-vis) absorption spectroscopy suggested that purified nsp13 expressed in Expi293F cells harbored  Fe–S clusters. Mössbauer spectrum analysis was used to determine the type and stoichiometry of nsp13 and its components. The role of the Fe–S cluster in nsp13 was revealed by comparing variants with and without the cluster and those with and without zinc ions.

"Loss of the Fe–S cluster in nsp13C50S-C55S impaired the unwinding activity of the helicase, likely as a result of diminished binding of the variant to the substrate, whereas the absence of the zinc ions from either of the two metal-binding sites in the nsp13^C5S-C8S, nsp13^C26S-C29S, nsp13^C16S-C19S, and nsp13^H33S-^H39S variants did not affect the unwinding activity."

Of the three US Food and Drug Administration (FDA) drugs used as anti-viral to SARS-CoV-2 replication, at least one has been ineffective against novel drug-resistant strains. This is due to its mechanism of action targeting the RdRp, a rapidly evolving part of the SARS-CoV-2 genome.

This necessitates the development of anti-virals that target conserved RNA segments, one of which is TEMPOL. TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) is a stable nitroxyl antioxidant.

This research identified TEMPOL effectively inhibiting SARS-CoV-2 replication by targeting the RdRp and nsp13.

"We propose that TEMPOL could be regarded as an anti-viral that works through a different mechanism than other anti-virals; its likely low toxicity could make it attractive for use as an oral postexposure preventative treatment against SARS-CoV-2."

Conclusion

In the present study, researchers investigated the structure and function of nsp13, a nonstructural protein present in coronavirus strains. They utilized multiple spectrometric techniques to elucidate its structure and stoichiometry.

These analyses revealed that the nsp13 plays a vital yet underestimated role in SARSr‑CoV replication by remarkably increasing its RNA unwinding ability on accepting Fe-S from nsp12, the catalytic subunit of the RdRp complex.

Nsp13 is one of the most conserved regions of the SARSr‑CoV genome. This research identified and recommended TEMPOL, a stable nitroxyl antioxidant that inhibits the nsp13, as an effective broad-spectrum anti-viral, with its low toxicity to humans making it an attractive candidate in the oral postexposure intervention against SARS-CoV-2.