COVID-19 News: Mutations In Loop 2 Of SARS-CoV-2 Macrodomain 2 In Nsp3 Can Enhance ADP-Ribose Binding And Lead To More Virulent Strains!

COVID-19 News: The ongoing battle against the COVID-19 pandemic has driven scientists worldwide to delve deeper into the intricate mechanisms of the SARS-CoV-2 virus, searching for clues that could unlock potential vulnerabilities. A recent collaborative study by researchers from the University of Kansas, USA, Oklahoma State University, USA, and the College of Veterinary Medicine in Stillwater, Oklahoma, USA, that is covered in this COVID-19 News report has shed light on a critical aspect of the virus - Macrodomain 2 within the nonstructural protein 3 (nsp3). This Macrodomain, referred to as Mac1, has been identified as an ADP-ribosylhydrolase that plays a pivotal role in binding and hydrolyzing mono-ADP-ribose from target proteins. Previous research has highlighted the significance of Mac1 in virus replication and pathogenesis.

SARS-CoV-2 I1153A and F1154A have increased ADP-ribose binding. (A) SARS-CoV-2 Mac1 protein was incubated with free ADP-ribose and binding affinity was measured by isothermal calorimetry. (B) An ADP-ribosylated peptide was incubated with indicated macrodomains at increasing concentrations and Alphacounts were measured. (C) WT, I1153A, and F1154A SARS-CoV-2 Mac1 proteins were incubated with MARylated PARP10 CD in vitro at an [E]/[S] molar ratio of 1:5 for the indicated times at 37°C. ADP-ribosylated PARP10 CD was detected by IB with anti-ADP- ribose binding reagent (MAB1076; MilliporeSigma) while total PARP10 CD protein levels were detected by IB with GST antibody. The reaction with PARP10 CD incubated alone at 37°C was stopped at 0 or 30 min. The data in panels A, B, and C show one experiment representative of three independent experiments. (D) The level of de-MARylation was measured by quantifying relative band intensity (ADP-ribose/GST-PARP10) using ImageJ software. Intensity values were plotted and fitted to a nonlinear regression curve; error bars represent standard deviations.

The Investigation into Mac1
To comprehend how specific biochemical activities within Mac1 impact coronavirus (CoV) replication, the study focused on highly conserved regions, including the glycine-isoleucine-phenylalanine (GIF) motif. Mutations involving isoleucine and phenylalanine residues were introduced, resulting in altered recombinant Mac1 proteins and CoVs, such as murine hepatitis virus (MHV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
 
The research revealed that the phenylalanine (F-A) mutant proteins exhibited defects in ADP-ribose binding and/or hydrolysis, leading to attenuated replication and pathogenesis in cell culture and mice. In contrast, isoleucine (I-A) mutations demonstrated normal enzyme activity but enhanced ADP-ribose binding. Surprisingly, despite increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 were highly attenuated, indicating that the isoleucine residue functions as a gate controlling ADP-ribose binding for efficient virus replication. These findings provide valuable insights into how macrodomains regulate ADP-ribose binding and hydrolysis to promote viral replication and pathogenesis.
 
Coronaviruses and Their Noteworthy Outbreaks
Coronaviruses, bel onging to the family Coronaviridae, have been significant pathogens in both human and veterinary settings. Three major outbreaks have marked recent history: severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002-2003, Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, and the emergence of SARS-CoV-2 in late 2019, causing the COVID-19 pandemic. These viruses have genomes ranging from 26-32 kb, encoding conserved structural proteins, accessory proteins, and non-structural proteins (nsps), with nsp3 being the largest and hosting multiple modular domains, including the critical Mac1.
 
The Crucial Role of Mac1 in CoV Infection
Mac1, a conserved domain present in all CoVs, exhibits a highly conserved structure with central β-sheets surrounded by α-helices. Functionally, Mac1 has been demonstrated to reverse host ADP-ribosylation of target proteins. ADP-ribosylation is a common posttranslational modification catalyzed by ADP-ribosyltransferases (ARTs), crucial for cellular stress responses, including viral infections.
 
Various studies have underlined the critical role of Mac1 in CoV, alphavirus, and Hepatitis E virus infections. Deletion or point mutations of the conserved macrodomain have been shown to render these viruses sensitive to host PARP-mediated antiviral functions. However, the specific residues contributing to Mac1's biochemical functions and their correlation with CoV replication and pathogenesis have been relatively unexplored.
 
Biochemical Activities of Mac1 Residues
Earlier studies primarily focused on mutations involving a highly conserved asparagine residue, revealing its impact on Mac1 deMARylating activity. The mutation led to poor virus replication in mice, emphasizing the significance of this residue in countering PARP activity. Additionally, mutations in residues D1022A, H1045A, G1130V, D1329A, and G1439V were evaluated, highlighting their roles in ADP-ribose binding and hydrolase activity. Notably, the isoleucine and phenylalanine residues in loop 2 of Mac1 drew attention due to their conservation and positioning within the ADP-ribose binding pocket.
 
The Unveiling of Isoleucine and Phenylalanine Mutations
In the recent study, researchers investigated mutations involving the isoleucine and phenylalanine residues in loop 2 of CoV Mac1. Contrary to expectations, the I-A mutation resulted in enhanced ADP-ribose binding, while the F-A mutation exhibited defects in binding and/or hydrolysis, leading to attenuated virus replication. The isoleucine residue, positioned strategically in the ADP-ribose binding domain, acted as a gate controlling ADP-ribose binding, ensuring optimal ADP-ribosylhydrolase activity.
 
Insights from SARS-CoV-2 Mutations
The study further explored the impact of I-A and F-A mutations in SARS-CoV-2 Mac1. Soluble mutant proteins were produced and analyzed for ADP-ribose binding ability. Both I1153A and F1154A SARS-CoV-2 Mac1 proteins exhibited increased ADP-ribose binding compared to the wild-type protein. Interestingly, while F1154A had reduced enzymatic activity, I1153A demonstrated robust enzymatic activity, indicating a complex interplay between ADP-ribose binding and enzyme activity.
 
Increased ADP-Ribose Binding Sensitivity
The enhanced ADP-ribose binding of I1153A and F1154A mutants in SARS-CoV-2 had significant implications for virus sensitivity to interferon-gamma (IFN-γ). In the presence of IFN-γ, both mutants showed a substantial decrease in replication compared to the wild-type virus. This heightened sensitivity to IFN-γ suggested that increased ADP-ribose binding negatively affected Mac1's ability to counter IFN-mediated antiviral responses.
 
Attenuation in Animal Models
To validate the impact of enhanced ADP-ribose binding on viral replication and pathogenesis in vivo, researchers infected K18-ACE2 mice with SARS-CoV-2 I1153A and F1154A mutants. Surprisingly, both mutants were extremely attenuated, causing no weight loss or lethal disease in mice. Viral titers were significantly reduced, and histological analysis revealed diminished signs of disease in the lungs. Moreover, mice infected with the mutants exhibited elevated levels of IFN-I, IFN-III, ISG15, and CXCL-10 mRNA, reminiscent of the effects observed with the Mac1 deletion virus.
 
Molecular Dynamics Unveiling the Isoleucine Gate
To gain insights into how the isoleucine residue functions as a gate controlling ADP-ribose binding, molecular dynamics simulations were performed. The simulations revealed that the isoleucine residue acts as a dynamic gate, allowing the entry of ADP-ribose into the binding pocket. The I1153A mutation, causing enhanced ADP-ribose binding, disrupted the dynamics of the gate, resulting in prolonged interactions with ADP-ribose. While this was beneficial in biochemical assays, it proved detrimental to virus replication, highlighting the delicate balance required for optimal Mac1 function during infection.
 
Conservation Across Viruses
Mac1 is not unique to CoVs; it is found in various RNA viruses, including Togaviruses, Rubiviruses, and Hepatitis E virus. Its conserved nature emphasizes its crucial role in viral infections. The study expands our understanding of how mutations in conserved regions, such as loop 2, can have profound effects on viral replication and pathogenesis across different viruses.
 
Potential Therapeutic Implications
Understanding the intricate relationship between Mac1 mutations and SARS-CoV-2 virulence opens new avenues for therapeutic interventions. Targeting Mac1 and its ADP-ribosylhydrolase activity may offer a novel approach to attenuate viral replication. Small molecules or peptides designed to modulate ADP-ribose binding without compromising enzymatic activity could serve as potential inhibitors, contributing to the development of antiviral strategies.
 
Conclusion
In conclusion, this comprehensive study provides a detailed exploration of the role of Mac1 mutations, specifically isoleucine and phenylalanine residues in loop 2, in regulating ADP-ribose binding and enzymatic activity. The delicate balance between these biochemical functions emerged as a crucial determinant of viral replication and pathogenesis. The findings not only contribute to our understanding of the molecular mechanisms underlying SARS-CoV-2 infection but also offer insights that can be harnessed for the development of targeted therapeutic interventions. As the world continues to grapple with the COVID-19 pandemic, unraveling the mysteries of SARS-CoV-2 at the molecular level becomes increasingly vital for devising effective strategies to mitigate the impact of the virus.
 
The study findings were published on a preprint server and are current being peer reviewed.
https://www.biorxiv.org/content/10.1101/2024.01.03.574082v1
 
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