Macro1 domain residue F156: A hallmark of SARS-CoV-2 de-MARylation specificity

Area of Science:

• Virology
• Molecular Biology
• Immunology

Background:

• Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is an RNA virus encoding non-structural proteins, including nsp3.
• The nsp3 protein contains a conserved Macro1 domain involved in ADP-ribosylation.
• Viral Macro domains bind to ADP-ribose moieties and possess hydrolase activity regulating innate immunity and interferon responses.

Purpose of the Study:

• To investigate the role of specific amino acid residues in the SARS-CoV-2 nsp3 Macro1 domain.
• To elucidate the impact of mutations on ADP-ribose binding and de-MARylation activity.
• To understand differences in host interferon response regulation between SARS-CoV-2 and SARS-CoV.

Main Methods:

• Bioinformatic and structural analyses to identify key residues (F156 in SARS-CoV-2, N157 in SARS-CoV).
• Site-directed mutagenesis to create alanine substitutions at these key positions.
• Biochemical assays to assess ADP-ribose binding and de-MARylation (hydrolysis) activity.

Main Results:

• Mutations slightly altered ADP-ribose binding affinity.
• Alanine substitution at F156 in SARS-CoV-2 abolished de-MARylation activity.
• The same substitution in SARS-CoV reduced de-MARylation activity by 70% and hampered poly-ADP-ribose binding.

Conclusions:

• The investigated residues are critical for the de-MARylation function of the nsp3 Macro1 domain.
• Differences in these residues may contribute to the distinct host interferon responses induced by SARS-CoV-2 and SARS-CoV.
• Targeting these enzymatic activities could offer therapeutic strategies against coronaviruses.