Biochemical and genetic analyses of murine hepatitis virus Nsp15 endoribonuclease

Area of Science:

• Virology
• Molecular Biology
• Enzymology

Background:

• Murine hepatitis virus (MHV) is a significant pathogen.
• The Nsp15 endoribonuclease (mNsp15) is essential for viral replication.
• Understanding mNsp15's enzymatic activity is key to controlling MHV infection.

Purpose of the Study:

• To investigate the relationship between mNsp15 endoribonuclease activity and MHV infection.
• To characterize the impact of mutations in the mNsp15 catalytic pocket on its function.
• To assess the role of mNsp15 in viral RNA synthesis and replication.

Main Methods:

• Molecular modeling to compare mNsp15 with SARS-CoV orthologs.
• Site-directed mutagenesis to alter key residues in the mNsp15 catalytic pocket (H262, H277, G275).
• Generation and characterization of mutant MHV viruses (vH262A, vH277A, vG275A, vH262P+H277A).
• Plaque assays, plaque-forming unit (PFU) enumeration, and analysis of viral RNA and protein levels.
• In trans expression experiments to assess rescue of viral RNA synthesis.

Main Results:

• Mutations in the mNsp15 catalytic pocket reduced endoribonuclease activity and uridylate cleavage specificity.
• Mutant viruses with catalytic site mutations (vH262A, vH277A, vG275A, vH262P+H277A) were viable but showed reduced PFU and viral RNA/protein accumulation.
• A mutation at a non-catalytic residue (D324A) resulted in an insoluble protein and inability to recover the virus.
• In trans expression of wild-type mNsp15 partially restored RNA synthesis in a mutant virus.

Conclusions:

• mNsp15's endoribonuclease activity is essential for optimal MHV replication.
• Specific catalytic residues and their enzymatic function are critical for viral propagation.
• mNsp15 plays a vital role in the synthesis of viral subgenomic RNAs and spike protein.
• Targeting mNsp15 activity represents a potential strategy for antiviral development against MHV.