In vitro replication of mouse hepatitis virus strain A59

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Mouse hepatitis virus (MHV) is a significant pathogen causing various diseases in mice.
• Understanding the molecular mechanisms of MHV replication is crucial for developing antiviral strategies.

Purpose of the Study:

• To establish an in vitro system for studying MHV strain A59 replication.
• To investigate the role of protein synthesis and specific viral proteins in MHV genomic RNA replication.

Main Methods:

• Development of an in vitro replication system using lysolecithin-treated cell extracts from MHV-infected cells.
• Incorporation of radiolabeled UMP into RNA and analysis by hybridization, gel electrophoresis, and density gradient centrifugation.
• Inhibition studies using puromycin and antisera against MHV proteins.

Main Results:

• A linear rate of radiolabeled UMP incorporation into genome-sized RNA was observed in vitro.
• In vitro RNA synthesis correlated with in vivo uridine incorporation and was sensitive to puromycin, indicating a requirement for ongoing protein synthesis.
• Antiserum against the MHV nucleocapsid (N) protein significantly inhibited RNA synthesis, while antisera against glycoproteins E1 and E2 did not.
• In vitro synthesized RNA formed ribonucleoprotein complexes similar to authentic MHV nucleocapsids.

Conclusions:

• Ongoing protein synthesis is essential for mouse hepatitis virus genomic RNA replication.
• The MHV N protein plays a critical role in the replication process.