Coronavirus JHM: coding assignments of subgenomic mRNAs

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Murine hepatitis virus (MHV) JHM infection disrupts host cell protein synthesis.
• Understanding viral protein synthesis is crucial for comprehending viral replication and pathogenesis.

Purpose of the Study:

• To identify and characterize virus-specific polypeptides synthesized during MHV JHM infection.
• To determine the messenger RNA (mRNA) molecules responsible for encoding these viral proteins.
• To elucidate the independent translation and gene order of MHV JHM viral proteins.

Main Methods:

• Hypertonic shock to inhibit and then reinitiate protein synthesis in infected cells.
• In vitro translation of polyadenylated RNA from infected cells.
• Immunoprecipitation and peptide mapping to identify and compare polypeptides.
• Two-dimensional polyacrylamide gel electrophoresis for polypeptide analysis.
• Isolation and in vitro translation of specific viral RNA fractions.

Main Results:

• Six virus-specific polypeptides (150K, 65K, 60K, 30K, 23K, 14K) were identified.
• In vitro translation products corresponded to several of these viral polypeptides (120K, 60K, 30K, 23K, 14K).
• Specific mRNAs were assigned to encode individual viral polypeptides, with mRNA 3 encoding the 120K polypeptide, mRNA 7 encoding the 60K, mRNA 2 encoding the 30K, mRNA 6 encoding the 23K, and mRNAs 4 or 5 encoding the 14K.
• These findings demonstrate that viral polypeptides are encoded by distinct subgenomic mRNAs and translated independently.

Conclusions:

• MHV JHM infection utilizes separate subgenomic mRNAs for the synthesis of its specific polypeptides.
• Each viral mRNA is translated independently, contributing to the overall viral protein production.
• The identified coding functions and RNA relationships allow for the deduction of the MHV JHM gene order.