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Characterization and translation of transmissible gastroenteritis virus mRNAs
Abstract:
Three protein species were identified in purified transmissible gastroenteritis virus particles (strain Purdue). They are thought to represent constituents of the peplomer (E2; molecular weights of 280,000 and 240,000), the envelope (E1; molecular weights of 28,000, 31,500, and 33,000), and the nucleocapsid (N; molecular weight of 48,000). In infected cells, proteins with molecular weights of 195,000 (E2), 48,000 (N), and 28,000 (E1) were detected. Tunicamycin, an inhibitor of N glycosylation, prevented the appearance of polypeptides with molecular weights of 195,000 and 28,000 in infected cells; instead, proteins with molecular weights of 160,000 and 25,000 were observed. One minor and five major mRNA species were detected in porcine cells after infection. Their size was determined to be 23.6 kilobases (kb) (RNA1), 8.4 kb (RNA3), 3.8 kb (RNA4), 3.0 kb (RNA5), 2.6 kb (RNA6), and 1.9 kb (RNA7). The RNAs were translated in vitro. RNA7 was shown to code for the N protein. Although complete separation of RNA6 could not be achieved, it was shown to encode an unglycosylated (molecular weight of 25,000) precursor of E1 (molecular weight of 28,000). RNA4 was translated into a nonstructural protein with a molecular weight of 24,000. Translation of RNA3 resulted in proteins with molecular weights of 250,000 and 130,000 and smaller molecules which could be precipitated with a monoclonal antibody directed against E2.
Insights
This study identifies key proteins in transmissible gastroenteritis virus (TGEV) and maps their corresponding genes on viral RNA. Understanding these viral protein structures is crucial for developing effective TGEV diagnostics and therapeutics.
Area of Science:
- Virology
- Molecular Biology
- Protein Chemistry
Background:
- Transmissible gastroenteritis virus (TGEV) is a significant pathogen in swine.
- Understanding the structural proteins and gene expression of TGEV is essential for controlling the disease.
Purpose of the Study:
- To identify and characterize the protein species present in purified TGEV particles.
- To determine the molecular weights and potential functions of TGEV structural proteins.
- To map the viral genes responsible for encoding these proteins using mRNA analysis and in vitro translation.
Main Methods:
- Purification and analysis of TGEV particles to identify constituent proteins.
- Detection of viral proteins in infected cells using molecular weight analysis.
- Treatment with tunicamycin to assess the role of N-glycosylation in protein processing.
- Isolation and size determination of viral mRNA species.
- In vitro translation of viral RNA to identify protein-coding sequences.
Main Results:
- Three main protein species (E2, E1, N) were identified in purified TGEV particles.
- In infected cells, E2, N, and E1 proteins were detected, with tunicamycin affecting E2 and E1 glycosylation.
- Six major mRNA species (RNA1, RNA3, RNA4, RNA5, RNA6, RNA7) were identified in infected porcine cells.
- RNA7 codes for the nucleocapsid (N) protein, and RNA6 encodes a precursor to the envelope (E1) protein.
- RNA4 translated to a nonstructural protein, and RNA3 produced proteins associated with the peplomer (E2) and other viral components.
Conclusions:
- The study successfully identified major structural proteins of TGEV and their corresponding mRNA molecules.
- The findings provide a foundation for understanding TGEV gene expression and protein synthesis.
- This knowledge is critical for the development of antiviral strategies and improved diagnostics for TGEV infections.