Translation of three mouse hepatitis virus strain A59 subgenomic RNAs in Xenopus laevis oocytes
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Abstract:
We have purified the seven virus-specific RNAs which were previously shown to be induced in Sac(-) cells upon infection with mouse hepatitis virus strain A59 (W. J. M. Spaan, P. J. M. Rottier, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 108:424-434, 1981). The individual RNAs, prepared by agarose gel electrophoresis of the polyadenylated RNA fraction from infected cells, were obtained pure, except for the preparations of RNAs 4, 5, and 6, which contained some contamination of RNA 7. The RNAs were microinjected into Xenopus laevis oocytes, and after incubation of these cells in the presence of [35S]methionine, the proteins synthesized were analyzed by polyacrylamide gel electrophoresis. Whereas no translation products of RNAs 1, 2, 4, and 5 were detected, the synthesis of virus-specific polypeptides coded by RNAs 3, 6, and 7 was observed. RNA 7 (0.6 X 10(6) daltons) directed the synthesis of a 54,000-molecular-weight polypeptide which comigrated with viral nucleocapsid protein and which was immunoprecipitated by antiserum from mice that had been infected with the virus. RNA 6 (0.9 X 10(6) daltons) directed the synthesis of three polypeptides with molecular weights of 24,000, 25,500, and 26,500, which migrated with the same electrophoretic mobilities as three low-molecular-weight virion polypeptides. After injection of RNA 3 (3.0 X 10(6) daltons), a polypeptide with a molecular weight of about 150,000 was immunoprecipitated. This polypeptide had no counterpart in the virion, but comigrated with a virus-specific glycoprotein present in infected cells which is immunoprecipitated by a rabbit antiserum against the mouse hepatitis virus strain A59 structural proteins. This antiserum could also immunoprecipitate the translation products of RNAs 3, 6, and 7. These results indicate that RNAs 3, 6, and 7 encode viral structural proteins. The significance of the data with respect to the strategy of coronavirus replication is discussed.
Insights
This study identified three specific RNAs (3, 6, and 7) from mouse hepatitis virus that encode viral structural proteins. These findings offer insights into coronavirus replication strategies.
Area of Science:
- Virology
- Molecular Biology
- RNA Biology
Background:
- Mouse hepatitis virus (MHV) strain A59 infection induces seven virus-specific RNAs in Sac(-) cells.
- Purification of these individual RNAs was achieved using agarose gel electrophoresis.
Purpose of the Study:
- To determine which of the purified virus-specific RNAs encode viral proteins.
- To investigate the role of specific RNAs in the synthesis of viral structural proteins.
- To elucidate aspects of coronavirus replication strategy.
Main Methods:
- Microinjection of purified RNAs into Xenopus laevis oocytes.
- In vitro protein synthesis in oocytes using [35S]methionine.
- Analysis of synthesized proteins by polyacrylamide gel electrophoresis and immunoprecipitation.
- Use of specific antisera for immunoprecipitation of viral proteins.
Main Results:
- RNAs 3, 6, and 7 directed the synthesis of virus-specific polypeptides.
- RNA 7 encoded a 54,000-dalton polypeptide identified as viral nucleocapsid protein.
- RNA 6 produced three low-molecular-weight polypeptides found in the virion.
- RNA 3 synthesized a 150,000-dalton glycoprotein, a virus-specific protein in infected cells.
Conclusions:
- RNAs 3, 6, and 7 are responsible for encoding structural proteins of mouse hepatitis virus.
- The findings contribute to understanding the replication strategy of coronaviruses.