Related Experiment Videos
Virus-specific early RNA in 3T6 cells infected by a tsA mutant of polyoma virus
1Tumor Virology Laboratory, Salk Institute, San Diego, California 92112, USA.
Abstract:
The accumulation of virus-specific early RNA in mouse 3T6 cells infected by wild type polyoma virus or by a tsA mutant, tsA25E, was measured by hybridization of cytoplasmic RNA to radiolabeled "early" strand polyoma DNA. Cells infected by the tsA25E mutant accumulated approximately 20 times more virus-specific early RNA during the early phase of lytic infection than did wild type-infected cells at both the permissive and the nonpermissive temperature under identical conditions of infection and hybridization. Cells infected by the tsA25E mutant at the permissive temperature continued to accumulate virus-specific early RNA during the late phase of infection after being shifted to the nonpermissive temperature to block further viral DNA replication. A mixed infection of cells by wild-type polyoma and tsA25E showed that the overproduction of early RNA by the tsA mutant alone could be suppressed by coinfection with the wild type. The results suggest that the A gene product of polyoma regulates transcription of early RNA, as has been suggested for SV40 (Reed et al., 1976) and that the wild-type A-gene product overcomes the effect of the temperature-sensitive A-gene product.
Insights
Polyoma virus tsA mutant infection leads to significantly higher early RNA accumulation. Wild-type virus coinfection suppresses this overproduction, indicating the A gene regulates early RNA transcription.
Area of Science:
- Virology
- Molecular Biology
- Gene Regulation
Background:
- Polyoma virus is a DNA tumor virus with distinct early and late gene products.
- The A gene product is implicated in viral DNA replication and transcription regulation.
- Temperature-sensitive mutants, like tsA25E, are crucial for studying essential viral gene functions.
Purpose of the Study:
- To investigate the role of the polyoma virus A gene in regulating early RNA transcription.
- To quantify and compare early RNA accumulation in cells infected with wild-type versus tsA mutant polyoma viruses.
- To determine if wild-type virus can suppress the aberrant RNA accumulation caused by the tsA mutant.
Main Methods:
- Quantitative hybridization of cytoplasmic RNA to radiolabeled early strand polyoma DNA.
- Infection of mouse 3T6 cells with wild-type polyoma virus and the tsA25E mutant under permissive and nonpermissive conditions.
- Analysis of RNA accumulation during early and late phases of infection, including temperature shift experiments.
- Mixed infection experiments involving both wild-type and tsA25E mutant viruses.
Main Results:
- Cells infected with tsA25E mutant accumulated approximately 20 times more virus-specific early RNA than wild-type infected cells.
- Early RNA accumulation by tsA25E continued even after a shift to nonpermissive temperature, blocking DNA replication.
- Coinfection with wild-type polyoma virus suppressed the overproduction of early RNA by the tsA mutant.
Conclusions:
- The polyoma virus A gene product plays a critical role in regulating the transcription of early viral RNA.
- The tsA25E mutation leads to a loss of this regulatory function, resulting in excessive early RNA production.
- Wild-type A gene product can overcome the defect caused by the temperature-sensitive A gene product, suggesting a dominant effect.