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Properties and kinetics of development of Rous sarcoma virus-infected cells evidenced by methylene blue staining
Abstract:
Three different kinds of areas of infected cells corresponding to different focus formation stages can be evidenced by methylene blue (MB) staining in cultures of chick embryo (CE) fibroblasts infected at low multiplicity with the temperature-sensitive (ts) mutant of Rous sarcoma virus (RSV), FU19, which transforms these fibroblasts at 37 degrees but not at 41 degrees. These are: (a) areas of MB-stainable cells with transformed phenotype (STP areas=foci); (b) areas of MB-stainable cells with normal phenotype (SNP areas), and (c) areas of MB-unstainable cells with normal phenotype (USNP areas). DNA and RNA synthesis and virus production were followed in these various stages at 37 degrees and at 41 degrees. The results show that when cultures are shifted from 37 degrees to 41 degrees, virus production in the SNP and USNP areas which arise by phenotypic reversion of STP foci remains comparable to that of the latter foci. On the contrary, DNA and RNA synthesis are markedly reduced in SNP and USNP areas, DNA synthesis falling down to the level of uninfected cells, and RNA synthesis remaining somewhat higher. The kinetics of development of SNP and STP areas in cultures infected with FU19 and with the parental virus SR4 were also compared. The results confirm that SNP areas are precursors of STP areas but that this passage occurs at a slower rate in cultures infected with FU19.
Insights
Temperature-sensitive Rous sarcoma virus (RSV) FU19 shows that cells reverting to a normal phenotype still produce virus, but with reduced DNA and RNA synthesis. This highlights distinct cellular responses during viral infection and transformation.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Chick embryo fibroblasts infected with Rous sarcoma virus (RSV) exhibit distinct cellular phenotypes.
- Temperature-sensitive (ts) RSV mutants, like FU19, allow for conditional studies of viral transformation.
Purpose of the Study:
- To investigate the characteristics of Rous sarcoma virus (RSV)-infected chick embryo fibroblast (CEF) cultures at different temperatures.
- To analyze DNA and RNA synthesis, and virus production in relation to cellular phenotype during RSV infection.
Main Methods:
- Methylene blue (MB) staining to differentiate cell areas based on phenotype (transformed, normal stained, normal unstained).
- Infection of CEF with ts-RSV mutant FU19 at permissive (37°C) and non-permissive (41°C) temperatures.
- Monitoring of DNA synthesis, RNA synthesis, and virus production.
Main Results:
- Three distinct areas were observed: MB-stainable transformed phenotype (STP), MB-stainable normal phenotype (SNP), and MB-unstainable normal phenotype (USNP).
- Upon temperature shift from 37°C to 41°C, SNP and USNP areas (phenotypic reversion) maintained comparable virus production to STP areas.
- DNA and RNA synthesis were significantly reduced in SNP and USNP areas at 41°C, with DNA synthesis dropping to uninfected cell levels.
Conclusions:
- Phenotypic reversion in ts-RSV infected cells does not abolish virus production.
- Reduced nucleic acid synthesis in reverted cells suggests a complex interplay between viral transformation and cellular metabolism.
- SNP areas are confirmed precursors to STP areas, with FU19 exhibiting a slower transition rate.