Regulation of sugar transport in chick embryo fibroblasts infected with a temperature-sensitive mutant of RSV

Abstract

Chick embryo fibroblasts, infected with a temperature-sensitive mutant of the Rous Sarcoma virus Ts-68 and cultured at the permissive temperature (37°C) for transformation, exhibit an increased V max for sugar transport, compared to that of uninfected fibroblasts and infected fibroblasts cultured at the nonpermissive temperature (41°C) for transformation. Studies were initiated to determine which events in the regulation of transport could account for these differences. Three experimental models were used which result in an increase in the V max for sugar transport: growing the cells in the absence of glucose; shifting the virus-infected cells from 41°C to 37°C; and adding fetal calf serum to serum-deprived cultures. Culturing Ts-68-infected cells at 41°C or 37°C in glucose-free medium for 30 hr resulted in a similar induction of the synthesis of the sugar transport system. This result established that the synthesis of the transport system was not temperature-sensitive in infected cells maintained at 41°C, nor was there a difference in the rate of its degradation. The half-life ( t 1 2 ) of the transport system for cells grown at 37°C was about 5.2 to 6 hr, and was the same for both virus-transformed and uninfected fibroblasts. The t 1 2 for cells cultured at 41°C was also similar for uninfected and infected cells, and was between 3.1 and 3.4 hr. Experiments using cordycepin demonstrated that a metabolic step blocked by this inhibitor was required for the enhancement of sugar transport in Ts-68-infected fibroblasts following a shift in culture temperature from 41°C to 37°C. Actinomycin D was without effect. Ts-68-infected cells cultured at 41°C and uninfected cells both exhibited a rapid decay of transport activity when fetal calf serum was removed from the medium and a biphasic increase in activity following its addition. The rate of sugar transport in Ts-68-infected cells cultured at 37°C was not affected by the removal or addition of serum, indicating that one aspect of transformation is the loss in the ability of cells to effect post-transcriptional and post-translational regulation of the number of sugar transport sites.