Cycloheximide inhibition of delayed early gene expression in baculovirus-infected cells

Abstract

The baculovirus protein IE I is required for the transactivation of many early viral genes in transient expression assays. However, cycloheximide inhibition studies have failed to reveal a dependence of early gene transcription on expression of IE I in infected cells. It is shown here that synthesis of lEl was not effectively inhibited by the addition of 100 mg/ml cycloheximide, the concentration routinely used in these studies. However, when cycloheximide was added at 250 mg/ml, IEI synthesis was repressed to less than five percent of control levels. These more stringent conditions were used to discriminate between immediate early and delayed early genes. Transcription of three immediate early genes (ie1, ie2, ie0) was increased by the addition of high concentrations of cycloheximide. However, transcription of three other early genes (39k, p35, and lef-3), which are known to be dependent upon IEI transactivation, was significantly reduced by the addition of 250 mg/ml cycloheximide. Immunoblot analyses also revealed a difference between the immediate early and delayed early class of viral genes. Synthesis of IEI, IE2, and IEO was resistant to cycloheximide treatment, while translation of SSB/LEF-3, and pp3l was strongly inhibited even at the lower concentration of cycloheximide. Although cycloheximide was shown to be useful in defining early temporal classes, it induced apoptosis in both uninfected and infected Sf9 cells when used at the inhibitory concentration.