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

Abstract

The baculovirus protein IE1 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 IE1 in infected cells. We show here that synthesis of IE1 was not effectively inhibited by the addition of 100 μg/ml cycloheximide, the concentration routinely used in these studies. However, when cycloheximide was added at 250 μg/ml, IE1 synthesis was repressed to less than 5% 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, and 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 on IE1 transactivation, was significantly reduced by the addition of 250 μg/ml cycloheximide. Immunoblot analyses also revealed a difference between the immediate and delayed early class of viral genes. Synthesis of IE1, IE2, and IE0 was resistant to cycloheximide treatment, while translation of SSB/LEF-3 and pp31 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.