Characterization of the immediate-early functions of pseudorabies virus

The immediate-early transcripts of pseudorabies virus have been located in a region of the genome situated internally within the inverted repeat between map positions 0.99 and 0.95. A single immediate-early transcript (approximately 6 kb) can be detected both in the cytoplasmic and nuclear fractions of infected, cycloheximide-treated cells. Analysis of the proteins synthesized after removal of cycloheximide from infected cells or after translation in vitro of the RNA isolated from these cells revealed the presence of a single protein (180K) not present in similarly treated, uninfected cells. That this is a virus protein and is specified by the immediate-early region of the genome was shown by selection and translation of mRNA hybridizing with virus DNA from the appropriate region of the genome. The effects of infection of cells with a temperature-sensitive mutant (tsG1) defective in the 180K protein were studied. At the nonpermissive temperature only immediate-early RNA was transcribed and only one virus protein, the 180K protein was synthesized. Inhibition of cellular protein and DNA synthesis was also observed. After shift down of tsG1-infected cells from the nonpermissive to the permissive temperature at 3 hr post infection, a transition to early RNA transcription occurred. However, if the shift down was delayed until 5 hr post infection, transcription of the virus genome was completely inhibited and an abortive infection ensued. Shift of the mutant-infected cells from the permissive to the nonpermissive temperature resulted in a decrease in the rate of accumulation of early and late transcripts, and a resumption of the synthesis of immediate-early RNA and protein. From these as well as from previous results, it is concluded that pseudorabies virus codes for a single multifunctional immediate-early protein which is essential for the transcription of immediate-early to early RNA and is required for the continuous transcription of early (and late) RNA. The immediate-early protein is also self-regulatory; the presence of the functional immediate-early protein represses the transcription of its RNA. In addition, the immediate-early protein of pseudorabies virus appears to play a direct role, under certain conditions, in the inhibition of cellular macromolecular synthesis.