Analysis of the in vitro translation products of the equine herpesvirus type 1 immediate early mRNA

Equine herpesvirus type 1 (EHV-1) gene expression is coordinately regulated in an α, β, γ fashion. Viral α gene products include a 6.0-kb immediate early (IE) mRNA species (W. L. Gray et al., 1987, Virology 158, 79-87) and at least four closely related IE polypeptides (IEPs) (G. B. Caughman et al., 1985, Virology 145, 49-61). In this report, we describe results obtained from a series of in vitro translation experiments which were performed in an effort to characterize the IEPs and identify the mechanism by which individual IE protein species are generated. Our data indicate that a family of IEPs is generated in vitro from the 6.0-kb mRNA size class and that these IEPs correspond in overall size and antigenicity to those synthesized in infected cells. Using time-course/pulse-chase analyses, we show that production of three of the major IEPs [IE1′ (193 kDa), IE3′ (166 kDa), and IE4′ (130 kDA)] occurs concomitantly, that none of these protein species can be chased completely into another, and that at least two additional minor species appear to be processed following synthesis. Finally, we show that the 6.0-kb mRNA species isolated during early or late stages of the infection cycle can be translated to yield all of the major IE proteins, indicating that production of the family of IEPs is not dependent upon accumulation of the IE mRNA which occurs during a cycloheximide blocked infection cycle. The implications of these findings are discussed as they relate to the origin and production of the IEPs both in vivo and in vitro.