Characterization of equine herpesvirus type 1 immediate early proteins

EHV-1 immediate early (IE) gene expression in lytic infection results in the production of four high mol wt immediate early polypeptides (IEPs), designated IE1, IE2, IE2, and IE4; however, IE transcription is limited to the synthesis of a single 6-kb mRNA. Together, these findings raised questions as to whether the four IEPs were related products of the same gene. In the present study the IEPs were characterized with respect to their structural similarities, antigenic relatedness, and postsynthetic modifications. IE1 was the most abundant IEP, in that it accounted for approximately 80% of the IEP-incorporated radiolabel in infected rabbit kidney cells labeled under IE conditions with [35S]methionine or 14C-labeled amino acid mixtures. IE1 also was the major phosphorylated species. Limited proteolytic digestion of isolated radiolabeled IEP bands with Staph V8 protease yielded virtually identical fragment profiles in SDS-PAGE, as did digestions with chymotrypsin and N-chlorosuccinimide. Monospecific rabbit antisera raised against each of the four isolated IEPs reacted with all the IEP species in immunoblotting assays. Pulse-chase experiments indicated that all the IEPs were detectable immediately after a 15-min pulse and that several alterations in the IEP profile occurred during subsequent chase periods. Thus, the EHV-1 IEPs are closely related structurally and antigenically and appeared to be either produced simultaneously or processed to yield the individual forms immediately.