Complementation of a replication-defective mutant of equine herpesvirus type 1 by a cell line expressing the...

Equine herpesvirus type 1 (EHV-1) possesses a sole, diploid immediate-early (IE) gene that encodes a major regulatory protein of 1487 amino acids capable of modulating expression of both early and late EHV-1 promoters and capable of trans-repressing its own promoter. In this study, a rabbit kidney cell line (IE13.1) that constitutively expresses the EHV-1 IE protein was generated by cotransfection of rabbit kidney (RK-13) cells with the viral IE gene and a neomycin resistance marker. The IE protein expressed by this cell line was shown (1) to be expressed by and to localize to the nucleus of virtually all cells as demonstrated by indirect immunofluorescence, (2) to be the full-size IE polypeptide as judged by Western immunoblot analyses with an anti-IE protein-specific antibody, and (3) to be functional as shown by the transactivation of two representative EHV-1 early promoters linked to the chloramphenicol acetyltransferase reporter gene in transient transfection assays. The IE13.1 cell line was able to complement a recombinant virus in which both copies of the IE gene were replaced by insertion of the Escherichia coli lacZ gene. This IE deletion mutant, designated KyADeltaIE, was not able to replicate in equine, rabbit, or mouse cells but was capable of replication in the IE13.1 cells that provided the IE protein in trans. Rescue of the KyADeltaIE virus was achieved by recombination with a marker plasmid that harbors the wild-type IE gene, and the rescued virus (KyADeltaIER) was able to grow on noncomplementary cells. Overall, these results offer direct evidence that the IE gene is essential for EHV-1 replication and provide reagents useful for the analysis of IE protein function.