Identification of recombinant viruses by RFLP and Western blotting. (a) Purified DNA from EHV-1, EHV-1gH4, EHV-1gH4Kan and EHV-1gHS440A (left panel) as well as EHV-4, EHV-4gH1Kan and EHV-4gH1 (right panel) were digested with Sal I and Sac II. Fragments in the mutants that appeared as a consequence of the deletion or insertion of gH sequence are marked by arrows. (b) Amino acid sequence alignment of EHV-1 and EHV-4 gHs’ N-terminal part. The alignment begins with aa49 to aa 327 (H1 domain homologue, that binds gL, in HSV-2). (c) Parental and mutant viruses as well as complementing RK13/gH1 cells express gH at similar levels. Cell lysates were prepared either from infected FHK cells or from RK13/gH1 and proteins were separated by SDS-10%-PAGE. The blots were incubated with EHV-1 polyclonal anti-gH antibody and detected with anti-rabbit IgG peroxidase conjugate. For RK13/gH1 cells (left panel), EHV-1 and related mutants (middle panel), and EHV-4 and related mutants (right panel), two bands of approximately 125 and 115kD were detected that are not present in mock-infected cells. β-actin was used as a loading control.