FIG 4.

A3 is an early gene encoding a 93-kDa glycoprotein secreted during virus infection. (A) Determination of the A3 kinetic class of transcription. BT cells were infected with AlHV-1 and treated with CHX or PAA or left untreated (−). At 24 h p.i., the expression of ORF73 (IE gene), ORF09 (DPOL, E gene), ORF22 (gH, L gene), or A3 was determined by using a reverse transcription-PCR approach, as described in Materials and Methods. (B) Recombineering methodology used to insert a carboxy-terminal hIgG1 Fc fragment tag to the A3 gene and produce the BAC A3Fc strain. (C) The BAC constructs were analyzed by Southern blotting after EcoRI restriction. Probes are indicated: galK, entire galK coding sequence; A3, entire A3 coding sequence. (D) AlHV-sema kinetics of expression after infection with the BAC A3Fc virus (MOI = 0.01). Infected BT cells were fixed and permeabilized at the given time points postinfection before staining with Alexa Fluor 488-nm anti-human IgG1 goat polyserum for AlHV-sema-Fc detection or MAb 15-A primary antibody (specific to gp115 viral complex), followed by Alexa Fluor 488-nm goat anti-mouse IgG secondary antibody. (E) Supernatants were collected and analyzed by immunoblotting with anti-human IgG1 polyserum.