Expression analysis of recombinant herpes simplex virus type 1 DNase

Expression of recombinant herpes simplex virus type 1 (HSV-1) deoxyribonucl ease (DNase) was analyzed in BHK-21 cells, a standard cell line for virus p ropagation, by using mammalian cell expression systems based on vaccinia vi rus and on Semliki Forest virus (SFV)(1). Although the establishing of reco mbinant vaccinia virus failed due to the apparent toxicity of the herpesvir al enzyme, soluble and functional HSV-1 DNase was efficiently expressed in BHK-21 cells by the vaccinia virus/T7 RNA polymerase hybrid system as well as by recombinant Semliki Forest virus. Using rabbit antiserum ExoC, direct ed against the C-terminal residues 503-626, or mouse monoclonal antibody (M Ab) Q1, raised against the type 2 enzyme, a major 85-kDa protein with the i dentical size of the enzyme from HSV-1-infected cells was identified to be induced in both expression systems. With recombinant SFV functional HSV-1 D Nase coincided with the overproduction of a single major 85-kDa protein rea ching an optimum between 16 h and 36 h after infection. At later times of i nfection the enzymatic activity vanished. Thus, recombinant SFV may be an a ppropriate expression vector for biochemical studies of the enzyme when (i) packaged recombinant virus particles are used for infection and (ii) infec tion does not exceed 24 h, Due to the limitations of transient expression s ystems, the vaccinia/T7 RNA polymerase hybrid system is suited for expressi on analysis on a small scale, and for studying intracellular interactions o f the enzyme as demonstrated by immunofluorescence microscopy studies. Usin g vector pTM1, recombinant HSV-1 DNase was efficiently overproduced in BHK- 21 cells at 6 h after transfection and was shown to colocalize with the cel lular chromatin at sites apparently distinct from the bulk of the herpesvir al replication sites the way it is observed for the enzyme of lytically inf ected cells. The deleting of the 123 C-terminal amino acid residues did not alter this nuclear localization of HSV-1 DNase, suggesting that the latter sequences and other herpesviral factors are not required for the chromatin association.