Expression analysis of recombinant herpes simplex virus type 1 DNase.
Expression of recombinant herpes simplex virus type 1 (HSV-1) deoxyribonuclease (DNase) was analyzed in BHK-21 cells, a standard cell line for virus propagation, by using mammalian cell expression systems based on vaccinia virus and on Semliki Forest virus (SFV)1. Although the establishing of recombinant vaccinia virus failed due to the apparent toxicity of the herpesviral 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, directed against the C-terminal residues 503-626, or mouse monoclonal antibody (MAb) Q1, raised against the type 2 enzyme, a major 85-kDa protein with the identical size of the enzyme from HSV-1-infected cells was identified to be induced in both expression systems. With recombinant SFV functional HSV-1 DNase coincided with the overproduction of a single major 85-kDa protein reaching an optimum between 16 h and 36 h after infection. At later times of infection the enzymatic activity vanished. Thus, recombinant SFV may be an appropriate expression vector for biochemical studies of the enzyme when (i) packaged recombinant virus particles are used for infection and (ii) infection does not exceed 24 h. Due to the limitations of transient expression systems, the vaccinia/T7 RNA polymerase hybrid system is suited for expression analysis on a small scale, and for studying intracellular interactions of the enzyme as demonstrated by immunofluorescence microscopy studies. Using 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 cellular chromatin at sites apparently distinct from the bulk of the herpesviral replication sites the way it is observed for the enzyme of lytically infected 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.
Kehm
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Forschungsschwerpunkt Genomforschung und Bioinformatik, Deutsches Krebsforschungszentrum, Heidelberg, FRG.
Göksu
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MA
Knopf
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CW
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004261:DNA Replication; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D003851:Deoxyribonucleases / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D004720:Endonucleases; D005092:Exonucleases; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D012672:Semliki forest virus / Q000201:enzymology / Q000235:genetics; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics; D014764:Viral Proteins
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D003851:Deoxyribonucleases; D004720:Endonucleases; D005092:Exonucleases; C020104:endoexonuclease