Selective assay for herpes simplex viruses expressing thymidine kinase

Abstract

A technique for selecting herpes simplex viruses expressing the viral thymidine kinase (TK+) from a population of predominantly TK-viruses was developed. This was accomplished by infecting TKcells and incubating the cultures under a liquid overlay medium containing methotrexate. Since the TKcells survive in this medium for only a limited period of time, it was necessary to add fresh uninfected TKcells 48 h after infection. The technique allowed the detection and quantitation of the TK+ virus fraction in mixtures of TK+ and TK-viruses where the TK+ fraction was present in frequencies as low as 10-. It was also used to estimate reversion frequencies and to obtain and analyze TK+ revertants from TKmutant strains of herpes simplex virus type 1.

Cell lines containing varicella-zoster virus open reading frame 62 and expressing the "IE" 175 protein complement ICP4 mutants of herpes simplex virus type 1

“…Fold increases for F114 cells were 458 at 24 h and 2.3 x 104 at 48 h. Although the degree of complementation was less for the VZV-derived cell lines than that for E5 cells, significant a Each of the indicated cell lines was seeded in duplicate into 60-mmdiameter dishes and infected with serial dilutions of tsB21 or d120. Following virus adsorption for 1 h, the cells were overlaid with maintenance medium supplemented with 0.2% human immune serum globulin (6) and incubated for 7 days at 39°C for tsB21 or at 37°C for d120, after which the cells were fixed and stained with 0.1% crystal violet.…”

Activation of the major immediate early gene of human cytomegalovirus by cis-acting elements in the promoter-regulatory sequence and by virus-specific trans-acting components

“…The HF strain of HSV-1 was obtained from G. Cohen (University of Pennsylvania, Philadelphia, Pa.) and assayed for infectious virus as previously described (6). Human 143 TK-cells (5) were obtained from C. Croce (Wistar Institute, Philadelphia, Pa.).…”

“…For the transformation of HeLa cells with the pCAT plasmid DNAs (Fig. 1), 5 ,ug of a pCAT plasmid with 20 ,ug of sonicated DNA as carrier was used as described above, analyze the effect of the HCMV major IE promoter-regulatory region on gene expression. Construction of the various chimeric plasmids is described in the text.…”

Human Herpesvirus 8-Encoded Thymidine Kinase and Phosphotransferase Homologues Confer Sensitivity to Ganciclovir

“…TK activity of the HHV-8 TK. HAT selection in TK(Ϫ) cells and incorporation of [ 3 H]thymidine were utilized to assess HHV-8 ORF 21 TK activity in mammalian cells (12,37,54). When plasmids encoding the HHV-8 ORF 21 sequence were transfected into TK(Ϫ) cell lines, including mouse LTK(Ϫ) and the human osteosarcoma 143B, no survival was observed in HHV-8 TK-transformed cells, whereas TK(Ϫ) cells transfected with plasmids carrying either the EBV or HSV TK gene survived selection in HAT medium and grew as HAT-resistant colonies within 2 to 3 weeks of transfection (Table 3).…”