Phosphorylation and cytotoxicity of therapeutic nucleoside analogues: a comparison of alpha and gamma herpesvirus thymidine kinase suicide genes.
Thymidine kinase (TK) genes from three alpha-herpesviruses (i.e., human herpes simplex type 1, varicella-zoster virus, equid herpesvirus 4) and two y-herpesviruses (i.e., Epstein-Barr virus and Saimiri herpesvirus 2) were cloned in expression vectors based on zeocin resistance by complementation of a TK-defective Escherichia coli strain. In vivo complementation of an appropriate yeast strain and in vitro enzymatic measurements demonstrated that all viral TKs possess a second phosphorylating activity corresponding to the thymidylate kinase function in contrast to the E coli TK, which is deprived of this activity. When expressed in an engineered E coli strain rendered resistant to purine and pyrimidine nucleoside analogs, the viral TKs sensitize host bacteria to 3'-azido-3'-deoxythymidine (AZT), 3'-deoxy-2',3'-didehydrothymidine (D4T), dideoxyinosine, or fluorodeoxyuridine (5-FUdR). The extent of activation of all these analogs, in this bacterial assay, was found to be greatly superior for the two gamma-virus TKs, compared to the alpha-virus TKs, including the reference suicide gene, HSV1-TK. TK from the two gamma-Epstein-Barr and Saimiri 2 viruses were also found to be more efficient in sensitizing murine melanoma B16 tumor cells to pyrimide nucleoside analogs.
Cazaux
C
C
Laboratoire de Microbiologie et de Génétique, Université Paul Sabatier, Toulouse, France.
Tiraby
M
M
Loubiere
L
L
Haren
L
L
Klatzmann
D
D
Tiraby
G
G
D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival; D003001:Cloning, Molecular; D016049:Didanosine / Q000378:metabolism* / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005467:Floxuridine / Q000378:metabolism* / Q000494:pharmacology; D005822:Genetic Vectors; D006564:Herpesviridae / Q000201:enzymology*; D009154:Mutation; D010766:Phosphorylation; D011995:Recombination, Genetic; D018119:Stavudine / Q000378:metabolism* / Q000494:pharmacology; D013937:Thymidine Kinase / Q000235:genetics*; D014407:Tumor Cells, Cultured; D015215:Zidovudine / Q000378:metabolism* / Q000494:pharmacology
D000998:Antiviral Agents; D005467:Floxuridine; D015215:Zidovudine; D018119:Stavudine; D013937:Thymidine Kinase; D016049:Didanosine