Cell-free synthesis of enzymatically active vaccinia virus thymidine kinase

Mouse L cells that lacked thymidine kinase (L_tk^? cells) were infected with wild-type vaccinia virus or with a mutant that was deficient in the viral thymidine kinase. Cytoplasmic messenger RNA preparations were isolated 2 hr after infection and translated in a messenger RNA-dependent reticulocyte lysate. Both messenger RNA preparations directed the efficient synthesis of early vaccinia virus polypeptides. Furthermore, translation of the messenger RNA from cells infected with wild-type virus resulted in a substantial increase in the thymidine kinase activity of the lysate which was prevented by inhibitors of cell-free protein synthesis. Translation of the messenger RNA from cells infected with the viral tk^? mutant, or from uninfected L_tk^? cells, did not increase the thymidine kinase activity of the extract. The enzyme synthesized in vitro was compared with that isolated from vaccinia virus-infected cells. The two enzymes were indistinguishable with respect to their rates of thermal inactivation, their sedimentation coefficients in glycerol gradients, and their relative electrophoretic mobilities in nondenaturing polyacrylamide gels. We conclude that active viral thymidine kinase can be synthesized de novo in a cell-free system directed by early messenger RNA from vaccinia virus-infected cells.