The Herpes Simplex Virus 1 U S 3 Protein Kinase Blocks Caspase-Dependent Double Cleavage and Activation of the Proapoptotic Protein BAD

Abstract

ABSTRACT An earlier report showed that the U S 3 protein kinase blocked the apoptosis induced by the herpes simplex virus 1 (HSV-1) d 120 mutant at a premitochondrial stage. Further studies revealed that the kinase also blocks programmed cell death induced by the proapoptotic protein BAD. Here we report the effects of the U S 3 protein kinase on the function and state of a murine BAD protein. Specifically, (i) in uninfected cells, BAD was processed by at least two proteolytic cleavages that were blocked by a general caspase inhibitor. The untreated transduced cells expressed elevated caspase 3 activity. (ii) In cells cotransduced with the U S 3 protein kinase, the BAD protein was not cleaved and the caspase 3 activity was not elevated. (iii) Inasmuch as the U S 3 protein kinase blocked the proapoptotic activity and cleavage of a mutant (BAD3S/A) in which the codons for the regulatory serines at positions 112, 136, and 155 were each replaced with alanine codons, the U S 3 protein kinase does not act by phosphorylation of these sites nor was the phosphorylation of these sites required for the antiapoptotic function of the U S 3 protein kinase. (iv) The U S 3 protein kinase did not enable the binding of the BAD3S/A mutant to the antiapoptotic proteins 14-3-3. Finally, (v) whereas cleavage of BAD at ASP56 and ASP61 has been reported and results in the generation of a more effective proapoptotic protein with an M r of 15,000, in this report we also show the existence of a second caspase-dependent cleavage site most likely at the ASP156 that is predicted to inactivate the proapoptotic activity of BAD. We conclude that the primary effect of U S 3 was to block the caspases that cleave BAD at either residue 56 or 61 predicted to render the protein more proapoptotic or at residue 156, which would inactivate the protein.