In Transduced Cells, the U S 3 Protein Kinase of Herpes Simplex Virus 1 Precludes Activation and Induction of Apoptosis by Transfected Procaspase 3

Abstract

ABSTRACT The U S 3 protein kinase of herpes simplex virus 1 blocks apoptosis induced by replication-incompetent virus mutants, proapoptotic members of the Bcl-2 family of proteins, and by a variety of other agents that act at the premitochondrial level in the proapoptotic cascade. To define the role of U S 3 in blocking apoptosis at the postmitochondrial level, we investigated the U S 3 protein kinase in transduced cells that were either transfected with a plasmid encoding procaspase 3 or superinfected with a proapoptotic mutant virus lacking the gene encoding the infected cell protein no. 4. (i) We show that U S 3 blocks the proteolytic cleavage that generates active caspase 3 from the transfected zymogen procaspase 3, concomitant with inhibition of apoptosis. (ii) Studies based on detection of fluorescence emitted upon cleavage of a synthetic caspase 3 substrate showed that expression of the U S 3 kinase and appearance of the cleaved substrate were mutually exclusive. (iii) An affinity-purified glutathione S -transferase (GST)-U S 3 fusion protein, but not the inactive GST-U S 3(K220N) protein, phosphorylated procaspase 3 in vitro. The studies published earlier on the effect of U S 3 on the upstream regulatory proteins and current studies suggest that the U S 3 protein kinase may act on several proteins in the proapoptotic cascade to enable the virus to complete its replication.