Neoplastic transformation, cellular localization and protein kinase activity of the RR1 subunit of herpes simplex virus type 2 (ICP10)

Abstract

The large subunit of the herpes simplex virus (HSV) Type 1 and Type 2 ribonucleotide reductase (ICP6 and ICP10, respectively) is a chimaera consisting of a protein kinase (PK) domain at the amino terminus and a ribonucleotide reductase domain at the carboxy terminus. The present work was performed in order to further characterize this PK activity and determine its biological significance. Computer-assisted analyses of ICP10 and ICP6 in comparison to other, known protein kinases suggest that they are members of a previously unidentified sub-family of serine/threonine (Ser/Thr) growth factor receptors. Using cells infected with laboratory and patient isolates of HSV, ICP10 was demonstrated to possess a Ser/Thr specific auto- and transphosphorylating kinase activity. ICP6 exhibited a similar PK activity, but was considerably weaker. Cells that constitutively express ICP10 or a library of ICP10 mutants illustrated a direct relationship between ICP10 PK activity and its ability to transform cells as determined by anchorage-independent growth. All mutations which abolish PK activity resulted in a concomitant abrogation in its transforming potential. Cells transfected to constitutively express ICP10 demonstrated its internalization from the membrane and degradation in lysosomes in a manner characteristic of a typical activated growth factor receptor. ICP10 was also found bound to cytoskeletal actin, similar to epidermal growth factor receptor. A PK-negative mutant of ICP10, deleted in the transmembrane domain was recoevered from the cytosol, but did not localize to the membrane, nor was it found in endosomes or lysosomes, nor in association with actin. In ICP10 constitutively expressing cells, Raf-1 and Erk evidenced decreased electrophoretic mobility consistent with an activated state, suggesting that ICP10 PK may cause transformation through modulation of the MAP kinase pathway. It is suggested that the PK domains of ICP6 and ICP10 were cellular genes integrated into the genome of an ancestral herpes simplex virus and subsequently conserved to promote viral replication and/or reactivation from latency.