INTRACELLULAR INTERNALIZATION AND SIGNALING PATHWAYS TRIGGERED BY THELARGE SUBUNIT OF HSV-2 RIBONUCLEOTIDE REDUCTASE (ICP10)

The targe subunit of the HSV-2 ribonucleotide reductase (RR) (ICP10) i s a chimera consisting of a serine threonine (Ser/Thr) protein kinase domain at the amino terminus and the RR domain at the carboxy terminus . Transformed human cells that constitutively express ICP10 (JHLa1) we re stained with anti-LA-1 antibody (recognizes ICP10 amino acids 13-26 ) and immunogold-conjugated goat anti-rabbit IgG and were examined by electron microscopy. ICP10-associated gold particles were observed on the cell surface and in structures with ultrastructural characteristic s of endocytic vesicles, multivesicular bodies, and lysosomes, consist ent with endocytic internalization. ICP10 was also associated with the cytoskeleton fraction of JHLaf cells and, at least in part, it coloca lized with actin filaments. This was evidenced by immunoprecipitation of [S-35]methionine-labeled cell fractions and immunofluorescent stain ing of Triton-treated cells with anti-LA-1 antibody and phalloidin. En docytic localization of gold particles was not seen in cells that cons titutively express the ICP10 transmembrane (TM)-deleted mutant p139(TM ) (JHL15). p139(TM) did not associate with the cytoskeleton and was al most entirely localized within the cytoplasm. raf and Erk evidenced de creased mobility consistent with an activated state in JHLa1, but not JHL15, cells, and chloramphenicol acetyl transferase (CAT) expression from a c-fos/cat hybrid construct was significantly increased in JHLa1 but not JHL15 cells. The data indicate that effector molecules downst ream of ras are activated in JHLa1 cells and the ICP10 TM segment play s a critical role in ICP10 intracellular localization and its ability to activate signaling pathways. This behavior is analogous to that of an activated growth factor receptor kinase. (C) 1995 Academic Press, I nc.