IDENTIFICATION OF DOMAINS WITHIN THE HUMAN CYTOMEGALOVIRUS MAJOR IMMEDIATE-EARLY 86-KILODALTON PROTEIN AND THE RETINOBLASTOMA PROTEIN REQUIRED FOR PHYSICAL AND FUNCTIONAL INTERACTION WITH EACH OTHER

The human cytomegalovirus major immediate-early 86-kDa protein (IE2 86 ) plays an important role in the trans activation and regulation of HC MV gene expression, Previously, we demonstrated that IE2 86 contains t hree regions (amino acids [aa] 86 to 135, 136 to 290, and 291 to 364) that can independently bind to in vitro-translated Rb when IE2 86 is p roduced as a glutathione S-transferase fusion protein (M. H. Sommer, A . L. Scully, and D. H. Spector, J. Virol. 68:6223-6231, 1994). In this report, we have elucidated the regions of Rb involved in binding to I E2 86 and have further analyzed the functional nature of the interacti on between these two proteins, We find that two domains on Rb, the A/B pocket and the carboxy terminus, can each independently form a comple x with IE2 86, In functional assays, we demonstrate that IE2 86 and an other IE protein, IE1 72, can counter the enlarged flat cell phenotype , but not the G(1)/S block, which results from expression of wild-type Rb in the human osteosarcoma cell line Saos-2. Mutational analysis re veals that there are two domains on IE2 86 that can independently affe ct Rb function, One region (aa 241 to 369) includes the major Rb-bindi ng domain, while the second maps to the amino-terminal region (aa 1 to 85) common to both IE2 86 and IE1 72. These data show that Rb and IE2 86 physically and functionally interact,vith each other via at least two separate domains and provide further support for the hypothesis th at IE2 86 may exert its pleiotropic effects through the formation of m ultimeric protein complexes.