THE EPSTEIN-BARR VIRUS-ENCODED NUCLEAR ANTIGEN EBNA-5 ACCUMULATES IN PML-CONTAINING BODIES

EBNA-5 is one of the Epstein-Barr virus (EBV)-encoded nuclear proteins required for immortalization of human B lymphocytes. In the nuclei of EBV-transformed lymphoblastoid cell lines EBNA-5 is preferentially ta rgetted to distinct nuclear foci. Previously we have shown (W. Q. Jian g, L. Szekely, V. Wendel-Hansen, N. Ringertz, G. Klein, and A. Rosen, Exp. Cell Res. 197:314-318, 1991) that the same foci also contained th e retinoblastoma (Rb) protein. Using a similar double immunofluorescen ce technique, we now show that these foci colocalize with nuclear bodi es positive for PML, the promyelocytic leukemia-associated protein. Ar tificial spreading of the chromatin by exposure to the forces of fluid surface tension disrupts this colocalization gradually, suggesting th at the bodies consist of at least two subcomponents, Heat shock or met abolic stress Induced by high density leads to the release of EBNA-5 f rom the PML-positive nuclear bodies and induces it to translocate to t he nucleoli. In addition to their presence in nuclear bodies, both pro teins are occasionally present in nuclear aggregates and doughnut-like structures in which PML is concentrated in an outer shell. Nuclear bo dies with prominent PML staining are seen in resting B lymphocytes. Th is staining pattern does not Change upon EBV infection. In freshly inf ected cells EBNA-5 antigens are first distributed throughout the nucle oplasm. After a few days intensely staining foci develop. These foci c oincide with PML-positive nuclear bodies. At a later stage and in esta blished lymphoblastoid cell lines EBNA-5 is almost exclusively present in the PML-positive nuclear foci. The colocalization is restricted to EBV-infected human lymphoblasts. The data presented indicate that the distinct EBNA-5 foci are not newly formed structures but the result o f translocation of the viral protein to a specialized domain present a lready in the nuclei of uninfected cells.