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The Epstein-Barr virus (EBV)-encoded, nuclear matrix-associated EBNA-5 protein is preferentially localized within distinct nuclear blobs in EBV-immortalized lymphoblastoid cell lines. We have previously found that the same blobs also contain retinoblastoma (Rb) protein. We now show that they contain hsp70 protein as well. Both EBNA-5 and hsp70 translocate to the nucleolus under cell density congestion or after heat shock. Both proteins relocate to their original position upon the re-establishment of normal physiological conditions. EBNA-5 is tightly bound to the nuclear matrix. The translocated EBNA-5 is also tightly associated with matrix structures, as shown by sequential elution-based cell fractionation. The Rb protein does not translocate to the nucleolus. The virally encoded EBNA-1, -2, -3 and -6, and cellular PCNA, snRNP and cyclin E are not affected either. The translocation of EBNA-5 to the nucleolus is not species- or cell type-specific since stress conditions induced the same phenomenon in EBNA-5-transfected human, mouse and rat cells of different tissue origins.

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 targetted to distinct nuclear foci. Previously we have shown (W. Q. Jiang, L. Szekely, V. Wendel-Hansen, N. Ringertz, G. Klein, and A. Rosén, Exp. Cell Res. 197:314-318, 1991) that the same foci also contained the retinoblastoma (Rb) protein. Using a similar double immunofluorescence technique, we now show that these foci colocalize with nuclear bodies positive for PML, the promyelocytic leukemia-associated protein. Artificial spreading of the chromatin by exposure to the forces of fluid surface tension disrupts this colocalization gradually, suggesting that the bodies consist of at least two subcomponents. Heat shock or metabolic stress induced by high cell density leads to the release of EBNA-5 from the PML-positive nuclear bodies and induces it to translocate to the nucleoli. In addition to their presence in nuclear bodies, both proteins are occasionally present in nuclear aggregates and doughnut-like structures in which PML is concentrated in an outer shell. Nuclear bodies with prominent PML staining are seen in resting B lymphocytes. This staining pattern does not change upon EBV infection. In freshly infected cells EBNA-5 antigens are first distributed throughout the nucleoplasm. After a few days intensely staining foci develop. These foci coincide with PML-positive nuclear bodies. At a later stage and in established 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 of translocation of the viral protein to a specialized domain present already in the nuclei of uninfected cells.

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Abstract. We describe the dynamic organization of pre-mRNA splicing factors in the intact polytene nuclei of the dipteran Chironomus tentans. The snRNPs and an SR non-snRNP splicing factor are present in excess, mainly distributed throughout the interchromatin. Approximately 10% of the U2 snRNP and an SR nonsnRNP splicing factor are associated with the chromosomes, highly enriched in active gene loci where they are bound to RNA. We demonstrate that the splicing factors are specifically recruited to a defined gene upon induction of transcription during physiological conditions. Concomitantly, the splicing factors leave gene loci in which transcription is turned off. We also demonstrated that upon general transcription inhibition, the splicing factors redistribute from active gene loci to the interchromatin. Our findings demonstrate the dynamic intranuclear organization of splicing factors and a tight linkage between transcription and the intranuclear organization of the splicing machinery.