Topographical study of intranuclear distribution and compartementalization of viral and cellular proteins

Inst för cell- och molekylärbiologi / Dept of Cell and Molecular Biology 1996-09-20

The nuclei of eukaryotic cells contain a number of specialized domains involved in DNA replication, transcription, pre-mRNA splicing and ribosome assembly. In addition to these structures, nuclear bodies of unknown function have been described. In the present study, we have used a topographical approach, a combination of immuno-fluorescence and fluorescence digital imaging microscopy (FDIM), to analyze nuclear bodies in cells transformed by Epstein-Barr virus (EBV) and simian virus 40 (SV40). EBV-transformed Iymphoblastoid cell lines (LCLs) express six EBV-encoded nuclear antigens EBNA-1 to -6, the functions of which are not completely known. Detailed analysis of the nuclear patterns of EBNA-1, -2, -3 and -5 has revealed that they differ in their intranuclear distribution. EBNA-5 forms a limited number of large, globular foci, a pattern unique among the EBNAs. The subsequent analysis of the EBNA-5 foci in relation to other nuclear constituents, shows that a subfraction of the retinoblastoma protein (Rb) is concentrated in the EBNA-5 foci in the EBV-transformed LCL IB4. The heat shock protein hsp70 is also present in these foci. Double immunostaining of EBNA-5 and the promyelocytic leukemia associated proteins (PML) in IB4 cells demonstrates that the EBNA-5 foci are surrounded by a PML containing shell (PML body). Moreover, in EBV-infected B Iymphocytes, EBNA-5 accumulates in pre-existing PML bodies. Upon metabolic stress, EBNA-5 is dissociated from PML bodies and translocates to nucleoli. Based on these data, we suggest that in EBV-mediated transformation, EBNA-5 may be involved in the deregulation of cell growth control through: (1) sequestering Rb, and/or (2) targeting PML bodies. We have investigated the possible connection between the PML bodies and another viral transforming protein, the SV40-encoded large T antigen (SV40T), in a conditionally SV40T-immortalized human cell line, IDH4. Nearly all PML bodies are surrounded by 1-3 SV40T positive foci, suggesting t

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SEKIU oai:dspace.kib.ki.se:10616/43072
91-628-2181-4
19961011jian
1457949466

KAROLINSKA INSTITUTET UNIV
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