We have reported in the accompanying paper that the BFRF1 protein of Epstein-Barr virus (EBV) is important for efficient primary viral envelopment and egress (A. Farina, R. Feederle, S. Raffa, R. Gonnella, R. Santarelli, L. Frati, A. Angeloni, M. R. Torrisi, A. Faggioni, and H.-J. Delecluse, J. Virol. 79:3703-3712). Here we describe the characterization of the product of the EBV BFLF2 gene, which belongs to a family of conserved herpesviral genes which include the UL31 genes of herpes simplex virus and of pseudorabies virus and whose products are known to interact with UL34, the positional homolog of BFRF1. BFLF2 is an early transcript and is expressed in a variety of cell lines upon EBV lytic cycle activation. Western blotting of purified virion preparations showed that BFLF2 is a component of intracellular virions but is absent from mature extracellular virions. Coimmunoprecipitation experiments indicated that BFLF2 interacts with BFRF1, which was confirmed by immunofluorescence confocal microscopy showing that the two proteins colocalize on the nuclear membrane not only upon cotransfection in epithelial cells but also during viral replication. In cells carrying an EBV mutant with the BFRF1 gene deleted (293-BFRF1-KO cells) BFLF2 expression was low, and it was restored to wild-type levels upon treatment of the cells with the proteasome inhibitor MG132. Furthermore, recomplementing the 293-BFRF1-KO cells by BFRF1 transfection restored BFLF2 expression to the wild-type level. In addition, when expressed alone BFLF2 was localized diffusely inside the nucleus, whereas in the presence of BFRF1 the two proteins colocalized at the nuclear rim. Finally, 293 epithelial cells transfected with either protein or cotransfected were analyzed by electron microscopy to investigate potential alterations in the morphology of the nuclear membrane. The ultrastructural analysis revealed that (i) BFRF1 caused duplications of the nuclear membrane, similar to those reported to occur during the course of herpesviral replication, and (ii) while BFLF2 alone did not cause any apparent alteration, coexpression of the two proteins dramatically induced profound convolutions of the duplicated nuclear membrane. Both biochemical and morphological analysis showed association of the BFRF1-BFLF2 complex with a component of the nuclear lamina, lamin B. Taken together, these results and those of the accompanying paper (Farina et al., J. Virol. 79:3703-3712) indicate an important role of BFRF1 and BFLF2 in the early steps of EBV maturation at the nuclear membrane.

Gonnella, R., Farina, A., Santarelli, R., Raffa, S., Feederle, R., Bei, R., et al. (2005). Characterization and intracellular localization of the Epstein-Barr virus protein BFLF2: Interactions with BFRF1 and with the nuclear lamina. JOURNAL OF VIROLOGY, 79(6), 3713-3727 [10.1128/JVI.79.6.3713-3727.2005].

Characterization and intracellular localization of the Epstein-Barr virus protein BFLF2: Interactions with BFRF1 and with the nuclear lamina

BEI, ROBERTO;MODESTI, ANDREA;
2005

Abstract

We have reported in the accompanying paper that the BFRF1 protein of Epstein-Barr virus (EBV) is important for efficient primary viral envelopment and egress (A. Farina, R. Feederle, S. Raffa, R. Gonnella, R. Santarelli, L. Frati, A. Angeloni, M. R. Torrisi, A. Faggioni, and H.-J. Delecluse, J. Virol. 79:3703-3712). Here we describe the characterization of the product of the EBV BFLF2 gene, which belongs to a family of conserved herpesviral genes which include the UL31 genes of herpes simplex virus and of pseudorabies virus and whose products are known to interact with UL34, the positional homolog of BFRF1. BFLF2 is an early transcript and is expressed in a variety of cell lines upon EBV lytic cycle activation. Western blotting of purified virion preparations showed that BFLF2 is a component of intracellular virions but is absent from mature extracellular virions. Coimmunoprecipitation experiments indicated that BFLF2 interacts with BFRF1, which was confirmed by immunofluorescence confocal microscopy showing that the two proteins colocalize on the nuclear membrane not only upon cotransfection in epithelial cells but also during viral replication. In cells carrying an EBV mutant with the BFRF1 gene deleted (293-BFRF1-KO cells) BFLF2 expression was low, and it was restored to wild-type levels upon treatment of the cells with the proteasome inhibitor MG132. Furthermore, recomplementing the 293-BFRF1-KO cells by BFRF1 transfection restored BFLF2 expression to the wild-type level. In addition, when expressed alone BFLF2 was localized diffusely inside the nucleus, whereas in the presence of BFRF1 the two proteins colocalized at the nuclear rim. Finally, 293 epithelial cells transfected with either protein or cotransfected were analyzed by electron microscopy to investigate potential alterations in the morphology of the nuclear membrane. The ultrastructural analysis revealed that (i) BFRF1 caused duplications of the nuclear membrane, similar to those reported to occur during the course of herpesviral replication, and (ii) while BFLF2 alone did not cause any apparent alteration, coexpression of the two proteins dramatically induced profound convolutions of the duplicated nuclear membrane. Both biochemical and morphological analysis showed association of the BFRF1-BFLF2 complex with a component of the nuclear lamina, lamin B. Taken together, these results and those of the accompanying paper (Farina et al., J. Virol. 79:3703-3712) indicate an important role of BFRF1 and BFLF2 in the early steps of EBV maturation at the nuclear membrane.
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore MED/04 - Patologia Generale
English
Con Impact Factor ISI
benzyloxycarbonylleucylleucylleucinal; lamin B; unclassified drug; virus protein; virus protein BFLF2; article; cell activation; cell nucleus membrane; cellular distribution; confocal microscopy; controlled study; electron microscopy; epithelium cell; Epstein Barr virus; genetic transfection; Herpes simplex virus; human; human cell; immunofluorescence; immunoprecipitation; nuclear lamina; priority journal; Pseudorabies herpetovirus; virion; virus characterization; virus replication; Blotting, Western; Cell Line; Cysteine Proteinase Inhibitors; Dendritic Cells; Epithelial Cells; Gene Deletion; Genetic Complementation Test; Herpesvirus 4, Human; Humans; Immunoprecipitation; Lamin Type B; Leupeptins; Membrane Proteins; Microscopy, Confocal; Microscopy, Fluorescence; Nuclear Envelope; Nuclear Lamina; Open Reading Frames; Phosphoproteins; Protein Binding; Recombinant Proteins; Viral Proteins; Virion; Virus Replication
Gonnella, R., Farina, A., Santarelli, R., Raffa, S., Feederle, R., Bei, R., et al. (2005). Characterization and intracellular localization of the Epstein-Barr virus protein BFLF2: Interactions with BFRF1 and with the nuclear lamina. JOURNAL OF VIROLOGY, 79(6), 3713-3727 [10.1128/JVI.79.6.3713-3727.2005].
Gonnella, R; Farina, A; Santarelli, R; Raffa, S; Feederle, R; Bei, R; Granato, M; Modesti, A; Frati, L; Delecluse, H; Torrisi, M; Angeloni, A; Faggioni, A
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2108/30498
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