Intracellular localization of the Epstein‐Barr virus BFRF1 gene product in lymphoid cell lines and oral hairy leukoplakia lesions

Abstract

A novel protein encoded by the BFRF1 gene of the Epstein-Barr virus was identified recently [Farina et al. (2000) J Virol 74:3235-3244], which is antigenic "in vivo" and expressed early in the viral replicative cycle. In the present study, its subcellular localization was examined in greater detail comparing Epstein-Barr virus (EBV) induced producing and nonproducing cell lines by immunofluorescence: in 12-0-tetradecanoyl phorbol-13-acetate (TPA)-induced Raji and B95-8 cells, as well as in anti-IgG-stimulated Akata cells, the protein appeared to be localized over the cell nuclear membrane. A similar nuclear membrane localization was observed in epithelial cells of oral hairy leukoplakia, a pathological manifestation of permissive EBV infection. In contrast, upon transfection of BFRF1 in the EBV-negative Burkitt's lymphoma cell line DG75, the protein was localized predominantly over the plasma membrane. The membrane localization was abolished when DG75 cells were transfected with a C-terminal deletion mutant of BFRF1 lacking the transmembrane domain. Because induced Raji cells do not produce virus, the above observations indicate that the nuclear membrane localization is not associated with viral production, but requires the expression of EBV genes, and suggest that additional proteins, expressed early during viral lytic infection, might be necessary to target the protein to the nuclear membrane. Immunogold electron microscopy on ultrathin cryosections of induced B95-8 cells showed that BFRF1 on the nuclear membranes was concentrated over multilayered domains representing areas of active viral replication or at the sites of viral budding, suggesting that BFRF1 is involved in the process of viral assembly.

Characterization and Intracellular Localization of the Epstein-Barr Virus Protein BFLF2: Interactions with BFRF1 and with the Nuclear Lamina

“…The latter behavior was considered further evidence in favor of the envelopment-de-envelopment-reenvelopment process of alphaherpesviruses. Similarly, our recent results (16) and our previous observation by immunoelectron microscopy showing BFRF1 labeled intracellular virions but unlabeled extracellular virions (15) suggest that BFRF1 is not a component of the mature virions.…”

“…Concerning UL34 localization, our present confocal and previous immunoelectron microscopic analyses (15) clearly defined the unequivocal predominant nuclear membrane localization of BFRF1. Previous apparently conflicting results regarding the localization of BFRF1 homologs in other herpesviruses might simply reflect differences in the visualization methods (conventional versus confocal fluorescence) or be due to different viral strains or various target cell lines used.…”

“…Since by immunofluorescence, and previously by immunoelectron microscopy for BFRF1 (15), we have demonstrated the localization of BFRF1 on the nuclear membrane and since we show here that BFLF2, when coexpressed with BFRF1, acquires a similar nuclear membrane localization, we analyzed whether the single or double expression of the two proteins might induce structural alterations of the nuclear membrane. To this end, we used untransfected 293 cells and 293 cells transfected with either BFRF1, BFLF2, BFRF1 and BFLF2, or a vector control.…”

“…Based upon its intracellular localization (15) and the functional analysis of a viral mutant with BFRF1 deleted (16), we showed that BFRF1 is involved in viral envelopment and egress, and we suggested that, in analogy with other herpesviruses, it might interact with the EBV homolog of the conserved herpesviral protein UL31, encoded by the BFLF2 gene. Such an interaction has been recently demonstrated by Lake and Hutt-Fletcher (24), who reported that BFRF1 and BFLF2 colocalize in cotransfected cells and that each protein influences the intracellular localization of the other.…”

Interferon Regulatory Factor 7 Is Negatively Regulated by the Epstein-Barr Virus Immediate-Early Gene,BZLF-1

“…Features of this infected tissue include parakeratosis of the superficial epithelial layer, acanthosis of the mid-stratum spinosum, and ballooning degeneration of koilocyte-like cells (24,62,79). Viral lytic cycle EBV proteins are detected in the spinosum of HLP, including BZLF-1, BRLF-1, and BHRF-1 gene products, as well as latent cycle genes such as LMP-1, LMP-2A, LMP-2B, EBV nuclear antigen 1 (EBNA-1), EBNA-2, and EBNA-LP (16,(61)(62)(63)(81)(82)(83)(84). Importantly, LMP-1 signaling seems intact in HLP lesions since the protein's associated cofactors, such as TRAFs, are found colocalized and downstream targets such as activated NF-B are detected in the nucleus (83).…”