The<i>BFRF1</i>Gene of Epstein-Barr Virus Encodes a Novel Protein

Farina, Antonella; Santarelli, Roberta; Gonnella, Roberta; Bei, Roberto; Muraro, Raffælla; Cardinali, Giorgia; Uccini, Stefania; Ragona, Giuseppe; Frati, Luigi; Faggioni, Alberto; Angeloni, Antonio

doi:10.1128/jvi.74.7.3235-3244.2000

Cited by 45 publications

(42 citation statements)

References 39 publications

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“…3A, BFLF2 expression was not affected by PAA. In addition, since BFLF2 is expressed in Raji cells, which harbor a defective viral strain which does not allow the expression of late EBV genes, we conclude that BFLF2, similarly to BFRF1 (14), is expressed as an early gene.…”

Section: Resultsmentioning

confidence: 83%

“…The generation of a specific MAb has allowed us to study the intracellular localization of the protein in virus-infected cells and its interaction with the recently described BFRF1 gene product (14), the UL34 positional homolog of EBV. Although the function of BFLF2 is still unknown, clues come from its intracellular localization and from its interaction with BFRF1, as well from analogies with other herpesviral homologs.…”

Section: Discussionmentioning

confidence: 99%

“…The CMV-BFRF1 construct was described elsewhere (14). One microgram of either the CMV-BFRF1 or CMV-BFLF2 construct was used to transfect 3 ϫ 10 4 293 cells by using the Fugene 6 kit (Roche) according to the manufacturer's instructions Production of a MAb against BFLF2.…”

Section: Methodsmentioning

confidence: 99%

“…We initially identified and characterized the product of the Epstein-Barr virus (EBV) BFRF1 gene, which is the positional homolog of the UL34 gene (14). 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.…”

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confidence: 99%

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

Gonnella

Farina

Santarelli

et al. 2005

J Virol

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105

135

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. 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.Two conserved herpesvirus proteins, designated UL34 and UL31, of herpes simplex virus (HSV) and pseudorabies virus (PrV) are involved in the early steps of viral maturation at the nuclear envelope (reviewed in reference 26). With many similarities and a few differences, accumulating evidence indicates that these proteins and their homologs play similar roles in nuclear egress of both alpha-and betaherpesviruses (9,17,23,30,33,34,36,37,40,48,50). The physical interaction between the two proteins appears to be important to facilitate virion envelopment at the inner nuclear membrane, a process which probably also involves nuclear lamina disruption, to allow nucleocapsids to gain access at the interior face of the nuclear envelope (28, 39).We initially identified and characterized the prod...

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Section: Resultsmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Gonnella

Farina

Santarelli

et al. 2005

J Virol

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105

135

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“…We have recently identified and characterized the product of the BFRF1 open reading frame (ORF), which is expressed early in the viral replication process (1,12). BFRF1 shows a degree of homology to UL34, and both proteins are located in the nuclear membrane of replicating cells, preferentially in areas where budding of the nucleocapsids underneath occurs (13,15,43).…”

mentioning

confidence: 99%

BFRF1 of Epstein-Barr Virus Is Essential for Efficient Primary Viral Envelopment and Egress

Farina

Feederle

Raffa

et al. 2005

J Virol

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101

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The molecular mechanisms that underlie maturation and egress of Epstein-Barr virus (EBV) virions are only partially characterized. We have recently shown that the BFRF1 gene, the EBV positional homolog of herpes simplex virus type 1 and pseudorabies virus UL34, is expressed early during EBV lytic replication and that it is found predominantly on the nuclear membrane (A. Farina, R. Santarelli, R. Gonnella, R. Bei, R. Muraro, G. Cardinali, S. Uccini, G. Ragona, L. Frati, A. Faggioni, and A. Angeloni, J. Virol. 74:3235-3244, 2000). These data suggest that the BFRF1 protein might be involved in viral primary envelopment. To precisely determine the function of this protein, we have constructed an EBV mutant devoid of the BFRF1 gene (BFRF1-KO). 293 cells carrying BFRF1-KO showed no differences in comparison with wild-type EBV in terms of DNA lytic replication or expression of late viral proteins upon induction of the lytic cycle. However, binding assays and infection experiments using cell lines or human cord blood lymphocytes showed a clear reduction in the viral mutant titers. Complementation experiments with BFRF1-KO and a BFRF1 expression vector restored viral titers to levels similar to those for the wild-type control, showing that the modifications that we introduced were limited to the BFRF1 gene. Electron microscopic observations showed that the reduction in viral titers was due to sequestration of EBV nucleocapsids in the nuclei of lytically induced cells. This suggests that BFRF1 is involved in transport of the maturing virion across the nuclear membrane. This hypothesis was further supported by the observation that BFRF1 is present in maturing intracellular virions but not in their extracellular counterparts. This implies that BFRF1 is a key protein for EBV maturation.Epstein-Barr virus (EBV) is one of the eight known human herpesviruses. This member of the gammaherpesvirus subfamily infects B lymphocytes, in which it establishes a latent infection characterized by the expression of a limited set of viral genes (25). Viral reactivation from the latent state either occurs spontaneously or is induced by a variety of different stimuli (11,30,32,49,55), leading to viral lytic replication and shedding of viral progeny. The EBV lytic program consists of the sequential activation of three distinct classes of viral genes: immediate early, early, and late. The two transactivators BZLF1 (ZEBRA) and BRLF1 (Rta) are immediate-early genes that can initiate the switch between latency and lytic replication (14,24,41). Early genes are frequently but not exclusively involved in viral DNA replication; these genes include, among many others, those for the viral DNA polymerase (31) and its processivity factor BMRF1 (5), the bcl-2 homolog BHRF1 (38), and the major DNA binding protein BALF2 (8).Late genes are known to encode predominantly structural proteins, such as gp350/220, the most abundant glycoprotein of the viral envelope. gp350/220 mediates the binding of the virus to its cognate receptor CR2 (50). Herpesvirus DNA repli...

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A proteome‐wide analysis unveils a core Epstein–Barr virus antibody signature of classic Hodgkin lymphoma across ethnically diverse populations

Sarathkumara,

Xian,

Liu

et al. 2024

Intl Journal of Cancer

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Epstein–Barr virus (EBV) is an oncogenic virus associated with various malignancies, including classical Hodgkin lymphoma (cHL). Despite its known association, the specific role of humoral immune response to EBV remains poorly characterized in cHL. To address this, we conducted a study using a custom protein microarray to measure the antibody responses in cHL patients and matched healthy controls recruited from an East‐Asian hospital‐based case–control study. We identified 16 IgG antibodies significantly elevated in EBV‐positive cHL compared with controls, defining an “East‐Asian antibody signature of EBV‐positive cHL.” We evaluated responses against these 16 antibodies in a distinct European population, leveraging data from our previous European cHL case–control study from the UK, Denmark, and Sweden. A subset of antibodies (14/16, 87.5%) from the “East‐Asian antibody signature of EBV‐positive cHL” exhibited significant associations with cHL in the European population. Conversely, we assessed the “European antibody signature of EBV‐positive cHL” identified in our prior study which consisted of 18 EBV antibodies (2 IgA, 16 IgG), in the East‐Asian population. A subset of these antibodies (15/18, 83.3%) maintained significant associations with cHL in the East‐Asian population. This cross‐comparison of antibody signatures underscores the robust generalizability of EBV antibodies across populations. Five anti‐EBV IgG antibodies (LMP‐1, TK, BALF2, BDLF3, and BBLF1), found in both population‐specific antibody signatures, represent a “core signature of EBV‐positive cHL.” Our findings suggest that the antibody responses targeting these core EBV proteins reflect a specific EBV gene expression pattern, serving as potential biomarkers for EBV‐positive cHL independent of population‐specific factors.

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TheBFRF1Gene of Epstein-Barr Virus Encodes a Novel Protein

Cited by 45 publications

References 39 publications

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

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

BFRF1 of Epstein-Barr Virus Is Essential for Efficient Primary Viral Envelopment and Egress

A proteome‐wide analysis unveils a core Epstein–Barr virus antibody signature of classic Hodgkin lymphoma across ethnically diverse populations

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