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
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DOI：
10.1128/jvi.79.6.3713-3727.2005
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发表时间：
2005-03-01
影响因子：
5.4
通讯作者：
Faggioni, A
中科院分区：
文献类型：
--
作者：
Gonnella, R;Farina, A;Faggioni, A
我们在随附的论文中报道了EB病毒（EBV）的BFRF 1蛋白对于有效的原发性病毒抑制和排出是重要的（A.法里纳河Feederle，S.拉法河贡内拉河桑塔雷利湖Frati，A. Angeloni，M. R. Torrisi，A. Faggioni和H.- J. Delecluse，J. Virol. 79：3703-3712）。在这里，我们描述了EBV BFLF 2基因的产物的表征，该基因属于保守的疱疹病毒基因家族，其包括单纯疱疹病毒和伪狂犬病病毒的UL 31基因，并且已知其产物与BFRF 1的位置同源物UL 34相互作用。BFLF 2是一种早期转录物，在EBV裂解周期激活后在多种细胞系中表达。纯化的病毒体制剂的蛋白质印迹法显示，BFLF 2是细胞内病毒体的组分，但不存在于成熟的细胞外病毒体中。免疫共沉淀实验表明，BFLF 2与BFRF 1相互作用，这是证实了免疫荧光共聚焦显微镜显示，这两种蛋白质共定位在核膜上，不仅在上皮细胞共转染，但也在病毒复制。在携带BFRF 1基因缺失的EBV突变体的细胞（293-BFRF 1-KO细胞）中，BFLF 2表达较低，并且在用蛋白酶体抑制剂MG 132处理细胞后，其恢复至野生型水平。此外，通过BFRF 1转染再补充293-BFRF 1-KO细胞使BFLF 2表达恢复至野生型水平。此外，当单独表达时，BFLF 2弥散定位于细胞核内，而在BFRF 1存在下，这两种蛋白质共定位于核边缘。最后，293上皮细胞转染蛋白质或共转染进行了分析，通过电子显微镜研究潜在的核膜形态的改变。超微结构分析显示，（i）BFRF 1引起的核膜的复制，类似于那些报道发生在疱疹病毒复制过程中，和（ii）虽然BFLF 2单独没有引起任何明显的改变，两种蛋白质的共表达显着诱导深刻的卷积的复制核膜。生物化学和形态学分析均显示BFRF 1-BFLF 2复合物与核纤层的组分核纤层蛋白B相关。总之，这些结果和所附论文的结果（Farina等人，J. Virol. 79：3703-3712）表明BFRF 1和BFLF 2在EBV在核膜成熟的早期步骤中的重要作用。
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.