The Epstein-Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation
The Epstein-Barr virus LMP1 amino acid sequence that engages tumor necrosis factor receptor associated factors is critical for primary B lymphocyte growth transformation
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DOI：
10.1073/pnas.94.4.1447
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发表时间：
1997-02-18
影响因子：
11.1
通讯作者：
Kieff, ED
中科院分区：
文献类型：
--
作者：
Izumi, KM;Kaye, KM;Kieff, ED
Epstein-Barr 病毒 (EBV) 潜伏膜蛋白 1 (LMP1) 对于将原代 B 淋巴细胞转化为类淋巴母细胞系至关重要。 LMP1 基因在 LMP1 羧基末端的近 45 个密码子后被截短的 EBV 重组体足以进行转化。近端 45 个残基包括一个与肿瘤坏死因子受体相关因子 (TRAF) 结合的结构域。我们通过测定删除 LMP1 密码子 185-211 的重组 EBV 基因组的转化能力，研究了 TRAF 结合结构域的重要性。该突变消除了酵母和淋巴母细胞中的 TRAF 关联，但不影响 LMP1 稳定性或定位。通过用重叠的 EBV 粘粒转染 P3HR-1 细胞来产生特异性突变的重组 EBV 基因组，原代 B 淋巴细胞的感染导致细胞系与 LMP1 Delta 185-211 EBV 重组体和 P3HR-1 EBV 共感染，P3HR-1 EBV 具有野生型 LMP1 基因，但由于另一次缺失而存在转化缺陷。尽管来自 5 个共感染细胞系的病毒制剂中野生型和突变型 LMP1 基因等摩尔混合物，但在原代 B 淋巴细胞转化后获得的 412 个细胞系中仅发现野生型 LMP1 基因。没有转化的细胞系仅具有LMP1 Delta 185-211基因。带有 Flag 标签的 LMP1 基因的 EBV 重组体从共感染的 P3HR-1 中平行传代分离。这些数据表明 LMP1 TRAF 结合域对于原代 B 淋巴细胞生长转化至关重要。
Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for transforming primary B lymphocytes into lymphoblastoid cell lines. EBV recombinants with LMP1 genes truncated after the proximal 45 codons of the LMP1 carboxyl terminus are adequate for transformation. The proximal 45 residues include a domain that engages the tumor necrosis factor receptor associated factors (TRAFs), We investigated the importance of the TRAF binding domain by assaying the transforming ability of recombinant EBV genomes with a deletion of LMP1 codons 185-211. This mutation eliminates TRAF association in yeast and in lymphoblasts but does not affect LMP1 stability or localization. Specifically mutated recombinant EBV genomes were generated by transfecting P3HR-1 cells with overlapping EBV cosmids, Infection of primary B lymphocytes resulted in cell lines that were coinfected with an LMP1 Delta 185-211 EBV recombinant and P3HR-1 EBV, which has a wild-type LMP1 gene but is transformation defective due to another deletion. Despite the equimolar mixture of wild-type and mutated LMP1 genes in virus preparations from five coinfected cell lines, only the wild-type LMP1 gene was found in 412 cell lines obtained after transformation of primary B lymphocytes. No transformed cell line had only the LMP1 Delta 185-211 gene. An EBV recombinant with a Flag-tagged LMP1 gene passaged in parallel segregated from the coinfecting P3HR-1. These data indicate that the LMP1 TRAF binding domain is critical for primary B lymphocyte growth transformation.