Changes of cell cycle distribution and nuclear structure in NK cells by EBV infection

虽然相当一部分自然杀伤(NK)细胞肿瘤具有EBV基因组，但目前还没有直接证据表明EBV在体外感染人NK细胞。在这项研究中，我们使用含有增强型绿色荧光(EGFP)的重组EBV证明了EBV进入NK细胞。48小时后，约30%的NK细胞系和40%以上的正常外周血NK细胞呈EGFP信号阳性。EBNAs和BHLFs的原位杂交结果显示，EBV感染早期潜伏感染和裂解感染并存。潜伏期EBV感染而不是溶解EBV感染的NK细胞呈现一种奇特的巨型。在G2/M转换过程中起重要作用的CDc2、Cyclin B和CDc25C蛋白水平升高，并部分迁移到细胞核，但它们的激活受到抑制。流式细胞仪分析显示EBV感染的NK细胞主要发生G2期停滞。尽管PML、SC35和HP1的核分布没有变化，但由肌动蛋白、微管蛋白和层蛋白组成的细胞骨架和核骨架发生了明显的重排，并检测到中心体数量的异常。我们建立了8个携带EBV的克隆，它们都表现为潜伏期类型I和II。这两个克隆在EBV相关的NK细胞肿瘤中都被识别。在携带EBV的NKL克隆中，细胞尺寸变大，微核形成频率增加，这是基因组不稳定的标志。此外，与亲本NK细胞系相比，这些克隆对各种化疗药物和细胞稀释具有抵抗力。

Although considerable part of natural killer (NK) cell neoplasms possess EBV genome, there has been no direct evidence that EBV infects human NK cells in vitro. In this study, we demonstrated EBV entry into the NK cells using enhanced green fluorescence (EGFP)-containing recombinant EBV. After 48 hr, about 30% of NK cell lines and more than 40% of normal peripheral blood NK cells were positive for EGFP signal. In situ hybridization for EBNAs and BHLFs showed that latent and lytic infections coexisted at the early phase of EBV infection. NK cells in latent but not lytic EBV infection tended to show a bizzare and giant shape. Cdc2, cyclin B, and Cdc25C, which are important for G2/M transition, increased in protein levels and partially migrated to the nucleus though their activation was inhibited. Flow cytometric analysis showed mainly G2 arrest in EBV-infected NK cells. Although nuclear distribution of PML, SC35, and HP1 was unchanged, cyto- and nuclear-skeletons made of actin, tubulin and lamin has apparently rearranged and abnormalities of centrosome numbers were detected. We established eight EBV-carrying clones, which showed both latency types I and II. Both of them are recognized in EBV-associated NK-cell neoplasms. The cell size became larger and the frequency of micronuclei formation, a hallmark of genomic instability, increased in EBV-carrying NKL clones. Furthermore, these clones were resistant to various chemotherapeutic agents and cell dilution compared to the parent NK cell lines.