Sustained expression of the novel EBV-induced zinc finger gene, ZNFEB, is critical for the transition of B lymphocyte activation to oncogenic growth transformation.

EBV is a human tumor virus that infects and establishes latency in the majority of humans worldwide. In vitro, EBV growth transforms primary B lymphocytes into lymphoblastoid cell lines with high efficiency. We have used cDNA subtraction cloning to identify cellular target genes required for growth transformation and identified a new C(2)H(2) (Krüppel-type) zinc finger gene, ZNF(EB), that is trans-activated early following EBV infection. In this study, we characterize ZNF(EB), including its intronless locus, and human and mouse protein variants. The gene is transiently expressed during normal lymphocyte activation, and its expression is sustained in EBV-positive but not EBV-negative B cell lines. There is limited expression in nonhemopoietic tissues. Its critical role in the growth transformation of B lineage cells is indicated by the abrogation of transformation with antisense strategies. ZNF(EB) maps to chromosome 18q12, a region with mutations in numerous, predominantly hemopoietic malignancies.     

Epstein-Barr virus mRNA export factor EB2 is essential for production of infectious virus

The splicing machinery which positions a protein export complex near the exon-exon junction mediates nuclear export of mRNAs generated from intron-containing genes. Many Epstein-Barr virus (EBV) early and late genes are intronless, and an alternative pathway, independent of splicing, must export the corresponding mRNAs. Since the EBV EB2 protein induces the cytoplasmic accumulation of intronless mRNA, it is tempting to speculate that EB2 is a viral adapter involved in the export of intronless viral mRNA. If this is true, then the EB2 protein is essential for the production of EBV infectious virions. To test this hypothesis, we generated an EBV mutant in which the BMLF1 gene, encoding the EB2 protein, has been deleted (EBV(BMLF1-KO)). Our studies show that EB2 is necessary for the production of infectious EBV and that its function cannot be transcomplemented by a cellular factor. In the EBV(BMLF1-KO) 293 cells, oriLyt-dependent DNA replication was greatly enhanced by EB2. Accordingly, EB2 induced the cytoplasmic accumulation of a subset of EBV early mRNAs coding for essential proteins implicated in EBV DNA replication during the productive cycle. Two herpesvirus homologs of the EB2 protein, the herpes simplex virus type 1 protein ICP27 and, the human cytomegalovirus protein UL69, only partly rescued the phenotype of the EBV(BMLF1-KO) mutant, indicating that some EB2 functions in virus production cannot be transcomplemented by ICP27 and UL69.     

Cell Type- and Tissue Context-dependent Nuclear Distribution of Human Ago2

Argonaute-2 protein (Ago2), a major component of RNA-induced silencing complex (RISC), has been viewed as a cytoplasmic protein. In this study, we demonstrated by immunofluorescence confocal microscopy that Ago2 is distributed mainly as a nuclear protein in primary human foreskin keratinocytes in monolayer cultures and their derived organotypic (raft) cultures, although it exhibits only a minimal level of nuclear distribution in continuous cell lines such as HeLa and HaCaT cells. Oncogenic human papillomavirus type 16 (HPV16) or type 18 (HPV18) infection of the keratinocytes does not affect the nuclear Ago2 distribution. Examination of human tissues reveals that Ago2 exhibits primarily as a nuclear protein in skin, normal cervix, and cervical cancer tissues, but not in larynx. Together, our data provide the first convincing evidence that the subcellular distribution of Ago2 occurs in a cell type- and tissue context-dependent manner and may correlate with its various functions in regulation of gene expression.     

The Epstein-Barr virus immediate-early protein BZLF1 induces expression of E2F-1 and other proteins involved in cell cycle progression in primary keratinocytes and gastric carcinoma cells

The Epstein-Barr virus (EBV) immediate-early protein BZLF1 mediates the switch between the latent and lytic forms of EBV infection and has been previously shown to induce a G(1)/S block in cell cycle progression in some cell types. To examine the effect of BZLF1 on cellular gene expression, we performed microarray analysis on telomerase-immortalized human keratinocytes that were mock infected or infected with a control adenovirus vector (AdLacZ) or a vector expressing the EBV BZLF1 protein (AdBZLF1). Cellular genes activated by BZLF1 expression included E2F-1, cyclin E, Cdc25A, and a number of other genes involved in cell cycle progression. Immunoblot analysis confirmed that BZLF1 induced expression of E2F-1, cyclin E, Cdc25A, and stem loop binding protein (a protein known to be primarily expressed during S phase) in telomerase-immortalized keratinocytes. Similarly, BZLF1 increased expression of E2F-1, cyclin E, and stem loop binding protein (SLBP) in primary tonsil keratinocytes. In contrast, BZLF1 did not induce E2F-1 expression in normal human fibroblasts. Cell cycle analysis revealed that while BZLF1 dramatically blocked G(1)/S progression in normal human fibroblasts, it did not significantly affect cell cycle progression in primary human tonsil keratinocytes. Furthermore, in EBV-infected gastric carcinoma cells, the BZLF1-positive cells had an increased number of cells in S phase compared to the BZLF1-negative cells. Thus, in certain cell types (but not others), BZLF1 enhances expression of cellular proteins associated with cell cycle progression, which suggests that an S-phase-like environment may be advantageous for efficient lytic EBV replication in some cell types.     

EB病毒诱发人B细胞淋巴瘤的分子病理特性

EB病毒 (EBV ,Epstein Barrvirus)与人类多种肿瘤有关 ,尤其是与鼻咽癌和淋巴瘤的关系密切。为此研究了EB病毒在huPBL SCID嵌合体小鼠体内诱发人B细胞淋巴瘤的分子特性及肿瘤发生机制。从健康成人外周血分离出淋巴细胞 ,将之移植到SCID小鼠腹腔内 ,实验感染EBV ,观察肿瘤的形成 ;采用单向免疫扩散法连续监测小鼠血清中人IgG的含量。分别用PCR方法检测肿瘤组织中是否存在人Alu序列 ,原位杂交检测肿瘤组织中EB病毒小RNA分子EBER 1;免疫组织化学方法检测人白细胞分化抗原 (CD4 5、CD2 0、CD4 5RO、CD3) ,病毒基因(LMP1、EBNA2、BZLF1)的表达 ,以及细胞瘤基因蛋白 (p5 3、C myc、Bcl 2、Bax)在诱发肿瘤中的表达情况。结果发现 ,实验中 34只感染EBV的huPBL SCID小鼠有 2 4只诱发出肿瘤 ,根据病理形态学特征、Alu PCR和免疫标志均证实诱发瘤是人源性B淋巴细胞肿瘤。原位分子杂交显示肿瘤细胞核内存在EBER 1,少数瘤细胞表达EB病毒BZLF1蛋白阳性 ,部分瘤细胞表达LMP1和EBNA2蛋白阳性。连续监测 12只huPBL SCID小鼠血清中人IgG含量 ,发现IgG水平随诱瘤时间延长和肿瘤生长有逐渐增高趋势。免疫组化显示诱发的 2 4例淋巴瘤组织p5 3、C myc、Bcl 2和Bax蛋白表达阳性率分别为 83.33%、10 0 %、95 .83%、91.6 7%。结果