Early events associated with infection of Epstein-Barr virus infection of primary B-cells.
Early events associated with infection of Epstein-Barr virus infection of primary B-cells.
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DOI：
10.1371/journal.pone.0007214
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发表时间：
2009-09-28
期刊：
影响因子：
3.7
通讯作者：
Robertson ES
中科院分区：
文献类型：
--
作者：
Halder S;Murakami M;Verma SC;Kumar P;Yi F;Robertson ES
爱泼斯坦巴尔病毒（EBV）与大量人类癌症的发展密切相关。为了开发用于监测与EBV感染相关的早期细胞和病毒事件的系统，将含有172-kb的爱泼斯坦巴尔病毒基因组BAC-EBV的自重组BAC命名为MD 1 BAC（Chen et al.，2005，J.Virology）通过同源重组引入绿色荧光蛋白（GFP）的表达盒，并将所得BAC克隆BAC-GFP-EBV转染到HEK 293 T上皮细胞系中。通过化学诱导剂从稳定的HEK 293 T BAC GFP EBV细胞系诱导得到的重组GFP EBV产生子代病毒，并将该病毒用于永生化人原代B细胞，如通过原代B细胞的绿色荧光和生长所监测的。使用流式细胞术通过B细胞表面抗原CD 5、CD 10、CD 19、CD 23、CD 39、CD 40、CD 44和细胞间增殖标记物Ki-67的表达来监测GFP EBV引起的感染、B细胞活化和细胞增殖。结果显示Ki-6 - 7显著增加，其在感染后6-7天继续增加。同样，CD 40信号显示逐渐增加，而CD 23信号在6-12小时增加，最多3天，然后下降。监测病毒基因表达模式显示裂解基因表达的早期爆发。在早期感染期间，裂解基因表达先于潜伏基因的这种上调强烈表明EBV以裂解基因表达的初始爆发感染原代B细胞，并且所得子代病毒能够感染新的原代B细胞。该过程对于在细胞转化之前建立潜伏期可能是关键的。可以进一步分析新感染的原代B细胞以研究由于EBV感染引起的B细胞活化。
Epstein Barr virus (EBV) is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology) was used to introduce an expression cassette of green fluorescent protein (GFP) by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6–7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6–12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection.
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影响因子：
5.4
作者：
COLBY, BM;SHAW, JE;PAGANO, JS
通讯作者：
PAGANO, JS
影响因子：
6.4
作者：
Gagro, A;Mccloskey, N;Gordon, J
通讯作者：
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影响因子：
5.4
作者：
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影响因子：
56.9
作者：
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通讯作者：
PRASHER, DC
DOI：
10.1073/pnas.1231363100
发表时间：
2003-06-24
影响因子：
11.1
作者：
Imadome, KI;Shirakata, M;Yamanashi, Y
通讯作者：
Yamanashi, Y