Nascimento R, Parkhouse R M E
Instituto Gulbenkian de Ciência, Apartado 14, Oeiras, Portugal.
J Gen Virol. 2007 May;88(Pt 5):1446-1453. doi: 10.1099/vir.0.82589-0.
The objective of this work was to identify novel viral 'evasion' genes without homology in the database through functional assays. Using this approach, the 'unassigned', conserved murine gammaherpesvirus ORF20 gene was shown to localize in the nucleus and to induce cell-cycle arrest followed by apoptosis in both mouse and human cells. Such growth-arrested cells did not express phospho-histone H3, demonstrating that the virus protein caused arrest at the G2 stage of the cell cycle. To characterize the mechanism further, Western blots of ORF20-recombinant lentivirus-infected cells were developed with antibodies to cyclin B1, Cdc2 and phospho-Tyr-15-Cdc2. This analysis revealed a relative increase in cyclin B and phospho-Tyr-15-Cdc2, from 24 to 72 h after infection with recombinant lentivirus. The demonstration that Cdc2 is in its inactive phosphorylated form and the clearly increased levels of cyclin B indicated that the virus gene blocks the progression of cells into mitosis by acting at the level of the Cdc2-cyclin B complex. To confirm this result, the Cdc2-cyclin B complex in ORF20-expressing cells was shown to be essentially without kinase activity. As the ORF20 gene is conserved in all herpesvirus, it may be presumed to have evolved to fulfil an important, as yet undefined, biological role in host-cell modification.
这项工作的目的是通过功能测定在数据库中鉴定出无同源性的新型病毒“逃避”基因。采用这种方法，未分类的保守小鼠γ疱疹病毒ORF20基因被证明定位于细胞核，并在小鼠和人类细胞中诱导细胞周期停滞，随后发生凋亡。这种生长停滞的细胞不表达磷酸化组蛋白H3，表明病毒蛋白导致细胞周期在G2期停滞。为了进一步表征其机制，用细胞周期蛋白B1、Cdc2和磷酸化酪氨酸-15-Cdc2抗体对感染ORF20重组慢病毒的细胞进行了蛋白质免疫印迹分析。该分析显示，感染重组慢病毒后24至72小时，细胞周期蛋白B和磷酸化酪氨酸-15-Cdc2相对增加。Cdc2处于无活性磷酸化形式以及细胞周期蛋白B水平明显升高的证明表明，病毒基因通过作用于Cdc2-细胞周期蛋白B复合物水平来阻断细胞进入有丝分裂的进程。为了证实这一结果，在表达ORF20的细胞中，Cdc2-细胞周期蛋白B复合物被证明基本没有激酶活性。由于ORF20基因在所有疱疹病毒中都是保守的，可以推测它已经进化到在宿主细胞修饰中发挥重要的、尚未明确的生物学作用。
