Noteborn Mathieu H M
Department of Molecular Cell Biology, Leadd BV and LUMC, Leiden, The Netherlands.
Vet Microbiol. 2004 Feb 4;98(2):89-94. doi: 10.1016/j.vetmic.2003.10.003.
In 1990, the chicken anemia virus (CAV) genome was cloned by us and proven to be representative for CAV isolates worldwide. This genome contains unique promoter/enhancer replication elements and genes. Upon infection of its target cells, CAV replicates via a double-stranded (ds) DNA intermediate. From this ds CAV molecule, a single mRNA is transcribed, which encodes for three distinct proteins VP1, VP2, and VP3 or apoptin. Its capsid contains only the VP1 protein. However, for the production of the neutralizing epitope, co-synthesis of VP1 and VP2 are needed. CAV genomes with mutations in the 12 bp insert of the promoter/enhancer region were shown to produce immunogenic functional CAV particles. Mutations in these and other regulatory elements of CAV might also decrease its virus load resulting in a reduced pathogenic effect. CAV causes fatal cytopathogenic effects in e.g. chicken thymocytes via apoptosis. Under in vitro conditions, CAV replicates only in transformed chicken cell lines, which indicates that at least a part of the CAV life-cycle requires transformed-like cellular events. In these transformed cell lines, the synthesis of the apoptin protein alone mimics the CAV-induced apoptosis, whereas the VP2 protein also harbors some apoptotic activity. Extensive studies on apoptin resulted in the characterization of domains essential for its apoptotic activity and nuclear localization, which seems to be related with its ability to induce apoptosis. Therefore, both VP2 and apoptin are of interest in reducing the pathogenicity of CAV infections. A series of biomedical studies on apoptin have been carried out in human cell systems, which are informative about the mechanism of CAV-induced apoptosis in chicken (transformed) cells. Synthesis of apoptin alone induces apoptosis in various human transformed and/or tumorigenic cell lines, but not in normal human diploid cells. A striking difference in the cellular localization of apoptin was observed in human normal diploid cells versus tumor cells. In all tumor cells, apoptin is located mainly in the heterochromatic regions of the nucleus, whereas in normal cells it is present in peri-nuclear structures. Apoptin contains a bipartite nuclear localization signal, and one domain that resemble a nuclear export signal. Elucidation of parts of the apoptin-induced apoptotic pathway revealed unique characteristics: apoptin-induced apoptosis is independent of the tumor suppressor p53. The anti-apoptotic protein Bcl-2 does not inhibit but even accelerates apoptin-induced apoptosis in tumor cells, whereas over expression of Bcl-2 in normal cells has no effect on the apoptin activity. Upstream caspases are not involved, whereas downstream caspase 3 is, but seems not to be essential. A number of novel proteins were shown to interact with apoptin in transformed cells. Future studies of apoptin, VP2 and related cellular proteins in chicken cells will unravel the regulatory aspects of CAV-induced apoptosis.
1990年，我们克隆了鸡贫血病毒（CAV）基因组，并证明其代表了全球范围内的CAV分离株。该基因组包含独特的启动子/增强子复制元件和基因。感染靶细胞后，CAV通过双链（ds）DNA中间体进行复制。从这个ds CAV分子转录出一条单一的mRNA，它编码三种不同的蛋白质VP1、VP2和VP3或凋亡素。其衣壳仅包含VP1蛋白。然而，为了产生中和表位，需要VP1和VP2共同合成。已证明启动子/增强子区域12 bp插入片段发生突变的CAV基因组能产生具有免疫原性的功能性CAV颗粒。CAV这些及其他调控元件的突变也可能降低其病毒载量，从而降低致病作用。CAV通过凋亡在例如鸡胸腺细胞中引起致命的细胞病变效应。在体外条件下，CAV仅在转化的鸡细胞系中复制，这表明CAV生命周期的至少一部分需要类似转化的细胞事件。在这些转化细胞系中，单独合成凋亡素蛋白可模拟CAV诱导的凋亡，而VP2蛋白也具有一定的凋亡活性。对凋亡素的广泛研究导致了对其凋亡活性和核定位所必需结构域的表征，这似乎与其诱导凋亡的能力有关。因此，VP2和凋亡素在降低CAV感染的致病性方面都具有研究价值。已经在人类细胞系统中对凋亡素进行了一系列生物医学研究，这些研究有助于了解CAV在鸡（转化）细胞中诱导凋亡的机制。单独合成凋亡素可在各种人类转化和/或致瘤细胞系中诱导凋亡，但在正常人类二倍体细胞中则不会。在人类正常二倍体细胞与肿瘤细胞中观察到凋亡素在细胞定位上存在显著差异。在所有肿瘤细胞中，凋亡素主要位于细胞核的异染色质区域，而在正常细胞中它存在于核周结构中。凋亡素包含一个双分核定位信号和一个类似于核输出信号的结构域。对凋亡素诱导的凋亡途径部分的阐明揭示了独特的特征：凋亡素诱导的凋亡独立于肿瘤抑制因子p53。抗凋亡蛋白Bcl-2不抑制反而加速肿瘤细胞中凋亡素诱导的凋亡，而在正常细胞中过表达Bcl-2对凋亡素活性没有影响。上游半胱天冬酶不参与，而下游半胱天冬酶3参与，但似乎不是必需的。已证明一些新蛋白在转化细胞中与凋亡素相互作用。未来对鸡细胞中凋亡素、VP2及相关细胞蛋白的研究将揭示CAV诱导凋亡的调控方面。
