Darbinyan Armine, Siddiqui Khwaja M, Slonina Dorota, Darbinian Nune, Amini Shohreh, White Martyn K, Khalili Kamel
Center for Neurovirology and Cancer Biology, College of Science and Technology, Temple University, 1900 N. 12th St., 015-96, Room 203, Philadelphia, PA 19122, USA.
J Virol. 2004 Aug;78(16):8593-600. doi: 10.1128/JVI.78.16.8593-8600.2004.
The late region of human neurotropic JC virus encodes a small 71-amino-acid agnoprotein that is also found in the polyomaviruses simian virus 40 and BK virus. Several functions of agnoprotein have been identified, including roles in regulating viral transcription and virion maturation. Earlier studies showed that agnoprotein expressed alone induced p21/WAF-1 expression and caused cells to accumulate in the G(2)/M stage of the cell cycle. Here we report that agnoprotein expression sensitized cells to the cytotoxic effects of the DNA-damaging agent cisplatin. Agnoprotein reduced the viability of cisplatin-treated cells and increased chromosome fragmentation and micronucleus formation. Whereas cisplatin-treated control cells accumulated in S phase, cells expressing agnoprotein did not, instead becoming aneuploid. Agnoprotein expression correlated with impaired double-strand-break repair activity in cellular extracts and reduced expression of the Ku70 and Ku80 DNA repair proteins. After agnoprotein expression, much of the Ku70 protein was located in the perinuclear space, where agnoprotein was also found. Results from binding studies showed an interaction of agnoprotein with Ku70 which was mediated by the N terminus. The ability of agnoprotein to inhibit double-strand break repair activity when it was added to cellular extracts was also mediated by the N terminus. We conclude that agnoprotein inhibits DNA repair after DNA damage and interferes with DNA damage-induced cell cycle regulation. Since Ku70 is a subunit of the DNA-dependent protein kinase that is responsible both for double-strand break repair and for signaling damage-induced cell cycle arrest, the modulation of Ku70 and/or Ku80 by agnoprotein may represent an important event in the polyomavirus life cycle and in cell transformation.
嗜人神经JC病毒的晚期区域编码一种由71个氨基酸组成的小ag蛋白，在多瘤病毒猴病毒40和BK病毒中也能发现该蛋白。已确定ag蛋白具有多种功能，包括在调节病毒转录和病毒粒子成熟方面发挥作用。早期研究表明，单独表达的ag蛋白可诱导p21/WAF-1表达，并使细胞在细胞周期的G(2)/M期积累。在此，我们报告ag蛋白的表达使细胞对DNA损伤剂顺铂的细胞毒性作用敏感。ag蛋白降低了顺铂处理细胞的活力，并增加了染色体片段化和微核形成。顺铂处理的对照细胞在S期积累，而表达ag蛋白的细胞则不然，反而变成了非整倍体。ag蛋白的表达与细胞提取物中双链断裂修复活性受损以及Ku70和Ku80 DNA修复蛋白表达降低相关。ag蛋白表达后，大部分Ku70蛋白位于核周空间，ag蛋白也存在于此。结合研究结果表明，ag蛋白与Ku70相互作用，该相互作用由N端介导。当将ag蛋白添加到细胞提取物中时，其抑制双链断裂修复活性的能力也由N端介导。我们得出结论，ag蛋白在DNA损伤后抑制DNA修复，并干扰DNA损伤诱导的细胞周期调控。由于Ku70是DNA依赖性蛋白激酶的一个亚基，负责双链断裂修复和信号损伤诱导的细胞周期停滞，因此ag蛋白对Ku70和/或Ku80的调节可能代表多瘤病毒生命周期和细胞转化中的一个重要事件。
