de La Fuente C, Santiago F, Chong S Y, Deng L, Mayhood T, Fu P, Stein D, Denny T, Coffman F, Azimi N, Mahieux R, Kashanchi F
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103, USA.
J Virol. 2000 Aug;74(16):7270-83. doi: 10.1128/jvi.74.16.7270-7283.2000.
Human T-cell lymphotropic virus type 1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). T-cell transformation is mainly due to the actions of the viral phosphoprotein Tax. Tax interacts with multiple transcriptional factors, aiding the transcription of many cellular genes. Here, we report that the cyclin-dependent kinase inhibitor p21/waf1 is overexpressed in all HTLV-1-infected cell lines tested as well as in ATL and HAM/TSP patient samples. Tax was found to be able to transactivate the endogenous p21/waf1 promoter, as detected by RNase protection, as well as activate a series of wild-type and 5'-deletion constructs linked to a luciferase reporter cassette. Wild-type but not a mutant form of Tax (M47) transactivated the p21/waf1 promoter in a p53-independent manner and utilized a minimal promoter that contained E2A and TATA box sequences. The p21/waf1 protein was reproducibly observed to be complexed with cyclin A/cdk2 and not with any other known G(1), S, or G(2)/M cyclins. Functionally, the association of p21/cyclin A/cdk2 decreased histone H1 phosphorylation in vitro, as observed in immunoprecipitations followed by kinase assays, and affected other substrates, such as the C terminus of Rb protein involved in c-Abl and histone deacetylase-1 (HDAC1) regulation. Interestingly, upon the use of a stress signal, such as gamma-irradiation, we found that the p21/cyclin A/cdk2 complex was able to block all known phosphorylation sites on the Rb molecule. Finally, using elutriated cell cycle fractions and a stress signal, we observed that the HTLV-1-infected T cells containing wild-type Tax, which had been in early or mid-G(1) phase prior to gamma-irradiation, arrested in G(1) and did not undergo apoptosis. This may be an important mechanism for an oncogenic virus such as HTLV-1 to stop the host at the G(1)/S boundary and to repair the damaged DNA upon injury, prior to S-phase entry.
人类嗜T细胞病毒1型（HTLV-1）与成人T细胞白血病（ATL）以及HTLV-1相关脊髓病/热带痉挛性截瘫（HAM/TSP）有关。T细胞转化主要归因于病毒磷蛋白Tax的作用。Tax与多种转录因子相互作用，有助于许多细胞基因的转录。在此，我们报告细胞周期蛋白依赖性激酶抑制剂p21/waf1在所有测试的HTLV-1感染细胞系以及ATL和HAM/TSP患者样本中均过度表达。通过核糖核酸酶保护检测发现，Tax能够反式激活内源性p21/waf1启动子，并且还能激活一系列与荧光素酶报告盒相连的野生型和5'-缺失构建体。野生型而非突变形式的Tax（M47）以不依赖p53的方式反式激活p21/waf1启动子，并利用了一个包含E2A和TATA盒序列的最小启动子。可重复性地观察到p21/waf1蛋白与细胞周期蛋白A/cdk2形成复合物，而不与任何其他已知的G1、S或G2/M细胞周期蛋白形成复合物。在功能上，如免疫沉淀后进行激酶分析所观察到的，p21/细胞周期蛋白A/cdk2的结合在体外降低了组蛋白H1的磷酸化，并影响了其他底物，例如参与c-Abl和组蛋白去乙酰化酶-1（HDAC1）调节的Rb蛋白的C末端。有趣的是，在使用应激信号（如γ射线照射）时，我们发现p21/细胞周期蛋白A/cdk2复合物能够阻断Rb分子上所有已知的磷酸化位点。最后，使用淘析的细胞周期组分和应激信号，我们观察到含有野生型Tax的HTLV-1感染T细胞，在γ射线照射前处于G1期早期或中期，在G1期停滞且未发生凋亡。这可能是诸如HTLV-1这样的致癌病毒在进入S期之前，使宿主在G1/S边界停滞并在损伤后修复受损DNA的重要机制。
