Rumlová Michaela, Křížová Ivana, Keprová Alena, Hadravová Romana, Doležal Michal, Strohalmová Karolína, Pichová Iva, Hájek Miroslav, Ruml Tomáš
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v,v,i,, IOCB & Gilead Research Center, Flemingovo nám, 2, 166 10 Prague, Czech Republic.
Retrovirology. 2014 May 20;11:37. doi: 10.1186/1742-4690-11-37.
Apoptosis is one of the presumptive causes of CD4+ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established.
Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein.
In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3.
细胞凋亡是人类免疫缺陷病毒(HIV)感染及发展为获得性免疫缺陷综合征(AIDS)过程中CD4⁺T细胞耗竭的假定原因之一。然而,HIV-1在此过程中的精确作用仍未明确。HIV-1蛋白酶(PR)被认为是一个可能的因素,但HIV-1 PR酶活性与细胞凋亡之间的直接联系尚未确立。
在此,我们表明活性HIV-1 PR的表达通过线粒体凋亡途径诱导HeLa和HEK-293细胞死亡。这一结论基于对HIV-1 PR在线粒体中直接定位的体内观察,线粒体是触发细胞凋亡的关键因素。此外,我们观察到HIV-1 PR浓度依赖性地降低线粒体膜电位,以及HIV-1 PR在激活半胱天冬酶9、聚(ADP-核糖)聚合酶(PARP)裂解和DNA片段化中的作用。此外,体外数据表明HIV-1 PR介导线粒体蛋白Tom22、电压依赖性阴离子通道(VDAC)和腺嘌呤核苷酸转运体(ANT)的裂解,导致凋亡诱导因子(AIF)和热休克蛋白60(Hsp60)的释放。通过酵母双杂交筛选,我们还鉴定了一种新的HIV-1 PR相互作用伙伴,乳腺癌相关蛋白3(BCA3)。我们发现BCA3加速p53对bax启动子的转录活性,从而提高促凋亡蛋白Bax的细胞水平。
总之,我们的结果描述了HIV-1 PR参与细胞凋亡,这是由HIV-1 PR对线粒体膜完整性的直接作用或其与细胞蛋白BCA3的相互作用引起的。