Peng Ke, Muranyi Walter, Glass Bärbel, Laketa Vibor, Yant Stephen R, Tsai Luong, Cihlar Tomas, Müller Barbara, Kräusslich Hans-Georg
Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany.
Gilead Sciences Inc., Foster City, United States.
Elife. 2014 Dec 17;3:e04114. doi: 10.7554/eLife.04114.
The steps from HIV-1 cytoplasmic entry until integration of the reverse transcribed genome are currently enigmatic. They occur in ill-defined reverse-transcription- and pre-integration-complexes (RTC, PIC) with various host and viral proteins implicated. In this study, we report quantitative detection of functional RTC/PIC by labeling nascent DNA combined with detection of viral integrase. We show that the viral CA (capsid) protein remains associated with cytoplasmic RTC/PIC but is lost on nuclear PIC in a HeLa-derived cell line. In contrast, nuclear PIC were almost always CA-positive in primary human macrophages, indicating nuclear import of capsids or capsid-like structures. We further show that the CA-targeted inhibitor PF74 exhibits a bimodal mechanism, blocking RTC/PIC association with the host factor CPSF6 and nuclear entry at low, and abrogating reverse transcription at high concentrations. The newly developed system is ideally suited for studying retroviral post-entry events and the roles of host factors including DNA sensors and signaling molecules.
从HIV-1进入细胞质到逆转录基因组整合的步骤目前尚不清楚。这些步骤发生在定义不明确的逆转录和预整合复合物（RTC、PIC）中，涉及多种宿主和病毒蛋白。在本研究中，我们报告了通过标记新生DNA并结合病毒整合酶的检测对功能性RTC/PIC进行定量检测。我们发现，在源自HeLa的细胞系中，病毒CA（衣壳）蛋白仍与细胞质RTC/PIC相关，但在核PIC中丢失。相反，在原代人巨噬细胞中，核PIC几乎总是CA阳性，表明衣壳或衣壳样结构进入细胞核。我们进一步表明，靶向CA的抑制剂PF74表现出双峰机制，在低浓度下阻断RTC/PIC与宿主因子CPSF6的结合以及核进入，在高浓度下则废除逆转录。新开发的系统非常适合研究逆转录病毒进入后的事件以及包括DNA传感器和信号分子在内的宿主因子的作用。
