Viral Mutation Section and Viral Recombination Section, HIV Drug Resistance Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702.
Proc Natl Acad Sci U S A. 2013 Dec 3;110(49):E4780-9. doi: 10.1073/pnas.1315996110. Epub 2013 Nov 18.
Human cytidine deaminases APOBEC3F (A3F) and APOBEC3G (A3G) are host factors that incorporate into virions and restrict virus replication. We labeled HIV-1 particles with yellow fluorescent protein (YFP)-tagged APOBEC3 proteins and examined their association with preintegration complexes (PICs) in infected cells. Labeling of PICs with A3F-YFP, and to a lesser extent A3G-YFP, could be used to visualize PICs in the nuclei, which was dependent on nuclear pore protein Nup153 but not TNPO3. We show that reverse transcription is not required for nuclear import of PICs, indicating that a viral core uncoating event associated with reverse transcription, and the central DNA flap that forms during reverse transcription, are not required for nuclear import. We also quantify association of cytoplasmic PICs with nuclear envelope (NE) and report that capsid mutations that increase or decrease core stability dramatically reduce NE association and nuclear import of PICs. In addition, we find that nuclear PICs remain close to the NE and are not distributed throughout the nuclei. These results provide tools for tracking retroviral PICs in infected cells and reveal insights into HIV-1 replication.
人类胞苷脱氨酶 APOBEC3F（A3F）和 APOBEC3G（A3G）是整合到病毒颗粒中并限制病毒复制的宿主因子。我们用黄色荧光蛋白（YFP）标记的 APOBEC3 蛋白标记 HIV-1 颗粒，并在感染细胞中检查它们与整合前复合物（PIC）的关联。用 A3F-YFP 标记 PIC，在较小程度上用 A3G-YFP 标记，可用于可视化核内的 PIC，这依赖于核孔蛋白 Nup153，但不依赖于 TNPO3。我们表明，PIC 的核内输入不需要逆转录，这表明与逆转录相关的病毒核心脱壳事件以及在逆转录过程中形成的中央 DNA 瓣对于核内输入不是必需的。我们还定量了细胞质 PIC 与核膜（NE）的关联，并报告说，增加或减少核心稳定性的衣壳突变会极大地减少 NE 关联和 PIC 的核内输入。此外，我们发现核内 PIC 仍然靠近 NE，而不是分布在整个核内。这些结果为跟踪感染细胞中的逆转录病毒 PIC 提供了工具，并揭示了 HIV-1 复制的见解。
