Laboratory of Molecular Human Retrovirology, University of Manitoba, Winnipeg, MB, Canada.
Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, MB, Canada.
Virol J. 2019 Apr 2;16(1):42. doi: 10.1186/s12985-019-1134-8.
HIV integrase (IN) and its cellular cofactors, including lens-epithelium-derived growth factor (LEDGF/p75), Ku70, p300, and Rad52, are subject to small ubiquitin-like modifier (SUMO) modification. In addition to covalent SUMOylation, SUMO paralogs can also noncovalently bind proteins through SUMO-interacting motifs (SIMs). However, little is known about whether HIV IN contains SIMs and the roles of these motifs.
We searched for the amino acid sequence of HIV IN and investigated three putative SIMs of IN: SIM1 72VILV75, SIM2 200IVDI203 and SIM3 257IKVV260. Our mutational analysis showed that 200IVDI203 and 257IKVV260 are two bona fide SIMs that mediate IN-SUMO noncovalent interactions. Additionally, a cell-based SUMOylation assay revealed that IN SIMs negatively regulate the SUMOylation of IN, as well as the interaction between IN and SUMO E2 conjugation enzyme Ubc9. Conversely, IN SIMs are required for its interactions with LEDGF/p75 but not with Ku70. Furthermore, our study reveals that SIM2 and SIM3 are required for the nuclear localization of IN. Finally, we investigated the impact of IN SIM2 and SIM3 on HIV single cycle replication in CD4 C8166 T cells, and the results showed that viruses carrying IN SIM mutants are replication defective at the steps of the early viral life cycle, including reverse transcription, nuclear import and integration.
Our data suggested that the IN-SUMO interaction constitutes a new regulatory mechanism of IN functions and might be important for HIV-1 replication.
HIV 整合酶（IN）及其细胞辅助因子，包括晶状体上皮衍生生长因子（LEDGF/p75）、Ku70、p300 和 Rad52，都受到小泛素样修饰物（SUMO）的修饰。除了共价 SUMO 化，SUMO 同源物还可以通过 SUMO 相互作用基序（SIM）非共价结合蛋白质。然而，目前尚不清楚 HIV IN 是否含有 SIM 以及这些基序的作用。
我们搜索了 HIV IN 的氨基酸序列，并研究了 IN 的三个假定 SIM：SIM1 72VILV75、SIM2 200IVDI203 和 SIM3 257IKVV260。我们的突变分析表明，200IVDI203 和 257IKVV260 是两个真正的 SIM，介导 IN-SUMO 非共价相互作用。此外，基于细胞的 SUMOylation 测定显示，IN SIM 负调控 IN 的 SUMO 化以及 IN 与 SUMO E2 连接酶 Ubc9 的相互作用。相反，IN SIM 对于其与 LEDGF/p75 的相互作用是必需的，但不是与 Ku70 的相互作用。此外，我们的研究表明，SIM2 和 SIM3 对于 IN 的核定位是必需的。最后，我们研究了 IN SIM2 和 SIM3 对 CD4 C8166 T 细胞中 HIV 单周期复制的影响，结果表明，携带 IN SIM 突变体的病毒在早期病毒生命周期的步骤中复制缺陷，包括逆转录、核输入和整合。
我们的数据表明，IN-SUMO 相互作用构成了 IN 功能的新调节机制，可能对 HIV-1 复制很重要。
