Institut Cochin, Université de Paris, INSERM U1016, Paris, France.
PLoS Pathog. 2021 Oct 26;17(10):e1009609. doi: 10.1371/journal.ppat.1009609. eCollection 2021 Oct.
Human Immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) succeed to evade host immune defenses by using their viral auxiliary proteins to antagonize host restriction factors. HIV-2/SIVsmm Vpx is known for degrading SAMHD1, a factor impeding the reverse transcription. More recently, Vpx was also shown to counteract HUSH, a complex constituted of TASOR, MPP8 and periphilin, which blocks viral expression from the integrated viral DNA. In a classical ubiquitin ligase hijacking model, Vpx bridges the DCAF1 ubiquitin ligase substrate adaptor to SAMHD1, for subsequent ubiquitination and degradation. Here, we investigated whether the same mechanism is at stake for Vpx-mediated HUSH degradation. While we confirm that Vpx bridges SAMHD1 to DCAF1, we show that TASOR can interact with DCAF1 in the absence of Vpx. Nonetheless, this association was stabilized in the presence of Vpx, suggesting the existence of a ternary complex. The N-terminal PARP-like domain of TASOR is involved in DCAF1 binding, but not in Vpx binding. We also characterized a series of HIV-2 Vpx point mutants impaired in TASOR degradation, while still degrading SAMHD1. Vpx mutants ability to degrade TASOR correlated with their capacity to enhance HIV-1 minigenome expression as expected. Strikingly, several Vpx mutants impaired for TASOR degradation, but not for SAMHD1 degradation, had a reduced binding affinity for DCAF1, but not for TASOR. In macrophages, Vpx R34A-R42A and Vpx R42A-Q47A-V48A, strongly impaired in DCAF1, but not in TASOR binding, could not degrade TASOR, while being efficient in degrading SAMHD1. Altogether, our results highlight the central role of a robust Vpx-DCAF1 association to trigger TASOR degradation. We then propose a model in which Vpx interacts with both TASOR and DCAF1 to stabilize a TASOR-DCAF1 complex. Furthermore, our work identifies Vpx mutants enabling the study of HUSH restriction independently from SAMHD1 restriction in primary myeloid cells.
人类免疫缺陷病毒 1 型和 2 型（HIV-1 和 HIV-2）通过利用其病毒辅助蛋白拮抗宿主限制因子，成功逃避宿主免疫防御。HIV-2/SIVsmm Vpx 已知可降解 SAMHD1，后者是阻碍逆转录的因子。最近，Vpx 也被证明可以拮抗 HUSH，这是一个由 TASOR、MPP8 和 peripherin 组成的复合物，可阻止整合的病毒 DNA 表达病毒。在经典的泛素连接酶劫持模型中，Vpx 将 DCAF1 泛素连接酶底物衔接蛋白与 SAMHD1 桥接，随后进行泛素化和降解。在这里，我们研究了 Vpx 介导的 HUSH 降解是否涉及相同的机制。虽然我们确认 Vpx 将 SAMHD1 桥接到 DCAF1，但我们表明 TASOR 可以在没有 Vpx 的情况下与 DCAF1 相互作用。尽管如此，在 Vpx 的存在下，这种关联得到了稳定，表明存在一个三元复合物。TASOR 的 N 端 PARP 样结构域参与 DCAF1 结合，但不参与 Vpx 结合。我们还对一系列 HIV-2 Vpx 点突变体进行了表征，这些突变体在降解 TASOR 时受损，但仍能降解 SAMHD1。Vpx 突变体降解 TASOR 的能力与它们增强 HIV-1 微小基因组表达的能力相关，这是意料之中的。引人注目的是，一些 Vpx 突变体在降解 TASOR 方面受损，但在降解 SAMHD1 方面不受损，它们与 DCAF1 的结合亲和力降低，但与 TASOR 的结合亲和力不变。在巨噬细胞中，Vpx R34A-R42A 和 Vpx R42A-Q47A-V48A 与 DCAF1 的结合严重受损，但与 TASOR 的结合不受影响，不能降解 TASOR，但能有效降解 SAMHD1。总之，我们的结果强调了 Vpx 与 DCAF1 之间牢固结合以触发 TASOR 降解的核心作用。然后，我们提出了一个模型，其中 Vpx 与 TASOR 和 DCAF1 相互作用，以稳定 TASOR-DCAF1 复合物。此外，我们的工作鉴定了 Vpx 突变体，这些突变体可在原代髓样细胞中独立于 SAMHD1 限制来研究 HUSH 限制。
