Ramirez Peter W, DePaula-Silva Ana Beatriz, Szaniawski Matt, Barker Edward, Bosque Alberto, Planelles Vicente
Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, 84112, USA.
Department of Immunology and Microbiology, Rush University Medical Center, Chicago, IL, 60612, USA.
Retrovirology. 2015 Jul 28;12:65. doi: 10.1186/s12977-015-0192-2.
Hijacking of the cullin-RING E3 ubiquitin ligase (CRL) machinery is a common mechanism employed by diverse groups of viruses for the efficient counteraction and degradation of host proteins. In particular, HIV-1 Vpu usurps the SCF(β-TrCP) E3 ubiquitin ligase complex to mark CD4 for degradation by the 26S proteasome. Vpu also interacts with and downmodulates a number of other host proteins, including the restriction factor BST-2. However, whether Vpu primarily relies on a cullin-dependent or -independent mechanism to antagonize its cellular targets has not been fully elucidated.
We utilized a sulphamate AMP analog, MLN4924, to effectively block the activation of CRLs within infected primary CD4(+) T cells. MLN4924 treatment, in a dose dependent manner, efficiently relieved surface downmodulation and degradation of CD4 by NL4-3 Vpu. MLN4924 inhibition was highly specific, as this inhibitor had no effect on Nef's ability to downregulate CD4, which is accomplished by a CRL-independent mechanism. In contrast, NL4-3 Vpu's capacity to downregulate BST-2, NTB-A and CCR7 was not inhibited by the drug. Vpu's from both a transmitted founder (T/F) and chronic carrier (CC) virus preserved the ability to downregulate BST-2 in the presence of MLN4924. Finally, depletion of cellular pools of cullin 1 attenuated Vpu's ability to decrease CD4 but not BST-2 surface levels.
We conclude that Vpu employs both CRL-dependent and CRL-independent modes of action against host proteins. Notably, we also establish that Vpu-mediated reduction of BST-2 from the cell surface is independent of β-TrCP and the CRL- machinery and this function is conserved by Vpu's from primary isolates. Therefore, potential therapies aimed at antagonizing the activities of Vpu may need to address these distinct mechanisms of action in order to achieve a maximal effect.
劫持cullin-RING E3泛素连接酶（CRL）机制是不同病毒群体用于有效对抗和降解宿主蛋白的常见机制。特别是，HIV-1 Vpu利用SCF(β-TrCP) E3泛素连接酶复合物标记CD4以便被26S蛋白酶体降解。Vpu还与许多其他宿主蛋白相互作用并下调其表达，包括限制因子BST-2。然而，Vpu主要是依赖cullin的机制还是不依赖cullin的机制来对抗其细胞靶点尚未完全阐明。
我们使用氨基磺酸酯AMP类似物MLN4924有效阻断感染的原代CD4(+) T细胞内CRL的激活。MLN4924处理以剂量依赖的方式有效缓解了NL4-3 Vpu对CD4的表面下调和降解。MLN4924的抑制作用具有高度特异性，因为该抑制剂对Nef下调CD4的能力没有影响，Nef下调CD4是通过不依赖CRL的机制实现的。相反，该药物并未抑制NL4-3 Vpu下调BST-2、NTB-A和CCR7的能力。来自传播奠基者（T/F）病毒和慢性携带者（CC）病毒的Vpu在存在MLN4924的情况下仍保留下调BST-2的能力。最后，cullin 1细胞池的消耗减弱了Vpu降低CD4的能力，但不影响BST-2的表面水平。
我们得出结论，Vpu对宿主蛋白采用依赖CRL和不依赖CRL的作用模式。值得注意的是，我们还确定Vpu介导的从细胞表面减少BST-2与β-TrCP和CRL机制无关，并且来自原代分离株的Vpu保留了该功能。因此，旨在对抗Vpu活性的潜在疗法可能需要针对这些不同的作用机制，以实现最大效果。
