Fab-based inhibitors reveal ubiquitin independent functions for HIV Vif neutralization of APOBEC3 restriction factors

The lentiviral protein Viral Infectivity Factor (Vif) counteracts the antiviral effects of host APOBEC3 (A3) proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs) to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity.

慢病毒蛋白病毒感染因子（Viral Infectivity Factor，Vif）可拮抗宿主APOBEC3（A3）蛋白的抗病毒效应，进而促进持续性HIV感染。Vif通过将A3限制因子招募至多蛋白泛素E3连接酶复合物，使其发生泛素化并经蛋白酶体降解。本研究通过针对Vif复合物的抗体抗原结合片段（Fabs）的详细结构-功能研究，揭示了Vif介导拮抗作用的非降解依赖机制。研究发现两种Fabs可通过两种截然不同的机制抑制Vif介导的A3中和作用：一是阻断A3的泛素转移过程，二是阻碍Vif E3复合物的组装。综合生化、细胞生物学与结构生物学研究结果显示，破坏Vif E3复合物组装可抑制A3的泛素化，但不足以恢复其被包装至病毒颗粒的能力及抗病毒活性。上述实验结果证实，Vif能够以非降解依赖的方式中和A3家族成员。此外，本研究凸显了Fabs作为功能探针的应用潜力，并阐明了Vif如何通过兼顾降解依赖与非降解依赖的多管齐下策略，抑制A3介导的先天免疫。