Zhang Zeli, Gu Qinyong, Jaguva Vasudevan Ananda Ayyappan, Jeyaraj Manimehalai, Schmidt Stanislaw, Zielonka Jörg, Perković Mario, Heckel Jens-Ove, Cichutek Klaus, Häussinger Dieter, Smits Sander H J, Münk Carsten
Clinic for Gastroenterology, Hepatology, and Infectiology, Medical Faculty, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany.
Department of Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany.
J Virol. 2016 Oct 28;90(22):10193-10208. doi: 10.1128/JVI.01497-16. Print 2016 Nov 15.
Lentiviruses have evolved the Vif protein to counteract APOBEC3 (A3) restriction factors by targeting them for proteasomal degradation. Previous studies have identified important residues in the interface of human immunodeficiency virus type 1 (HIV-1) Vif and human APOBEC3C (hA3C) or human APOBEC3F (hA3F). However, the interaction between primate A3C proteins and HIV-1 Vif or natural HIV-1 Vif variants is still poorly understood. Here, we report that HIV-1 Vif is inactive against A3Cs of rhesus macaques (rhA3C), sooty mangabey monkeys (smmA3C), and African green monkeys (agmA3C), while HIV-2, African green monkey simian immunodeficiency virus (SIVagm), and SIVmac Vif proteins efficiently mediate the depletion of all tested A3Cs. We identified that residues N/H130 and Q133 in rhA3C and smmA3C are determinants for this HIV-1 Vif-triggered counteraction. We also found that the HIV-1 Vif interaction sites in helix 4 of hA3C and hA3F differ. Vif alleles from diverse HIV-1 subtypes were tested for degradation activities related to hA3C. The subtype F-1 Vif was identified to be inactive for degradation of hA3C and hA3F. The residues that determined F-1 Vif inactivity in the degradation of A3C/A3F were located in the C-terminal region (K167 and D182). Structural analysis of F-1 Vif revealed that impairing the internal salt bridge of E171-K167 restored reduction capacities to A3C/A3F. Furthermore, we found that D101 could also form an internal interaction with K167. Replacing D101 with glycine and R167 with lysine in NL4-3 Vif impaired its counteractivity to A3F and A3C. This finding indicates that internal interactions outside the A3 binding region in HIV-1 Vif influence the capacity to induce degradation of A3C/A3F.
The APOBEC3 restriction factors can serve as potential barriers to lentiviral cross-species transmissions. Vif proteins from lentiviruses counteract APOBEC3 by proteasomal degradation. In this study, we found that monkey-derived A3C, rhA3C and smmA3C, were resistant to HIV-1 Vif. This was determined by A3C residues N/H130 and Q133. However, HIV-2, SIVagm, and SIVmac Vif proteins were found to be able to mediate the depletion of all tested primate A3C proteins. In addition, we identified a natural HIV-1 Vif (F-1 Vif) that was inactive in the degradation of hA3C/hA3F. Here, we provide for the first time a model that explains how an internal salt bridge of E171-K167-D101 influences Vif-mediated degradation of hA3C/hA3F. This finding provides a novel way to develop HIV-1 inhibitors by targeting the internal interactions of the Vif protein.
慢病毒进化出了Vif蛋白，通过将APOBEC3（A3）限制因子靶向蛋白酶体降解来对抗它们。先前的研究已经确定了人类免疫缺陷病毒1型（HIV-1）Vif与人APOBEC3C（hA3C）或人APOBEC3F（hA3F）界面中的重要残基。然而，灵长类动物A3C蛋白与HIV-1 Vif或天然HIV-1 Vif变体之间的相互作用仍知之甚少。在这里，我们报告HIV-1 Vif对恒河猴（rhA3C）、黑猩猩（smmA3C）和非洲绿猴（agmA3C）的A3C无活性，而HIV-2、非洲绿猴猴免疫缺陷病毒（SIVagm）和SIVmac Vif蛋白有效地介导了所有测试的A3C的消耗。我们确定rhA3C和smmA3C中的N/H130和Q133残基是这种HIV-1 Vif触发的对抗作用的决定因素。我们还发现hA3C和hA3F的螺旋4中HIV-1 Vif相互作用位点不同。测试了来自不同HIV-1亚型的Vif等位基因与hA3C相关的降解活性。发现F-1亚型Vif对hA3C和hA3F的降解无活性。在A3C/A3F降解中决定F-1 Vif无活性的残基位于C末端区域（K167和D182）。F-1 Vif的结构分析表明，破坏E171-K167的内部盐桥恢复了对A3C/A3F的还原能力。此外，我们发现D101也可以与K167形成内部相互作用。在NL4-3 Vif中用甘氨酸取代D101并用赖氨酸取代R167削弱了其对A3F和A3C的对抗活性。这一发现表明HIV-1 Vif中A3结合区域之外的内部相互作用影响诱导A3C/A3F降解的能力。
APOBEC3限制因子可作为慢病毒跨物种传播的潜在障碍。慢病毒的Vif蛋白通过蛋白酶体降解来对抗APOBEC3。在本研究中，我们发现猴源A3C，即rhA3C和smmA3C，对HIV-1 Vif具有抗性。这由A3C残基N/H130和Q133决定。然而，发现HIV-2、SIVagm和SIVmac Vif蛋白能够介导所有测试的灵长类动物A3C蛋白的消耗。此外，我们鉴定出一种天然HIV-1 Vif（F-1 Vif），其在hA3C/hA3F的降解中无活性。在这里，我们首次提供了一个模型来解释E171-K167-D101的内部盐桥如何影响Vif介导的hA3C/hA3F降解。这一发现为通过靶向Vif蛋白的内部相互作用开发HIV-1抑制剂提供了一种新方法。
