Nef Decreases HIV-1 Sensitivity to Neutralizing Antibodies that Target the Membrane-proximal External Region of TMgp41

Primate lentivirus nef is required for sustained virus replication in vivo and accelerated progression to AIDS. While exploring the mechanism by which Nef increases the infectivity of cell-free virions, we investigated a functional link between Nef and Env. Since we failed to detect an effect of Nef on the quantity of virion-associated Env, we searched for qualitative changes by examining whether Nef alters HIV-1 sensitivity to agents that target distinct features of Env. Nef conferred as much as 50-fold resistance to 2F5 and 4E10, two potent neutralizing monoclonal antibodies (nAbs) that target the membrane proximal external region (MPER) of TMgp41. In contrast, Nef had no effect on HIV-1 neutralization by MPER-specific nAb Z13e1, by the peptide inhibitor T20, nor by a panel of nAbs and other reagents targeting gp120. Resistance to neutralization by 2F5 and 4E10 was observed with Nef from a diverse range of HIV-1 and SIV isolates, as well as with HIV-1 virions bearing Env from CCR5- and CXCR4-tropic viruses, clade B and C viruses, or primary isolates. Functional analysis of a panel of Nef mutants revealed that this activity requires Nef myristoylation but that it is genetically separable from other Nef functions such as the ability to enhance virus infectivity and to downregulate CD4. Glycosylated-Gag from MoMLV substituted for Nef in conferring resistance to 2F5 and 4E10, indicating that this activity is conserved in a retrovirus that does not encode Nef. Given the reported membrane-dependence of MPER-recognition by 2F5 and 4E10, in contrast to the membrane-independence of Z13e1, the data here is consistent with a model in which Nef alters MPER recognition in the context of the virion membrane. Indeed, Nef and Glycosylated-Gag decreased the efficiency of virion capture by 2F5 and 4E10, but not by other nAbs. These studies demonstrate that Nef protects lentiviruses from one of the most broadly-acting classes of neutralizing antibodies. This newly discovered activity for Nef has important implications for anti-HIV-1 immunity and AIDS pathogenesis.

灵长类慢病毒的nef基因对于病毒在体内的持续复制以及艾滋病（AIDS）的加速进展不可或缺。在探究Nef提升无细胞病毒粒子（cell-free virions）感染性的机制过程中，我们对Nef与包膜蛋白（Env）之间的功能关联展开了研究。由于未能检测到Nef对病毒粒子结合的Env蛋白含量存在影响，我们转而通过考察Nef是否会改变HIV-1对靶向Env不同结构域的试剂的敏感性，来寻找其介导的定性变化。Nef可使病毒对2F5和4E10产生最高达50倍的抗性，这两种强效中和性单克隆抗体（neutralizing monoclonal antibodies，nAbs）靶向TMgp41的膜近端外部区域（MPER）。与之相反，Nef对MPER特异性单克隆抗体Z13e1、肽类抑制剂T20，以及一组靶向gp120的中和性单克隆抗体及其他试剂介导的HIV-1中和作用无影响。针对多种HIV-1及猴免疫缺陷病毒（Simian Immunodeficiency Virus，SIV）分离株的Nef，以及携带来自CCR5嗜性、CXCR4嗜性病毒、B型及C型亚型病毒或原代分离株Env的HIV-1病毒粒子，均观察到了对2F5和4E10中和作用的抗性。对一组Nef突变体的功能分析显示，该活性依赖于Nef的肉豆蔻酰化修饰，但在遗传上可与Nef的其他功能（如增强病毒感染性及下调CD4分子（CD4）的能力）相互分离。来自莫洛尼鼠白血病病毒（Moloney Murine Leukemia Virus，MoMLV）的糖基化Gag蛋白（Glycosylated-Gag）可替代Nef，赋予病毒对2F5和4E10的抗性，这表明该活性在不编码Nef的逆转录病毒中也具有保守性。鉴于已有研究表明2F5和4E10对MPER的识别依赖于膜环境，而Z13e1的MPER识别则不依赖膜环境，本研究数据与“Nef在病毒粒子膜环境中改变MPER的识别”这一模型相符。事实上，Nef与糖基化Gag蛋白均可降低2F5和4E10对病毒粒子的捕获效率，但对其他中和性单克隆抗体的病毒捕获效率无影响。本研究证实，Nef可使慢病毒免受一类最具广谱性的中和性抗体的攻击。此次新发现的Nef活性，对于抗HIV-1免疫及艾滋病发病机制研究具有重要意义。