Department of Molecular Medicine, University of Padua, Padua, Italy.
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA
J Virol. 2020 May 18;94(11). doi: 10.1128/JVI.02019-19.
The structural protein Gag is the only viral component required for retroviral budding from infected cells. Each of the three conserved domains-the matrix (MA), capsid (CA), and nucleocapsid (NC) domains-drives different phases of viral particle assembly and egress. Once virus assembly is complete, retroviruses, like most enveloped viruses, utilize host proteins to catalyze membrane fission and to free progeny virions. These proteins are members of the endosomal sorting complex required for transport (ESCRT), a cellular machinery that coats the inside of budding necks to perform membrane-modeling events necessary for particle abscission. The ESCRT is recruited through interactions with PTAP and LYPXnL, two highly conserved sequences named late (L) domains, which bind TSG101 and Alix, respectively. A TSG101-binding L-domain was identified in the p2 region of the feline immunodeficiency virus (FIV) Gag protein. Here, we show that the human protein Alix stimulates the release of virus from FIV-expressing human cells. Furthermore, we demonstrate that the Alix Bro1 domain rescues FIV mutants lacking a functional TSG101-interacting motif, independently of the entire p2 region and of the canonical Alix-binding L-domain(s) in FIV Gag. However, in contrast to the effect on human immunodeficiency virus type 1 (HIV-1), the CS double mutation, which disrupts both CCHC zinc fingers in the NC domain, does not abrogate Alix-mediated virus rescue. These studies provide insight into conserved and divergent mechanisms of lentivirus-host interactions involved in virus budding. FIV is a nonprimate lentivirus that infects domestic cats and causes a syndrome that is reminiscent of AIDS in humans. Based on its similarity to HIV with regard to different molecular and biochemical properties, FIV represents an attractive model for the development of strategies to prevent and/or treat HIV infection. Here, we show that the Bro1 domain of the human cellular protein Alix is sufficient to rescue the budding of FIV mutants devoid of canonical L-domains. Furthermore, we demonstrate that the integrity of the CCHC motifs in the Gag NC domain is dispensable for Alix-mediated rescue of virus budding, suggesting the involvement of other regions of the Gag viral protein. Our research is pertinent to the identification of a conserved yet mechanistically divergent ESCRT-mediated lentivirus budding process in general, and to the role of Alix in particular, which underlies the complex viral-cellular network of interactions that promote late steps of the retroviral life cycle.
结构蛋白 Gag 是逆转录病毒从受感染细胞出芽所必需的唯一病毒成分。三个保守结构域中的每一个——基质（MA）、衣壳（CA）和核衣壳（NC）结构域——驱动病毒粒子组装和出芽的不同阶段。一旦病毒组装完成，与大多数包膜病毒一样，逆转录病毒利用宿主蛋白催化膜分裂并释放子代病毒颗粒。这些蛋白是内体分选复合物必需的运输（ESCRT）的成员，该细胞机制在内出芽颈内涂覆，以执行粒子分离所必需的膜建模事件。ESCRT 通过与 PTAP 和 LYPXnL 的相互作用被募集，PTAP 和 LYPXnL 是两个高度保守的序列，分别命名为晚期（L）结构域，分别与 TSG101 和 Alix 结合。在猫免疫缺陷病毒（FIV）Gag 蛋白的 p2 区鉴定到 TSG101 结合 L 结构域。在这里，我们表明人蛋白 Alix 刺激 FIV 表达的人细胞中病毒的释放。此外，我们证明了 Alix Bro1 结构域可以挽救缺乏功能性 TSG101 相互作用基序的 FIV 突变体，而与整个 p2 区和 FIV Gag 中的经典 Alix 结合 L 结构域无关。然而，与对人类免疫缺陷病毒 1（HIV-1）的影响相反，CS 双突变（破坏 NC 结构域中的两个 CCHC 锌指）不会消除 Alix 介导的病毒拯救。这些研究为参与病毒出芽的慢病毒-宿主相互作用的保守和不同机制提供了深入了解。FIV 是非灵长类慢病毒，感染家猫并引起类似于人类艾滋病的综合征。基于其在不同分子和生化特性方面与 HIV 的相似性，FIV 代表了开发预防和/或治疗 HIV 感染策略的有吸引力的模型。在这里，我们表明人细胞蛋白 Alix 的 Bro1 结构域足以挽救缺乏经典 L 结构域的 FIV 突变体的出芽。此外，我们证明 Gag NC 结构域中的 CCHC 基序的完整性对于 Alix 介导的病毒出芽拯救是可有可无的，这表明 Gag 病毒蛋白的其他区域也参与其中。我们的研究与普遍存在的保守但机制上不同的 ESCRT 介导的慢病毒出芽过程有关，特别是与 Alix 在促进逆转录病毒生命周期后期步骤的复杂病毒-细胞相互作用网络中的作用有关。
