A Sphingomyelin Hydrolase Regulates the Late Stages of HIV Assembly and Budding

摘要 HIV组装发生在特定膜微域内的质膜(通常称为 作为脂肪大鼠)，由于包括PIP2在内的饱和脂质的丰富而表现出侧向活动能力降低， 神经酰胺、鞘磷脂和甾醇。HIV-1 Gag前体蛋白针对这些病毒的内叶 质膜微区和调节病毒粒子组装和萌发所需的基本事件。 然而，GAG调节这些微区形成的机制及其在这些微区中的作用 病毒组装中的微域目前尚不清楚。在这里，我们提供了GAG共同定位的第一个证据 利用鞘磷脂水解酶nSMase2(其催化从鞘磷脂形成神经酰胺)， NSMase2是HIV复制的关键调控因子。在初步实验中，我们发现这一点 酶是成功完成HIV组装和成熟后期所必需的。 NSMase2的药理抑制或基因缺失阻止了GAG的加工，并导致 产生形状不规则的未成熟非传染性病毒粒子。在人源化的HIV感染小鼠中，抑制 在大多数被测试的动物中，nSMase2使病毒载量线性下降到低于可检测的限度。 更重要的是，接受治疗后病毒载量低于可检测限度的动物没有表现出病毒。 在停止给药后出现反弹。这些研究的发现将增加我们的基本 通过首次描述nSMase2和神经酰胺在病毒复制中的作用来理解病毒复制周期 艾滋病毒复制。

ABSTRACT HIV assembly occurs at the plasma membrane within specialized membrane microdomains (commonly known as lipid rats) that exhibit reduced lateral mobility owing to an enrichment of saturated lipids including PIP2, ceramide, sphingomyelin, and sterols. The HIV-1 Gag precursor protein targets to the inner leaflet of these plasma membrane microdomains and regulates essential events required for virion assembly and budding. However, the mechanisms by which Gag regulates the formation of these microdomains and the role for these microdomains in viral assembly is currently unknown. Here we provide the first evidence that Gag co-localizes with the sphingomyelin hydrolase nSMase2 (which catalyzes the formation of ceramide from sphingomyelin), and that nSMase2 is a critical regulator of HIV replication. In preliminary experiments we discovered that this enzyme is required for the successful completion of the late stages of HIV assembly and maturation. Pharmacological inhibition or genetic deletion of nSMase2 prevented the processing of Gag and resulted in the production of irregularly shaped immature non-infectious virions. In humanized HIV infected mice, inhibition of nSMase2 produced a linear decrease of viral loads to below detectable limits in the majority of animals tested. More importantly, animals who achieved viral loads below detectable limits with treatment, did not exhibit viral rebound when drug administration was discontinued. The findings from these studies will increase our basic understanding of the viral replication cycle by describing for the first time a role for nSMase2 and ceramide in HIV replication.