Mechanistic studies of an unexpected HIV-1 Vpu function

说明（由申请人提供）：艾滋病毒-1感染已在世界范围内造成流行病，夺去了2 000多万人的生命，另有4 000多万人受到感染。除了在公共卫生方面的重要性外，HIV-1和其他逆转录病毒也已成为将外源基因送入靶细胞进行基因治疗和其他应用的重要工具。在组装过程中，HIV-1必须获得病毒表面糖蛋白以靶向宿主细胞并与宿主细胞融合。虽然HIV-1通常获得自己的表面糖蛋白，但它也可以与某些外来病毒糖蛋白组装成感染性病毒粒子，形成所谓的伪型病毒。然而，并非所有的外源糖蛋白都与HIV-1兼容。长臂猿白血病病毒（GaLV Env）的糖蛋白是与HIV-1不相容的糖蛋白的一个例子。HIV-1与GaLV Env的共表达基本上不会产生感染性假型病毒颗粒。GaLV中导致这种不相容的元件已被定位到糖蛋白的c端胞质尾部结构域（CTD）。我们最近发现HIV-1和GaLV Env之间的不相容性是由HIV-1辅助蛋白Vpu调节的。在缺乏Vpu的情况下，带有GaLV CTD的糖蛋白被整合到HIV-1颗粒中，并且这些颗粒具有传染性。然而，在Vpu存在的情况下，这些糖蛋白不被HIV-1颗粒结合，颗粒的感染性降低了50 - 100倍。在某些方面，这种现象类似于已知的针对宿主细胞蛋白CD4在内质网降解的Vpu活性。然而，GaLV Env限制的机制与目前关于如何限制CD4的模型不同。最重要的是，Vpu仅限制某些逆转录病毒获得具有GaLV CTD的糖蛋白，即使存在显著的表面糖蛋白表达，也会发生这种限制。虽然已知Vpu可以调节某些宿主细胞蛋白以增强病毒的传染性，但令人惊讶的是，Vpu特异性地调节来自外来病毒的表面糖蛋白，而这种病毒与Vpu的已知靶标没有明显的相似性。因此，我们假设Vpu对GaLV Env的限制是GaLV Env模仿细胞蛋白并“诱骗”Vpu靶向该蛋白的偶然效应。通过表征这种下调，我们可以(1)阐明Vpu在感染细胞中的作用，(2)确定Vpu靶向的MLV/GaLV Env中的精确基序，从而获得在计算机上识别天然Vpu靶标的必要信息。

DESCRIPTION (provided by applicant): HIV-1 infections have caused a worldwide pandemic that has claimed the lives of over 20 million people and infected over 40 million more. In addition to its importance in public health, HIV-1 and other retroviruses have also become important tools for delivery of foreign genes into target cells for gene therapy and other applications. During the assembly process HIV-1 must acquire a viral surface glycoprotein to target and fuse with host cells. Although HIV-1 usually acquires it own surface glycoprotein, it can also assemble into infectious virions with certain foreign viral glycoproteins to form what is called a pseudotyped virus. However, not all foreign glycoproteins are compatible with HIV-1. The glycoprotein from gammaretrovirus gibbon ape leukemia virus (GaLV Env) is an example of a glycoprotein that is not compatible with HIV-1. Co-expression of HIV-1 with GaLV Env yields essentially no infectious pseudotyped virus particles. The element in GaLV responsible for this incompatibility has been mapped to the C-terminal cytoplasmic tail domain (CTD) of the glycoprotein. We recently discovered that the incompatibility between HIV-1 and GaLV Env is modulated by the HIV-1 accessory protein Vpu. In the absence of Vpu, glycoproteins with a GaLV CTD are incorporated into HIV-1 particles, and the particles are infectious. However, in the presence of Vpu, these glycoproteins are not incorporated into HIV-1 particles, and the particle infectivity is reduced by 50 to 100 fold. In some ways this phenomenon is similar to the known Vpu activity of targeting the host cell protein CD4 for degradation at the endoplasmic reticulum. However, the mechanism of GaLV Env restriction is distinct from current models for how CD4 is restricted. Most nobly, Vpu only restricts certain retroviruses from acquiring glycoproteins with a GaLV CTD, and the restriction occurs even in presence of significant surface glycoprotein expression. Although Vpu is known to modulate certain host cell proteins to enhance viral infectivity, it is surprising that Vpu specifically modulates the surface glycoprotein from a foreign virus that displays no obvious similarity to Vpu's known targets. We therefore hypothesize that GaLV Env restriction by Vpu is an incidental effect resulting from GaLV Env mimicking a cellular protein and 'tricking' Vpu into targeting it. By characterizing this downmodulation, we can (1) elucidate the actions of Vpu in infected cells and (2) identify the precise motif in MLV/GaLV Env targeted by Vpu, yielding the necessary information to identify natural Vpu targets in silico. PUBLIC HEALTH RELEVANCE: HIV-1 infections have caused a worldwide pandemic that has claimed the lives of over 20 million people and infected over 40 million more. It has long been recognized that viruses, including HIV, have distinct mechanisms for modulating the host cell to facilitate virus assembly, but these mechanisms remain poorly understood. An understanding of what and how HIV-1 accessory proteins modulate proteins in the infected cell could lead to new targets for antiviral therapies.