CELLULAR RESTRICTIVE FACTOR TARGETED BY VIRAL PROTEIN X

描述(申请人提供)：HIV-2和SIVsm对髓系细胞的感染在反转录步骤中受到限制，除非表达病毒蛋白X(VPX)。VPX与Cul4a-DDB1-DCAF1泛素连接酶的结合是克服这一限制的重要因素。假设在VPX存在的情况下，一种细胞限制因子被迅速泛素化，并以蛋白酶体降解为目标。本项目的目标是确定VPX靶向的细胞限制因子(S)。这项研究将利用在以前的分子生物学筛选中确定的候选限制性因素，包括在酵母双杂交试验中确定的与VPX或DCAF1相互作用的蛋白质，通过2D差异凝胶电泳法发现的在感染HIV-2 VPX的髓系细胞中与野生型HIV-2相比过度表达的蛋白质，以及从293T细胞的功能筛选中确定的限制HIV感染的因素的cDNA克隆。我们还将根据许多家族成员限制HIV-1或SIV复制的数据，以及APOBEC3A与VPX结合的数据，检查APOBEC3家族成员的这些活动。目的1.分析限制HIV-2 VPX而不是野生型HIV-2能力的候选限制因素。潜在的限制因子cDNA将被检测为抑制HIV-2 VPX感染HeLa细胞的能力，但不是野生型HIV-2。然后，显示这种活性的cDNA将使用实时RT-PCR来检测它们对HIV-2逆转录的影响。目的2.检测蛋白酶体抑制剂和shRNA对DCAF1中VPX调节的限制因子表达的影响。用昆虫激素鼠李糖诱导293T细胞株VPX的表达，观察VPX、bortezomib和shRNA对DCAF1潜在限制因子表达水平的影响。针对目标1中确定的限制因子的shRNA将与dsRed标记一起在巨噬细胞中表达。将在dsRed阳性巨噬细胞中检测HIV-2gfp Vpx与HIV-2gfp的感染水平，以确定在没有Vpx的情况下shRNA是否解除了对HIV-2的限制。 公共卫生相关性：这项研究将确定限制艾滋病毒-2感染髓系细胞的细胞因子(S)，但可被病毒蛋白X(VPX)克服。假设VPX促进细胞限制因子的降解(S)。几个潜在的细胞限制因子已经从分子生物学筛选中被识别出来，它们对允许的HeLa细胞感染HIV-2的影响将在存在或不存在VPX的情况下进行检测。VPX克服的潜在限制因素将进一步研究VPX对其蛋白质稳定性的影响。此外，我们还将确定针对潜在限制因子的小干扰RNA(S)是否能克服缺乏VPX的HIV-2对髓系细胞的感染。这些实验对于确定一种新的HIV限制机制很重要。

DESCRIPTION (provided by applicant): Infection of myeloid cells by HIV-2 and SIVsm are restricted at the reverse transcription step unless viral protein X (Vpx) is expressed. Binding of Vpx to the Cul4a-DDB1-DCAF1 ubiquitin ligase was shown to be important to overcome this restriction. It is hypothesized that, in the presence of Vpx, a cellular restriction factor is rapidly ubiquitinated and targeted for proteasome degradation. The goal of this project is to identify cellular restrictive factor(s) targeted by Vpx. This study will utilize candidate restrictive factors identified in previous molecular biology screens, including proteins, identified in the yeast 2-hybrid assay, that interact with Vpx or DCAF1, proteins, found by 2D differential gel electrophoresis, that are over-expressed in myeloid cells infected with HIV-2 Vpx compared to wild type HIV-2, and cDNA clones identified from a functional screen in 293T cells for factors that restrict HIV infection in the absence but not the presence of Vpx. We will also examine members of the Apobec3 family for these activities, based on data that many family members restrict HIV-1 or SIV replication, and Apobec3A binds Vpx. Aim 1. Analyze candidate restriction factors for the ability to restrict HIV-2 Vpx but not wild type HIV-2. Potential restriction factor cDNAs will be examined for the ability to inhibit infection of HeLa cells by HIV-2 Vpx but not wild type HIV-2. The cDNAs showing this activity will then be examined for their effects on HIV-2 reverse transcription, using real time RT-PCR. Aim 2. Examine effects of a proteasome inhibitor and a shRNA to DCAF1 on expression of potential Vpx-regulated restriction factors. A 293T cell line in which Vpx expression is induced with insect hormone muristerone will be used to assess effects of Vpx, bortezomib, and shRNA to DCAF1 on the level of expression of potential restriction factors identified in Aim 1. Aim 3. Examine effects of shRNA to potential Vpx-regulated restriction factor on infection of macrophages. shRNAs to restriction factors identified in Aim 1 will be expressed in macrophages together with a dsRed marker. Levels of infection of HIV-2gfp Vpx compared to HIV-2gfp will be examined in dsRed positive macrophages, to determine if the shRNA relieves the restriction to HIV-2 in the absence of Vpx. PUBLIC HEALTH RELEVANCE: This study will identify cellular factor(s) that limit HIV-2 infection of myeloid cells, but are overcome by viral protein X (Vpx). It is hypothesized that Vpx promotes the degradation of the cellular restriction factor(s). Several potential cellular restriction factors have been identified from molecular biology screens, and their effects on HIV-2 infection of permissive HeLa cells will be examined in the presence or absence of Vpx. Potential restriction factors that are overcome by Vpx will be further examined for the effects of Vpx on their protein stability. In addition, we will determine if small interfering RNA to the potential restriction factor(s) overcomes the block to infection of myeloid cells by HIV-2 lacking Vpx. These experiments are important in identifying a novel mechanism of HIV restriction.