CELLULAR RESTRICTIVE FACTOR TARGETED BY VIRAL PROTEIN X

描述（由申请方提供）：除非表达病毒蛋白X（Vpx），否则HIV-2和SIVsm对骨髓细胞的感染仅限于逆转录步骤。Vpx与Cul 4a-DDB 1-DCAF 1泛素连接酶的结合对于克服这种限制是重要的。据推测，在Vpx的存在下，细胞限制因子被迅速泛素化，并被靶向蛋白酶体降解。本项目的目标是鉴定Vpx靶向的细胞限制性因子。本研究将利用在先前分子生物学筛选中鉴定的候选限制性因子，包括在酵母双杂交试验中鉴定的与Vpx或DCAF 1相互作用的蛋白质，通过2D差异凝胶电泳发现的蛋白质，与野生型HIV-2相比，在感染HIV-2 Vpx的骨髓细胞中过表达，以及从293 T细胞中的功能筛选中鉴定的cDNA克隆，所述功能筛选用于在Vpx不存在但不存在的情况下限制HIV感染的因子。我们还将检查Apobec 3家族成员的这些活动，基于许多家族成员限制HIV-1或SIV复制的数据，Apobec 3A结合Vpx。目标1.分析候选限制因子限制HIV-2 Vpx但不限制野生型HIV-2的能力。将检查潜在限制因子cDNA抑制HIV-2 Vpx而非野生型HIV-2感染HeLa细胞的能力。然后使用真实的时间RT-PCR检测显示这种活性的cDNA对HIV-2逆转录的影响。目标2.检查蛋白酶体抑制剂和DCAF 1的shRNA对潜在Vpx调节的限制因子表达的影响。将使用昆虫激素muristerone诱导Vpx表达的293 T细胞系来评估Vpx、硼替佐米和针对DCAF 1的shRNA对目标1中鉴定的潜在限制性因子表达水平的影响。目标3。检查shRNA对潜在的Vpx调节的限制因子对巨噬细胞感染的影响。针对Aim 1中鉴定的限制性因子的shRNA将与dsRed标记物一起在巨噬细胞中表达。将在dsRed阳性巨噬细胞中检查HIV-2gfp Vpx与HIV-2gfp相比的感染水平，以确定在Vpx不存在的情况下，shRNA是否减轻对HIV-2的限制。 公共卫生相关性：这项研究将确定限制HIV-2感染骨髓细胞的细胞因子，但被病毒蛋白X（Vpx）克服。假设Vpx促进细胞限制因子的降解。已经从分子生物学筛选中鉴定出几种潜在的细胞限制因子，并将在存在或不存在Vpx的情况下检查它们对HIV-2感染允许的HeLa细胞的影响。Vpx克服的潜在限制因素将进一步检查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是否能克服缺乏Vpx的HIV-2对骨髓细胞感染的阻断作用。这些实验对于确定HIV限制的新机制非常重要。

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.