NF Kappa Beta and the Regulation of HIV Latency

艾滋病毒的根除和感染患者的治愈已经失败，主要是因为艾滋病毒在静止的CD 4记忆T细胞中建立了一种潜伏的感染形式。尽管在感染的患者中可能总共仅存在10（6）-10（7）个潜伏感染的细胞，但是这些细胞的长寿命性质（T（1/2）为44个月）预测大约70年的抗病毒治疗将是有效的。 需要消融该储层。显然需要一种不同的策略来解决这个临床问题。一种方法可能涉及通过刺激HIV LTR的转录激活来从这些细胞中清除病毒。我们推测NF-κ B/Rel家族转录因子作为HIV潜伏期的生理拮抗剂起着关键作用。我们建议进行一系列全面的研究，探索艾滋病毒潜伏期的分子基础， NF-κ B/Rel蛋白的作用。这些研究将利用HIV潜伏期的新细胞模型，J-Lat细胞，来自感染患者的真正潜伏感染的CD 4记忆T细胞，以及慢病毒潜伏期的灵长类动物模型。这些研究将涉及与Eric Verdin博士（项目1）、Matija Peterlin博士（项目3）以及Tom North博士和Paul Luciw博士（核心B）的密切和积极合作，他们共同组成了我们的PPG团队。在具体目标1中，我们将确定转录因子NF-κ B/Rel家族的哪些成员作为潜伏期HIV的最有效激活剂，并将探索是否可以利用对NF-κ B/Rel作用调节的新见解来逆转HIV潜伏期。在特定目标2中，我们将使用染色质免疫沉淀（ChIP）测定来评估病毒潜伏期的分子基础，以鉴定在病毒潜伏期和HIV LTR κ B增强子的相反条件下与HIV LTR κ B增强子结合的细胞因子。 活跃的病毒复制在特定目标3中，我们将重点关注NF-κ B与PTEFb的有趣相互作用，PTEFb是被HIV达特劫持的细胞酶复合物，以促进高水平的病毒基因表达。这些研究将剖析NF-κ B与PTEFb组装的分子基础，并探索达特的初始产生是否取决于NF-κ B的这种作用。在具体目标4中，我们将推进我们对HIV潜伏期及其通过NF-κ B的潜在拮抗作用的研究，使其成为体内模型系统。我们将研究是否删除的kappaB增强子的HIV LTR的SHIV病毒增强恒河猴记忆CD 4 T细胞的潜伏感染，相反，是否组成型活性T细胞增强子的替代损害潜伏期。最后，我们将探讨是否已知激活和维持核NF-κ B作用的药物（prostratin和苯丁酸）降低了这些SHIV潜伏感染细胞的频率。这些研究有望推进我们对HIV潜伏期的分子、生物化学和细胞基础的理解，这反过来可能导致新的合理方法来解决这一困难的临床问题。

The eradication of HIV and cure of infected patients has failed chiefly because HIV establishes a latent form of infection in resting CD4 memory T-cells. Although a total of only 10(6)-10(7) latently infected cells may be present in infected patients, the long lived nature of these cells (T(1/2) of 44 months) predicts that approximately 70 years of antiviral therapy will be required for ablation of this reservoir. A different strategy is clearly required to attack this clinical problem. One approach could involve purging of the virus from these cells by stimulating transcriptional activation of the HIV LTR. We hypothesize that the NF-kappaB/Rel family of transcription factors plays a pivotal role as physiological antagonists of HIV latency. We propose to conduct a comprehensive set of studies exploring the molecular basis for HIV latency and the role played by NF-kappaB/Rel proteins. These studies will exploit a new cellular model of HIV latency, J-Lat cells, bona fide latently infected CD4 memory T cells from infected patients, and a primate model of lentiviral latency. These studies will involve close and active collaborations with Dr. Eric Verdin (Project 1) Dr. Matija Peterlin (Project 3) and Drs. Tom North and Paul Luciw (Core B) which together comprise our PPG team. In Specific Aim 1, we will determine which members of the NF-kappaB/Rel family of transcription factors function as the most potent activators of latent HIV and will explore whether new insights into the regulation of NF-kappaB/Rel action can be exploited to reverse HIV latency. In Specific Aim 2, we will assess the molecular basis of viral latency using chromatin immunoprecipitation (ChIP) assays to identify cellular factors that bind to the HIV LTR kappaB enhancers under the opposing conditions of viral latency and active viral replication. In Specific Aim 3, we will focus on the intriguing interplay of NF-kappaB with PTEFb, the cellular enzyme complex hijacked by HIV Tat to promote high level viral gene expression. These studies will dissect the molecular basis for NF-kappaB assembly with PTEFb and explore whether the initial production of Tat depends on this action of NF-kappaB. In Specific Aim 4, we will advance our studies of HIV latency and its potential antagonism by NF-kappaB into an in vivo model system. We will investigate whether deletion of the kappaB enhancers in the HIV LTR of SHIV viruses enhances latent infection of rhesus memory CD4 T-cells and conversely whether substitution of a constitutively active T cell enhancer impairs latency. Finally, we will explore whether agents known to activate and sustain nuclear NF-kappaB action (prostratin and phenylbutyrate) decrease the frequency of cells latently infected with these SHIVs. These studies promise to advance our understanding of the molecular, biochemical, and cellular basis for HIV latency, which in turn may lead to new rational approaches to solving this difficult clinical problem.