Human Retrovirus Effects on Blood Cells

该项目的目标是更好地了解宿主细胞和其他辅助因素对人类逆转录病毒复制和发病机制的影响。高效联合抗病毒药物疗法(HAART)有望将生产性艾滋病毒感染转变为慢性、非病毒症、非进展性疾病。然而，在一些患者中，观察到了有限但持续的病毒产生，在另一些患者中，在接受HAART治疗的HIV感染者中发现了潜伏感染的长寿外周T细胞，这些T细胞可以在正常免疫反应中重新激活。在开发针对艾滋病毒感染者的长期治疗方法时，有必要通过免疫疗法等辅助疗法来控制这些潜伏病毒的产生和/或根除这些潜伏的病毒。我们以前已经确定了两种激活潜伏的HIV前病毒表达的机制：免疫激活，这需要潜伏感染的细胞和未感染的活化的CD4+细胞之间的接触，以及低甲基化。我们最近证明，急性HIV感染增加了细胞甲基化基因的能力。HIV感染细胞中DNA甲基转移酶(DNMT)活性的增加可以通过HIV LTR的甲基化和随后的病毒基因表达沉默而导致潜伏库的增加。此外，DNMT活性的增加会导致细胞基因的异常甲基化，包括干扰素-伽马(干扰素-伽马)。干扰素-γ的这种高甲基化导致干扰素-γ的产生减少，1型免疫反应减弱，并增加了无细胞病毒粒子的传播。在试图了解甲基化在病毒和基因调控中的作用方面，我们在几个领域取得了进展。首先，HIV前病毒整合对于HIV感染并不是增加细胞甲基化基因能力所必需的。第二，在HIV感染淋巴细胞的过程中，DNMT-1表达增加，而DNMT3a和Dnmt3b表达增加。第三，在一些长期接受HAART的患者(49/96)中，CD4+T细胞产生干扰素-γ的能力大大减弱，这种能力可以通过干扰素-γ启动子的低甲基化来恢复。 使用低甲基化药物。对该启动子的亚硫酸氢盐基因组测序表明，在HAART治疗期间，干扰素-γ启动子可以保持高甲基化状态。第四，使用一种新开发的基于阵列的分析(差异甲基化杂交)，我们发现HIV感染者的CD4+T淋巴细胞有几个CpG岛，这些岛比正常对照组更广泛的甲基化。我们已经确定其中一个基因是p16(Ink4a)基因。 这表明通常低甲基化的CpG岛在HIV感染后可以被高甲基化。由于DNMT1是主要在HIV感染后上调的DNMT，最近的证据表明，除了它的酶功能外，它还可以与其他 包括TAT或VPR等转录因子的蛋白质可以以一种新的方式与含有DNMT-1转录调控失调的复合体结合。尽管人们对此知之甚少，但HTLV-1感染在淋巴增殖过程中触发的细胞事件对白血病的建立具有重要意义。我们最近鉴定了HTLV-1的一个感染性克隆以及一个敏感的表达细胞系，以研究这个感染性克隆的分子相互作用。包膜的HTLV-1表面单元已经与Ig分子融合，并被用于研究HIV-1受体的调节。 本项目前身为Z01 CM 09251-12 LLB。 艾滋病标题：艾滋病毒复制的负面调控-致病效应

The goal of this project is to better understand the effects of host cells and other co-factors on human retroviral replication and pathogenesis. Highly active combination antiviral drug therapy (HAART) holds promise to convert productive HIV infections into a chronic, non-viremic, non-progressive condition. However, in some patients, limited but continuous viral production has been observed and, in others, latently infected long-lived peripheral T cells which can be reactivated during a normal immune response have been identified in HAART-treated HIV infected individuals. In developing long term therapy for HIV infected individuals it will be necessary to control viral production from and/or eradicate these latent reservoirs through adjuvant therapies like immunotherapy. We have previously identified two mechanisms which activate expression of latent HIV provirus: immune activation, which requires contact between latently infected cells and uninfected activated CD4+ cells, and hypomethylation. We have recently demonstrated that acute HIV infection increases the cellular capacity to methylate genes. Increased DNA methyltransferase (DNMT) activity in HIV infected cells can result in an increase in the latent reservoir through methylation of the HIV LTR and the subsequent silencing of viral gene expression. In addition, increased DNMT activity can result in aberrant methylation of cellular genes including, interferon-gamma (IFN-gamma). This hypermethylation of IFN-gamma results in decreased production of IFN-gamma, decreased type 1 immune response and increased spread of cell free virions. In attempting to understand the role of methylation in the regulation of viral and gene regulation, we have made progress in several areas. First, HIV proviral integration is not necessary for HIV infection to increase the cellular capacity to methylate genes. Second, DNMT-1 but not DNMT3a and DNMT3b are increased during HIV infection of lymphoid cells. Third, in some (49/96) patients on long-term HAART, CD4+ T cells have greatly diminished ability to produce IFN-gamma which can be restored through hypomethylation of IFN-gamma promoter, using hypomethylating drugs. Bisulfite genomic sequencing of this promoter has shown that the IFN-gamma promoter can remain hypermethylated during HAART therapy. Fourth, using a newly developed array based assay (differential methylation hybridization), we have found that CD4+ T lymphocytes from HIV infected individuals have several CpG islands which are more extensively methylated than normal controls. We have identified one of these genes as the p16(Ink4a) gene indicating that CpG islands, normally hypomethylated, can be hypermethlated after HIV infection. Since DNMT1 is the DNMT that is predominantly upregulated following HIV infection and recent evidence suggests that, in addition to its enzymatic function, it can form complexes with other proteins including transcriptional factors such as Tat or Vpr can tether to complexes containing DNMT-1 dysregulate transcription control in a novel manner. Although largely unknown, the cellular events triggered by HTLV-1 infection in the lymphoproliferative process are of major importance to the establishment of leukemia. We have recently characterized an infectious clone of HTLV-1 as well as a susceptible expressing cell line to study the molecular interactions of this infectious clone. The HTLV-1 surface unit of the envelope has been fused to the Ig molecule and is being used to study the regulation of the HIV-1 receptor. This project was formerly Z01 CM 09251-12 LLB. AIDS Title: Negative Regulation of HIV replication - Pathogenic Effects