Reversal of epigenetic silencing rescues integrase-deficient HIV-1 replication

逆转表观遗传沉默可挽救整合酶缺陷的 HIV-1 复制

• 批准号: 10158875
• 负责人: BRYAN R. CULLEN
• 金额: $ 23.78万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-10 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10158875
• 关键词: Address; Antiviral Agents; Applications Grants; Area; CD4 Positive T Lymphocytes; Cell Line; Cell Nucleus; Cells; Characteristics; Chromatin; Circular DNA; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytoplasm; DNA; Data; Deposition; Detection; Drug Targeting; Enzymes; Epigenetic Process; Gene Expression; Gene Silencing; Genetic Screening; Genetic Transcription; Genome; Goals; Grant; HIV Integrase; HIV-1; HIV-1 integrase; Hepatitis B Virus; Herpesviridae; Herpesvirus 1; Human; Human T-Cell Leukemia Virus Type I tax Protein; Human T-lymphotropic virus 1; Immediate-Early Proteins; Immunologic Factors; Infection; Integrase; Laboratories; Length; Life; Life Cycle Stages; Light; Mediating; Mutagenesis; NF-kappa B; Nonstructural Protein; Nuclear; Pathway interactions; Patients; Pharmaceutical Preparations; Physiological; Play; Proteins; Proviruses; REL Protein; RNA; Regulation; Reporting; Retroviridae; Reverse Transcription; Role; Site; T-Lymphocyte; Taxes; Transcriptional Activation; Ubiquitination; Viral; Viral Proteins; base; blocking factor; chromatin modification; epigenetic silencing; extrachromosomal DNA; genome-wide; mutant; prevent; promoter; recruit; tax Gene Products; transcription factor; viral DNA

ABSTRACT After reverse transcription in the cytoplasm, retroviral DNAs enter the nucleus naked and are then detected by currently unknown innate immune factors and coated with repressive chromatin. However, retroviruses encode an integrase (IN) which allows the proviral DNA to be inserted into transcriptionally active areas of the host genome, which then results in the repressive chromatin marks on the retroviral DNA being replaced by the active marks characteristic of the flanking host chromatin. If IN activity is blocked either by mutagenesis or by a drug, then the unintegrated retroviral DNA remains covered with inhibitory chromatin and is transcriptionally silenced. This grant proposes has two goals. Firstly, I hypothesize that if unintegrated HIV-1 DNA is epigenetically silenced then it should be possible to activate that DNA, and rescue the replication of IN- HIV-1, by either directly activating the HIV-1 LTR promoter and/or by directly reversing the epigenetic silencing. We have now shown that expression of the HTLV-1 Tax protein, a potent activator of cellular NK-kB/Rel transcription factors, induces the recruitment of RelA and RelB to the NF-kB sites present in the HIV-1 LTR and also prevents or reverses the epigenetic silencing of unintegrated HIV-1 DNA, allowing IN- HIV-1 mutants to mount a robust, spreading infection in CEM-SS cells. It remains unclear whether transcriptional activation of the HIV-1 LTR occurs before or after the change in epigenetic marks on the viral DNA, and this is something we wish to address, including in primary T cells. We now report preliminary data, obtained in 293T cells, showing that the HSV-1 ICP0 protein can also rescue IN- HIV-1 gene expression, in this case by apparently directly regulating the epigenetic status of unintegrated HIV-1 DNA. We aim to extend this analysis to T cells to see if ICP0 is indeed rescuing IN- HIV-1 gene expression via a different mechanism than Tax. Finally, while the factors that recognize and silence IN- MLV have been identified by the Goff laboratory as the HUSH complex acting in concert with NP220, we show that these factors are not required to silence IN- HIV-1. A key goal of this application is therefore to perform an unbiased CRISPR/Cas genetic screen to identify the human innate immune factors that perform this task and then define their mechanism of action.

抽象的 在细胞质中逆转录后，逆转录病毒DNA裸露地进入细胞核，然后被检测到 由目前未知的先天免疫因素引起，并涂有抑制性染色质。然而，逆转录病毒 编码整合酶（IN），允许原病毒DNA插入到转录活性区域 宿主基因组，然后导致逆转录病毒 DNA 上的抑制性染色质标记被替换 侧翼宿主染色质的活性标记特征。如果 IN 活性被诱变或 药物，然后未整合的逆转录病毒 DNA 仍被抑制性染色质覆盖并被转录 沉默了。这笔赠款提议有两个目标。首先，我假设如果未整合的 HIV-1 DNA 是 表观遗传沉默，那么应该有可能激活该 DNA，并拯救 IN-HIV-1 的复制， 通过直接激活 HIV-1 LTR 启动子和/或直接逆转表观遗传沉默。我们 现在已经表明，HTLV-1 Tax 蛋白的表达是细胞 NK-kB/Rel 转录的有效激活剂 因子，诱导 RelA 和 RelB 募集到 HIV-1 LTR 中存在的 NF-kB 位点，并且还可以防止 或逆转未整合的 HIV-1 DNA 的表观遗传沉默，使 IN-HIV-1 突变体能够安装强大的、 CEM-SS 细胞中传播感染。目前尚不清楚 HIV-1 LTR 的转录激活是否 发生在病毒 DNA 表观遗传标记变化之前或之后，这是我们希望解决的问题， 包括原代T细胞。我们现在报告在 293T 细胞中获得的初步数据，表明 HSV-1 ICP0 蛋白还可以挽救 IN-HIV-1 基因表达，在这种情况下，显然是通过直接调节 未整合的 HIV-1 DNA 的表观遗传状态。我们的目标是将这种分析扩展到 T 细胞，看看 ICP0 是否确实是 通过与 Tax 不同的机制来拯救 IN-HIV-1 基因表达。最后，虽然认识到的因素 Goff 实验室已将 IN-MLV 和沉默 IN-MLV 确定为 HUSH 复合体，与 NP220，我们证明这些因素不需要沉默 IN-HIV-1。因此，该应用程序的一个关键目标是 进行公正的 CRISPR/Cas 遗传筛选，以确定发挥作用的人类先天免疫因子 这项任务然后定义他们的行动机制。