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

基本信息

批准号：
10369728
负责人：
BRYAN R. CULLEN
金额：
$ 19.74万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10369728
关键词：
Address 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

项目摘要

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进入核核，然后被检测到通过目前未知的先天免疫因子，并涂有抑制性染色质。但是，逆转录病毒编码一个集成酶（in），该集成酶允许病毒DNA插入宿主基因组，然后导致逆转录病毒DNA上的抑制性染色质标记被替代侧翼宿主染色质的活性标记特征。如果在活动中被诱变阻断或药物，然后，未整合的逆转录病毒DNA仍被抑制性染色质覆盖，并且是转录的沉默。该赠款提出有两个目标。首先，我假设如果没有集成的HIV-1 DNA为表观遗传沉默，然后应该有可能激活该DNA，并挽救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。我们现在已经表明了HTLV-1税收蛋白的表达，这是细胞NK-KB/rel转录的有效激活因子因素，诱导RELA和RELB募集到HIV-1 LTR中存在的NF-KB位点，也可以防止或逆转未集成的HIV-1 DNA的表观遗传沉默，使HIV-1突变体可以安装强大的，在CEM-SS细胞中传播感染。尚不清楚HIV-1 LTR的转录激活是否发生在病毒DNA上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。现在，我们报告了在293T细胞中获得的初步数据，表明HSV-1 ICP0蛋白还可以通过显然直接调节这种情况来挽救HIV-1基因表达未集成的HIV-1 DNA的表观遗传状态。我们旨在将此分析扩展到T细胞，以查看ICP0是否确实是通过与税收不同的机制来营救HIV-1基因表达。最后，而识别的因素戈夫实验室已经确定了寂静的In-mlv是与之共同行动 NP220，我们表明这些因素不需要沉默HIV-1。因此，此应用程序的关键目标是执行无偏见的CRISPR/CAS遗传筛选，以识别执行的人类先天免疫因素这项任务，然后定义他们的作用机理。