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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.

摘要在细胞质中反转录后，逆转录病毒DNA裸体进入细胞核，然后被检测到由目前未知的先天免疫因子和包被抑制性染色质。然而，逆转录病毒编码整合酶(IN)，它允许前病毒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具有侧翼宿主染色质特征的活性标记。如果IN活性被诱变或药物，那么未整合的逆转录病毒DNA仍然被抑制染色质覆盖，并在转录上沉默了。这项拨款提案有两个目标。首先，我假设如果未整合的HIV-1 DNA是表观遗传沉默，那么就有可能激活DNA，挽救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。我们现已证明，HTLV-1 Tax蛋白的表达是细胞NK-kB/Rel转录的有效激活因子因素，诱导RelA和RelB的募集到存在于HIV-1LTR中的NF-kB位点，并防止或逆转未整合的HIV-1DNA的表观遗传沉默，使IN-HIV-1突变体能够安装一个强大的，在CEM-SS细胞中传播感染。目前尚不清楚HIV-1 LTR的转录激活发生在病毒DNA的表观遗传标记变化之前或之后，这是我们希望解决的问题，包括在初级T细胞中。我们现在报告在293T细胞中获得的初步数据，显示HSV-1 ICP0蛋白还可以挽救IN-HIV-1基因的表达，在这种情况下，显然是通过直接调节非整合型HIV-1DNA的表观遗传学状态。我们的目标是将这一分析扩展到T细胞，看看ICP0是否真的是通过与税收不同的机制拯救IN-HIV-1基因表达。最后，虽然认识到的因素和Silent In-MLV已经被戈夫实验室确定为与 NP220，我们表明这些因素并不是沉默IN-HIV-1所必需的。因此，此应用程序的一个关键目标是进行无偏CRISPR/Cas基因筛查以确定人类先天免疫因子这项任务，然后定义他们的行动机制。