Allosteric integrase inhibitor effects on human T-cell leukemia virus infection

变构整合酶抑制剂对人T细胞白血病病毒感染的影响

• 批准号: 10451085
• 负责人: Sebla B. Kutluay
• 金额: $ 23.63万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-13 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10451085
• 关键词: Adult T-Cell Leukemia/Lymphoma; Affect; Anti-Retroviral Agents; Antiviral Agents; Antiviral Therapy; Binding; Biochemistry; C-terminal; Capsid; Catalytic Domain; Cells; Centrifugation; Clinical Trials; Collaborations; Complex; DNA; DNA Binding Domain; Data; Dendritic Cells; Development; Dinucleoside Phosphates; Disease; Drug resistance; Epithelial; Event; Exposure to; Goals; HIV Integrase Inhibitors; HIV-1; HIV-1 integrase; HIV-2; HTLV Infection; Human Activities; Human T-Cell Leukemia Viruses; Human T-lymphotropic virus 1; Infection; Inflammatory; Integrase; Integrase Inhibitors; Lamivudine; Ligation; Lymphocyte; Maps; Mediating; Molecular; Molecular Biology; Molecular Cloning; Morphogenesis; Morphology; Mutation; N-terminal; Nucleosides; Nucleotides; Pharmaceutical Preparations; Pharmacology; Primary Infection; Process; Protease Inhibitor; Proteins; RNA; RNA-Directed DNA Polymerase; Reaction; Recombinants; Research Personnel; Resistance; Resistance development; Retroviridae; Retroviridae Infections; Reverse Transcriptase Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Ribonucleoproteins; Route; Satellite Viruses; Sequence Analysis; Site; Specificity; Spinal Cord Diseases; Stavudine; Sucrose; Tenofovir; Therapeutic; Translating; Vaccines; Variant; Viral; Virion; Virus; Virus Assembly; Virus Diseases; Virus Inhibitors; Virus Replication; Zidovudine; base; crosslinking and immunoprecipitation sequencing; digital; drug sensitivity; experience; flexibility; genomic RNA; inhibitor; insight; integration site; lens; lens epithelium-derived growth factor; leukemia/lymphoma; next generation; non-nucleoside reverse transcriptase inhibitors; novel; particle; prevent; resistant strain; vector; viral DNA; viral RNA; viral transmission

Abstract – Allosteric Integrase Inhibitor Effects on Human T-Cell Leukemia Virus Infection Human T-cell leukemia virus type 1 (HTLV-1) causes myelopathy, adult T-cell leukemia-lymphoma, and other inflammatory disorders. Very limited information is available on effective antiviral agents against HTLV-1 and how and when to use them. A new class of antiviral agents are the allosteric integrase inhibitors or ALLINIs. ALLINIs do not inhibit the enzymatic function of HIV-1 integrase, but rather cause enhanced multimerization of the protein, preventing its interaction with lens epithelial derived factor (LEDGF) and genomic RNA (gRNA). Preliminary information shows activity of ALLINIs against HTLV-1 infection. Therefore we propose to examine the activity of these agents against HTLV -1 and their mechanism of action, with the following studies. Aim 1: Assess the activity of ALLINIs against HTLV-1 by examining activity in primary lymphocytes and dendritic cells, and by identifying and characterizing ALLINI-resistant variants of HTLV-1. We will use ALLINI- resistant HTLV-1 as a control in Aims 2 and 3. We will characterize the ALLINI-resistant HTLV-1 in order to identify the key IN residues that are responsible for drug-resistance through sequence analysis, site-directed mutational, and drug-sensitivity studies. Aim 2: Assess effect of ALLINIs on HTLV-1 genomic RNA packaging by examining the effect of ALLINIs on HTLV-1 IN binding to gRNA and effect on packaging of gRNA into virus particles. We will use CLIP-seq to examine IN binding to HTLV-1 gRNA, and determine if ALLINIs affect binding. We will determine the efficiency of gRNA packaging into virus particles and viral cores purified by sucrose gradient centrifugation and analyzed by digital droplet RT-PCR. Aim 3: Assess the effect of ALLINIs on cell-to-cell HTLV-1 infection by examining the effect of ALLINIs on early steps of infection, including reverse transcription and integration, and determine if ALLINIs alter integrase site specificity. We will determine the effect of ALLINIs on synthesis of early and late RT products, and on the efficiency of integration. Sites of integration in the presence or absence of ALLINI will be mapped by linker ligation-mediated PCR and next-generation sequence analysis. Completion of these studies will provide new insights into HTLV-1 replication and ALLINI activity.

摘要 – 变构整合酶抑制剂对人 T 细胞白血病病毒感染的影响 人类 T 细胞白血病病毒 1 型 (HTLV-1) 会导致脊髓病、成人 T 细胞白血病-淋巴瘤等 炎症性疾病。关于针对 HTLV-1 和 HTLV-1 的有效抗病毒药物的信息非常有限 如何以及何时使用它们。一类新型抗病毒药物是变构整合酶抑制剂或 ALLINI。 ALLINI 不会抑制 HIV-1 整合酶的酶功能，而是会导致 HIV-1 整合酶的多聚化增强 蛋白质，防止其与晶状体上皮衍生因子 (LEDGF) 和基因组 RNA (gRNA) 相互作用。 初步信息显示 ALLINI 具有抗 HTLV-1 感染的活性。因此我们建议审查 这些药剂抗HTLV -1 的活性及其作用机制，有如下研究。 目标 1：通过检查原代淋巴细胞和 树突状细胞，并通过鉴定和表征 HTLV-1 的 ALLINI 抗性变体。我们将使用ALLINI- 耐药 HTLV-1 作为目标 2 和 3 中的对照。我们将表征 ALLINI 耐药 HTLV-1，以便 通过序列分析、位点定向识别导致耐药性的关键 IN 残基 突变和药物敏感性研究。 目标 2：通过检查 ALLINI 对 HTLV-1 基因组 RNA 包装的影响来评估 ALLINI 对 HTLV-1 基因组 RNA 包装的影响 HTLV-1 IN 与 gRNA 结合并对 gRNA 包装到病毒颗粒中的影响。我们将使用 CLIP-seq 来 检查 IN 与 HTLV-1 gRNA 的结合，并确定 ALLINI 是否影响结合。我们将确定效率 gRNA 包装成病毒颗粒和病毒核心，通过蔗糖梯度离心纯化并分析 通过数字液滴 RT-PCR。 目标 3：通过检查 ALLINI 对细胞间 HTLV-1 感染的影响来评估 ALLINI 对细胞间 HTLV-1 感染的影响。 感染的早期步骤，包括逆转录和整合，并确定 ALLINI 是否改变整合酶 站点特异性。我们将确定 ALLINI 对早期和晚期 RT 产品合成的影响，以及对 整合效率。存在或不存在 ALLINI 时的整合位点将由链接器映射 连接介导的 PCR 和下一代序列分析。 这些研究的完成将为 HTLV-1 复制和 ALLINI 活性提供新的见解。