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）引起脊髓病、成人T细胞白血病-淋巴瘤和其他疾病。 炎症性疾病关于针对HTLV-1和HTLV-2的有效抗病毒剂的信息非常有限。 如何以及何时使用它们。一类新的抗病毒剂是变构整合酶抑制剂或ALLINI。 ALLINI不抑制HIV-1整合酶的酶功能，而是引起HIV-1整合酶的多聚化增强。 该蛋白质，阻止其与透镜上皮衍生因子（LEDGF）和基因组RNA（gRNA）的相互作用。 初步信息显示ALLINI抗HTLV-1感染的活性。因此，我们建议审查 这些药物对HTLV-1的活性及其作用机制，进行了以下研究。 目的1：通过检查原代淋巴细胞和淋巴细胞中的活性来评估ALLINI抗HTLV-1的活性。 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- 在目的2和3中作为对照的抗HTLV-1。我们将表征抗ALINI的HTLV-1，以便 通过序列分析、定点突变和基因测序，确定导致耐药性的关键IN残基， 突变和药物敏感性研究。 目的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的结合和对gRNA包装到病毒颗粒中的影响。我们将使用CLIP-seq 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 通过蔗糖梯度离心纯化gRNA包装到病毒颗粒和病毒核心中，并分析 通过数字液滴RT-PCR。 目的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 位点特异性我们将确定ALLINI对早期和晚期RT产物合成的影响，以及对 整合的效率。在存在或不存在ALLINI的情况下，整合位点将通过接头进行定位。 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。