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 人类T细胞白血病病毒1型(HTLV-1)可导致脊髓病、成人T细胞白血病-淋巴瘤等 炎症性疾病。关于有效的抗HTLV-1和抗病毒药物的信息非常有限 如何以及何时使用它们。一类新的抗病毒药物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整合酶多聚体的增强 蛋白质，阻止其与晶状体上皮衍生因子(LEDGF)和基因组RNA(GRNA)的相互作用。 初步信息显示，AlLINI具有抗HTLV-1感染的活性。因此，我们建议研究 这些药物对HTLV-1的活性及其作用机制，如下所述。 目的1：通过检测原代淋巴细胞和淋巴细胞的活性来评估Allini抗HTLV-1的活性。 通过鉴定和鉴定HTLV-1的耐阿利尼变异体。我们会用Allini- 抗性HTLV-1作为AIMS 2和3中的对照。我们将对耐阿利尼的HTLV-1进行特性分析，以便 通过序列分析、定点定位确定导致耐药的关键残基 突变和药物敏感性研究。 目的2：通过检测大蒜素对HTLV-1基因组RNA包装的影响，评估大蒜素对HTLV-1基因组RNA包装的影响。 HTLV-1与gRNA的结合及其对病毒颗粒包装的影响我们将使用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 用蔗糖梯度离心法将gRNA包装成病毒颗粒和病毒核心并进行分析 采用数字液滴RT-PCR技术。 目的3：通过检测大蒜素对细胞间HTLV-1感染的影响，评估大蒜素对细胞间HTLV-1感染的影响。 感染的早期步骤，包括反转录和整合，并确定ALLINI是否改变整合酶 地点专一性。我们将确定大蒜素对早期和晚期RT产物合成的影响，以及对 整合的效率。在Allini存在或不存在的情况下，整合位点将由Linker绘制 连接介导的聚合酶链式反应和下一代序列分析。 这些研究的完成将为HTLV-1复制和Allini活性提供新的见解。

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.