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

点击翻译按钮获取中文摘要

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.