Novel mechanism of integrase (IN) resistance to Dolutegravir through epistatic interactions between IN and the nucleocapsid and polypurine tract regions of HIV-1

项目总结 整合是HIV-1复制的关键，整合是由整合酶(IN)完成的。一类抑制血管紧张性疾病的药物 HIV整合酶的链转移(ST)功能，称为IN链转移抑制物(INSTI)，包括批准的 药物Raltegravir(Ral)、elvitegravir(EVG)(第1代)和Drotegravir(DTG)、Bictegravir(第2代) 代)。DTG具有比RAL或EVG更高的抗性遗传屏障，是世界推荐的 卫生组织作为低收入和中等收入国家一线治疗方案的替代方案 (LMIC)。DTG抗性的选择是罕见的，但确实存在，目前还不是很清楚。的确有 临床试验中基于DTG的治疗失败的越来越多的证据(Viking-3研究) 靶向IN基因突变。我们的主要假设是IN外的突变可以传递药物 通过我们称之为上位性的间接相互作用来抵抗IN靶向药物。科学的前提是 研究这些相互作用是基于两个关键证据的：第一，令人惊讶的初步 来自体外连续传代实验的数据，存在增加的DTG，一种DTG抗性 发现了位于IN外的突变。用重组病毒进行的实验证实对DTG的抗性 该突变在E157Q IN多态的存在下表现出更强的抗性。此外，深 测序分析表明，与野生型感染相比，含有突变的病毒导致了 更多的插入、删除和非规范的长末端重复序列(LTR)在2-LTR循环中结束并整合 病毒DNA。其次，最近的一项独立研究基于类似的系列通道实验，确定了变化 在耐DTG病毒中的3‘-多尿路(3’-PPT)的一般G-束区(Malet等人，2017年)。 随后，在一名DTG治疗失败的患者中，报告了不同的3‘-PPT变化。然而， 由于相互矛盾的假说，3‘-PPT突变导致DTG耐药的机制尚不清楚 缺乏实验验证。我们的假设是IN外的突变可以通过以下方式影响DTG抵抗 在INSTI结合位点改变LTR末端。这一假设将由一个专家团队进行测试，该团队包括 PiS Sarafianos(生化、病毒学耐药机制)、Pi Lyumkis(单粒子冷冻 Intasome/药物复合体)和Pi Hchiya(病毒学，耐药性)，由HIV in Hughes专家支持 (NCI)和Kvaratskhelia(科罗拉多州U)，使用病毒学、生化和结构工具来解决目标 通过上位性研究DTG抗性的病毒学、生物学和结构机制 互动。这些创新性的研究将有助于阐明INSTI耐药性的分子机制。 通过IN基因外的突变和影响INSTI结合位点的上位性相互作用。他们是 具有重要意义，并将有助于解释在IN没有突变的情况下基于DTG的方案临床失败的原因。

PROJECT SUMMARY Integration is essential for HIV-1 replication and is completed by integrase (IN). A class of drugs which inhibit the strand transfer (ST) function of HIV integrase, called IN strand transfer inhibitors (INSTIs), includes approved drugs raltegravir (RAL), elvitegravir (EVG) (1st generation) and dolutegravir (DTG), bictegravir (BIC) (2nd generation). DTG has a higher genetic barrier to resistance than RAL or EVG, and is recommended by the World Health Organization as an alternative to efavirenz in first-line regimens in low- and middle-income countries (LMICs). Selection for DTG resistance is rare, but does exist and is currently not well understood. There is mounting evidence for failure of DTG-based treatment in clinical trials (VIKING-3 study) in the absence of mutations in the targeted IN gene. Our overarching hypothesis is that mutations outside IN can impart drug resistance to IN-targeting drugs through indirect interactions that we call epistatic. The scientific premise for studying these interactions is soundly grounded on two key pieces of evidence: First, in surprising preliminary data from in vitro serial passage experiments in the presence of increasing amounts of DTG, a DTG resistance mutation located outside IN was discovered. Experiments with recombinant viruses validated DTG resistance of this mutation and showed enhanced resistance in the presence of the E157Q IN polymorphism. Moreover, deep sequencing analyses showed that compared to infection by wild-type, mutant–containing viruses resulted in more insertions, deletions, and non-canonical long terminal repeat (LTR) ends in 2-LTR circles and integrated viral DNA. Second, a recent independent study based on similar serial passage experiments, identified changes at the general G-tract region of the 3’-polypurine tract (3’-PPT) in a DTG-resistant virus (Malet et al., 2017). Subsequently, different 3’-PPT changes were reported in a patient that failed DTG therapy. However, the mechanism of DTG resistance through mutations at the 3’-PPT remains unclear due to conflicting hypotheses and lack of experimental validation. Our hypothesis is that mutations outside IN can affect DTG resistance by altering the LTR ends at the INSTI binding site. This hypothesis will be tested by a team of experts that includes PIs Sarafianos (biochemical, virological drug resistance mechanisms), PI Lyumkis (single particle cryo-EM on intasome/drug complexes) and PI Hachiya (virology, drug resistance) with the support by HIV IN experts Hughes (NCI) and Kvaratskhelia (U Colorado), using virological, biochemical, and structural tools to address the aims to investigate the virological, biological, and structural mechanisms of DTG resistance through epistatic interactions. These innovative studies will help elucidate the molecular mechanisms of INSTI resistance through epistatic interactions via mutations that are outside the IN gene and affect the INSTI-binding site. They are significant and will help explain clinical failures to DTG-based regimens in the absence of mutations in IN.